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Sample records for posttesticular sperm maturation

  1. Posttesticular sperm maturation, infertility, and hypercholesterolemia

    PubMed Central

    Whitfield, Marjorie; Pollet-Villard, Xavier; Levy, Rachel; Drevet, Joël R; Saez, Fabrice

    2015-01-01

    Cholesterol is a key molecule in the mammalian physiology of especial particular importance for the reproductive system as it is the common precursor for steroid hormone synthesis. Cholesterol is also a recognized modulator of sperm functions, not only at the level of gametogenesis. Cholesterol homeostasis regulation is crucial for posttesticular sperm maturation, and imbalanced cholesterol levels may particularly affect these posttesticular events. Metabolic lipid disorders (dyslipidemia) affect male fertility but are most of the time studied from the angle of endocrine/testicular consequences. This review will focus on the deleterious effects of a particular dyslipidemia, i.e., hypercholesterolemia, on posttesticular maturation of mammalian spermatozoa. PMID:26067871

  2. Post-Testicular Sperm Maturation: Centriole Pairs, Found in Upper Epididymis, are Destroyed Prior to Sperm’s Release at Ejaculation

    PubMed Central

    Simerly, C.; Castro, C.; Hartnett, C.; Lin, C. C.; Sukhwani, M.; Orwig, K.; Schatten, G.

    2016-01-01

    The fertilizing sperm’s lengthiest unchartered voyage is through the longest, least-investigated organ in a man’s body – the Epididymis. Over six meters long in men, ~80 meters in stallions and over one-hundred times a mouse’s body length, there are few functions known aside from sperm storage and nutrition. While spermatogenesis is completed in the testes, here we demonstrate sperm centriole reduction occurs within the epididymis. Investigations of GFP-CENTR mice and controls demonstrate both the presence of centriole pairs in the upper caput region of the epididymis and, the destruction, first, of the distal and, then, of the proximal centriole as the sperm transits to the cauda and vas deferens in preparation for its climactic release. These centrioles can neither recruit γ-tubulin nor nucleate microtubules when eggs are inseminated or microinjected, yet numerous maternally-nucleated cytasters are found. These sperm centrioles appear as vestigial basal bodies, destroyed in the mid-to-lower corpus. Post-testicular sperm maturation, in which sperm centrioles found in the caput are destroyed prior to ejaculation, is a newly discovered function for the epididymis. PMID:27534805

  3. Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals

    PubMed Central

    Shea, Jeremy M.; Boskovic, Ana; Derr, Alan G.; Bing, Xin Y.; Belleannee, Clemence; Kucukural, Alper; Serra, Ryan W.; Sun, Fengyun; Song, Lina; Carone, Benjamin R.; Ricci, Emiliano P.; Li, Xin Z.; Fauquier, Lucas; Moore, Melissa J.; Sullivan, Robert; Mello, Craig C.; Garber, Manuel; Rando, Oliver J.

    2016-01-01

    Several recent studies link parental environments to phenotypes in subsequent generations. Here, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein restriction in mice affects small RNA levels in mature sperm, with decreased let-7 levels and increased levels of 5’ fragments of glycine tRNAs. tRNA fragments are scarce in testicular sperm, but are gained as sperm mature in the epididymis. Epididymosomes – vesicles that fuse with sperm during epididymal transit – carry RNA payloads matching those of mature sperm, and deliver RNAs to immature sperm in vitro. Functionally, tRNA-Gly-GCC fragments repress genes associated with the endogenous retroelement MERVL, both in ES cells and embryos. Our results shed light on small RNA biogenesis, and its dietary regulation, during post-testicular sperm maturation, and link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo. PMID:26721685

  4. Monotremes provide a key to understanding the evolutionary significance of epididymal sperm maturation.

    PubMed

    Nixon, Brett; Ecroyd, Heath W; Dacheux, Jean-Louis; Jones, Russell C

    2011-01-01

    It has been widely accepted that mammalian spermatozoa are infertile when they leave the testes and require a period of maturation in both the epididymis and the female reproductive tract before acquiring the ability to fertilize an oocyte. However, the necessity for such a complex process of posttesticular sperm maturation appears to be unique to mammals because it is well established that these processes do not directly influence the fertilizing ability of the spermatozoa of birds, reptiles, and other lower vertebrates. Because of their key evolutionary position and form of reproduction, we contend that monotremes (platypus and echidna) provide a unique model for resolving why these processes are necessary. In the present review, we examine evidence that the epididymal maturation of monotreme spermatozoa is far less complex than in other mammals. However, a unique feature of the monotreme epididymis lies in its ability to promote the formation of elaborate sperm bundles that serve to greatly enhance the cells' motility. It is suggested that this intriguing cooperative strategy used by monotreme sperm represents an early form of epididymal maturation that appears to have been elaborated upon during the evolution of higher mammals, possibly as an adaptation for sperm competition.

  5. Olfactory receptors are displayed on dog mature sperm cells

    PubMed Central

    1993-01-01

    Olfactory receptors constitute a huge family of structurally related G protein-coupled receptors, with up to a thousand members expected. We have shown previously that genes belonging to this family were expressed in the male germ line from both dog and human. The functional significance of this unexpected site of expression was further investigated in the present study. We demonstrate that a few dog genes representative of various subfamilies of olfactory receptors are expressed essentially in testis, with little or no expression in olfactory mucosa. Other randomly selected members of the family show the expected site of expression, restricted to the olfactory system. Antibodies were generated against the deduced amino acid sequence of the most abundantly expressed olfactory receptor gene in dog testis. The purified serum was able to detect the gene product (DTMT receptor) in late round and elongated spermatids, as well as in the cytoplasmic droplet that characterizes the maturation of dog sperm cells, and on the tail midpiece of mature spermatozoa. Western blotting further confirmed the presence of a 40-kD immunoreactive protein in the membrane of mature sperm cells. Altogether , these results demonstrate that the main expression site of a subset of the large olfactory receptor gene family is not olfactory mucosa but testis. This expression correlates with the presence of the corresponding protein during sperm cell maturation, and on mature sperm cells. The pattern of expression is consistent with a role as sensor for unidentified chemicals possibly involved in the control of mammalian sperm maturation, migration, and/or fertilization. PMID:8253843

  6. Dual function of the selenoprotein PHGPx during sperm maturation.

    PubMed

    Ursini, F; Heim, S; Kiess, M; Maiorino, M; Roveri, A; Wissing, J; Flohé, L

    1999-08-27

    The selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) changes its physical characteristics and biological functions during sperm maturation. PHGPx exists as a soluble peroxidase in spermatids but persists in mature spermatozoa as an enzymatically inactive, oxidatively cross-linked, insoluble protein. In the midpiece of mature spermatozoa, PHGPx protein represents at least 50 percent of the capsule material that embeds the helix of mitochondria. The role of PHGPx as a structural protein may explain the mechanical instability of the mitochondrial midpiece that is observed in selenium deficiency.

  7. Is sperm motility maturation affected by static magnetic fields?

    PubMed Central

    Tablado, L; Pérez-Sánchez, F; Soler, C

    1996-01-01

    Kinematic parameters were evaluated in mouse epididymal extracts to monitor maturation of sperm movement in animals exposed to static magnetic fields using the Sperm-Class Analyzer computerized image analysis system. For this purpose, animals were exposed to a field of 0.7 T generated by a permanent magnet over 10 or 35 days for either 1 or 24 hr/day. The values of the motion endpoints were similar in animals used as controls and in those exposed to the nonionizing radiation, whatever the period of exposure or daily dosage. Changes in motility were observed in all groups: the percentage of total motile and progressive motile spermatozoa increased during passage through the epididymis, with major changes between the caput and corpus epididymides, and the pattern of swimming changed clearly towards more rapid and straighter trajectories. The processes of initiation of sperm motility and maturation of displacement patterns were not then affected by magnetic treatment. Moreover, it appears that sperm production is unaffected because no changes were observed in testicular or epididymal weights after exposure to static magnetic fields. Images Figure 1. Figure 2. Figure 3. PMID:8959411

  8. Sperm maturation in dogs: sperm profile and enzymatic antioxidant status in ejaculated and epididymal spermatozoa.

    PubMed

    Angrimani, D S R; Lucio, C F; Veiga, G A L; Silva, L C G; Regazzi, F M; Nichi, M; Vannucchi, C I

    2014-09-01

    Spermatozoa become more susceptible to the attack of reactive oxygen species during maturation. To avoid oxidative damage, the epididymis must provide the necessary antioxidant protection. The aim of this study was to compare the canine sperm profile and the enzymatic antioxidant status of the ejaculated fractions and samples collected from the different segments of the epididymis (caput, corpus and cauda). Five adult dogs were used, and after 1-3 weeks, subsequently to bilateral orchiectomy and epididymal storage, sperm samples were collected from the different segments of the epididymis. Samples were evaluated for conventional microscopy and computer-assisted motility analysis: sperm plasma membrane permeability and the activity of the antioxidant enzymes catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD). Samples collected from the caput and corpus showed lower values for most of the motility variables evaluated, indicating different levels of immaturity. Catalase activity was observed only in ejaculated samples. Conversely, GPx activity was higher in the cauda epididymidis. Correlations were found between SOD and GPx and SOD and sperm motility in the epididymal cauda and corpus, highlighting the importance of the enzymes for the protection of spermatozoa during the transit along the epididymis.

  9. Electron microscopic observation of the sagittal structure of Drosophila mature sperm.

    PubMed

    Yasuno, Yusaku; Yamamoto, Masa-Toshi

    2014-09-01

    Observation of sperm development and determination of their morphological characteristics are very important to the understanding of phylogenetic relationships and the study of sperm function during fertilization. Although ultrastructural studies of sperm development in the testes of the fruit fly Drosophila have been performed, there are few reports describing electron microscopic morphology of mature sperm, that is, those released from the testes to the seminal vesicles. Here, we present the first report of the sagittal organization of Drosophila sperm head and neck regions by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The head and tail structures of a mature sperm, for example, the acrosome, nucleus, and flagellum, were easy to distinguish by the morphological characteristics of the sperm surface by SEM. The morphological relationships between the surface and internal structures of mature sperm were confirmed by observing longitudinal sections with TEM. Our approach overcame the technical difficulties involved in sample preparation for electron microscopic observation of the Drosophila mature sperm head, and therefore, this study serves as an important foundation for future genetic dissection of sperm ultrastructure and function in male sterile mutants.

  10. Reactive oxygen species production and antioxidant enzyme activity during epididymal sperm maturation in Corynorhinus mexicanus bats.

    PubMed

    Arenas-Ríos, Edith; Rosado García, Adolfo; Cortés-Barberena, Edith; Königsberg, Mina; Arteaga-Silva, Marcela; Rodríguez-Tobón, Ahiezer; Fuentes-Mascorro, Gisela; León-Galván, Miguel Angel

    2016-03-01

    Prolonged sperm storage in the epididymis of Corynorhinus mexicanus bats after testicular regression has been associated with epididymal sperm maturation in the caudal region, although the precise factors linked with this phenomenon are unknown. The aim of this work is to determine the role of reactive oxygen species (ROS) and changes in antioxidant enzymatic activity occurring in the spermatozoa and epididymal fluid over time, in sperm maturation and storage in the caput, corpus and cauda of the bat epididymis. Our data showed that an increment in ROS production coincided with an increase in superoxide dismutase (SOD) activity in epididymal fluid and with a decrease in glutathione peroxidase (GPX) activity in the spermatozoa in at different time points and epididymal regions. The increase in ROS production was not associated with oxidative damage measured by lipid peroxidation. The results of the current study suggest the existence of a shift in the redox balance, which might be associated with sperm maturation and storage.

  11. Spermatogenesis, the mature sperm, and sperm egg association in Nematospiroides dubius

    NASA Technical Reports Server (NTRS)

    Clark, W. H., Jr.

    1974-01-01

    Nematode spermatogenesis was investigated using the strongyloid Nematospiroides dubius. The primary spermatocytes, development of spermatids, and changes in the sperm in the female tract are described.

  12. Genetic variation for resistance to high temperature stress of mature sperm – a study in Drosophila

    PubMed Central

    Barker, J. S. F.

    2017-01-01

    Genetic variation for resistance to heat stress has been found for a number of life-history components in Drosophila species. For male and female fertility (or sterility), stress resistance of the parents is confounded with stress resistance of the haploid gametes. Many genes are known to influence male fertility in Drosophila melanogaster. Some may carry temperature sensitive alleles that reduce fertility through effects on mature sperm when exposed to heat stress. In this study, sperm from each of 320 males were either not heat shocked (control) or exposed to a heat shock (36.9°C for 2 hours) either in the male testes or in the female reproductive tract. We did not detect any temperature sensitive sterility alleles. These results are relevant in relation to haploid gene expression and the findings of considerable amounts of mRNA in mature sperm, potentially important for sperm function and fertilization. PMID:28358879

  13. Glucose Regulated Protein 78 Phosphorylation in Sperm Undergoes Dynamic Changes during Maturation

    PubMed Central

    Lobo, Vivian; Rao, Parimala; Gajbhiye, Rahul; Kulkarni, Vijay; Parte, Priyanka

    2015-01-01

    GRP78, a resident endoplasmic reticulum (ER) chaperone involved in protein transport, folding and assembly, has been reported in sperm. It is shown to be localized in the neck region of human sperm. We have previously reported GRP78 to be less phosphorylated in asthenozoosperm.The present study aimed to determine whether sperm GRP78 undergoes phosphorylation changes during epididymal maturation and whether there are any differences in GRP78 phosphoforms in asthenozoosperm vis-à-vis normozoosperm. Testicular- and cauda epididymal- sperm from adult male Holtzman rats, and semen ejaculates collected from normal and asthenozoospermic individuals were investigated. DIGE carried out to determine phosphorylation of GRP78 in asthenozoosperm and normal sperm reveals a shift in the location of GRP78 of asthenozoosperm towards the alkaline pH, indicative of reduced GRP78 phosphorylation. Immunoprecipitation studies using antibodies specific to GRP78, serine-, threonine-, and tyrosine phosphorylation and Pan phospho antibody demonstrates GRP78 to be phosphorylated at all three residues in rat spermatozoa. Phosphatase assays using Calf intestinal alkaline phosphatase and Lambda protein phosphatase followed by nanofluidic proteomic immunoassay (NIA) show that in rat, GP4.96, GP4.94 and GP4.85 are the three phosphoforms in mature (caudal) sperm as against two phosphoforms GP4.96and GP4.94in immature (testicular) sperm. In mature human sperm GP5.04, GP4.96, and GP4.94were the 3 phosphoforms observed. GP4.94[P = 0.014]andGP5.04 [P = 0.02] are significantly reduced in asthenozoosperm. Ours is the first report indicating GRP78 in sperm to be phosphorylated at serine, threonine and tyrosine residues contrary to published literature reporting GRP78 not to be tyrosine phosphorylated. We report the presence of GRP78 phosphoforms in rat- and human- sperm and our data suggest that GRP78 phosphorylation in sperm undergoes spatial reorganization during epididymal maturation. Significant

  14. Normozoospermic versus teratozoospermic domestic cats: differential testicular volume, sperm morphometry, and subpopulation structure during epididymal maturation

    PubMed Central

    Gutiérrez-Reinoso, Miguel Angel; García-Herreros, Manuel

    2016-01-01

    Teratozoospermia (<40% morphologically normal spermatozoa/ejaculate) is a frequent phenomenon in feline species. This research was carried out to study the possible differences in testicular volume, differential sperm morphometric traits, and potential differences regarding the sperm subpopulational structure during epididymal sperm maturation in teratozoospermic feline donors. Epididymal sperm samples were collected from the caput (R1), corpus (R2), and cauda (R3) epididymidis in two donor groups (N: normozoospermic; T: teratozoospermic). Aliquots were assessed for concentration, viability, motility, and acrosomal integrity. Sperm morphometric descriptors from CASA-Morph analysis were analyzed by the Principal Component Analysis (PCA) and clustering analyses. Irrespective of the group analyzed, PCA revealed two Principal Components (PCs) for each epididymal region explaining more than the 93% of the variance. Surprisingly, the number of subpopulations remained constant in regions R1-R2-R3 irrespective of the donor group analyzed. However, the distribution of these subpopulations was found to be structurally different and strongly influenced by the epididymal region and the donor group. In conclusion, testicular morphometry and the sperm subpopulation structure were different in N and T donors. The alterations in subpopulations during epididymal maturation could be used as a potential clinical indicator of teratozoospermic individuals since an important influence of teratozoospermia on sperm subpopulation structure has been demonstrated. PMID:27624990

  15. Contribution of the secretory material of caecilian (amphibia: Gymnophiona) male Mullerian gland to motility of sperm: a study in Uraeotyphlus narayani.

    PubMed

    George, Jancy M; Smita, Mathew; Kadalmani, Balamuthu; Girija, Ramankutty; Oommen, Oommen V; Akbarsha, Mohammad A

    2005-02-01

    Caecilians are a unique group of limbless burrowing amphibians with discontinuous distribution. Several caecilian species are viviparous, and all practice internal fertilization. In amniotic vertebrates the sperm undergo post-testicular physiological maturation when they are initiated into motility under the influence of an epididymal secretion. Further, during ejaculation mammalian sperm are suspended in a fluid secreted by the male accessory sex glands, viz., prostate gland and seminal vesicles. Caecilians lack comparable glands, but still practice internal fertilization. Uniquely, male caecilians retain the Mullerian ducts in the adults as a pair of functional glands. It has long been hypothesized, based on indirect evidence, that the Mullerian gland would be a male accessory sex gland, secreting a fluid in which sperm are suspended during ejaculation and which would also provide nutritional support to the ejaculated sperm. In the present study, the secretory material of the Mullerian gland of Uraeotyphlus narayani was mixed with sperm obtained from the testis, and the changes in motility were recorded. Uraeotyphlus narayani sperm possess a perforatorium of the acrosome proceeding deep into the endonuclear canal of the nucleus. The midpiece is characterized by closely applied centrioles, the anterior ends of the axoneme and axial fiber, and a mitochondrial sheath. The long tail has an undulating membrane on one side, supported by the axoneme and an axial fiber. The live sperm possess a mitochondrial vesicle, also known as the cytoplasmic droplet, anywhere along the head and the midpiece, as in anuran sperm, which is shed from sperm that have ceased motility. Uraeotyphlus narayani sperm are motile the moment they are released directly from the testis, indicating that the sperm do not require post-testicular physiological maturation. On being mixed with the secretory material of the Mullerian gland, the spermatozoa are enhanced in speed as well as duration of

  16. Effect of dilution in sperm maturation media and time of storage on sperm motility and fertilizing capacity of cryopreserved semen of sex-reversed female rainbow trout.

    PubMed

    Judycka, Sylwia; Ciereszko, Andrzej; Dobosz, Stefan; Zalewski, Tomasz; Dietrich, Grzegorz J

    2017-05-01

    Masculinized females, also called neomales or sex-reversed females have a male phenotype but retain the female genotype (XX). Therefore, all spermatozoa produced in their functional testes carry an X chromosome, which is desired for the production of all-female rainbow trout populations. Semen of sex-reversed female rainbow trout is of low quality and in vitro maturation is required, which includes dilution of sperm suspensions with specially formulated maturation solutions. The aim of this study was to determine the effect of dilution in different maturation media on sperm quality (sperm motility characteristics and fertilizing capacity) of frozen/thawed sperm of sex-reversed female rainbow trout. The effect of time of post-thaw storage (0, 15, 60 and 120min) on semen quality was also tested. Sperm motility parameters and fertilization rate at the eyed and hatching stages were assessed for post-thaw semen diluted in different media. The cryopreservation procedure resulted in high post-thaw sperm motility of about 57% and did not differ from fresh semen. Unexpectedly, maturation media decreased sperm activation capacity immediately after dilution; however, sperm motility increased over time. Fertilization rates of frozen/thawed semen were high (71-87%) and did not differ significantly between experimental variants at any of tested periods of storage. Our results demonstrated that the effect of the maturation media on frozen/thawed sperm is different from that of fresh sperm. The progressive increase in post-thaw sperm motility in maturation media can potentially be applied to routine hatchery practice.

  17. Sperm nuclear expansion and meiotic maturation in normal and gynogenetic eggs of the scallop, Chlamys farreri

    NASA Astrophysics Data System (ADS)

    Pan, Ying; Li, Qi; Yu, Ruihai; Wang, Rucai

    2008-02-01

    Sperm nuclear expansion, meiosis and the association of the male and female pronuclei leading to the four-cell stage in normal Chlamys farreri eggs were observed under a fluorescence microscope. The effects of ultraviolet (UV) irradiation on the fertilizing sperm were also examined. Both normal and UV-irradiated sperm nuclei enlarged at three distinct phases (phase A, metaphase I; phase B, polar body formation; and phase C, female pronuclear development and expansion) that were temporally correlated with meiotic process of the maternal chromosomes. Sperm nuclei underwent a rapid, initial enlargement during phase A, but condensed slightly during phase B, then re-enlarged during phase C. The effects of UV irradiation were not apparent during transformation of the sperm nucleus into a male pronucleus, and there was not any apparent effect on meiotic maturation and development of the female pronucleus. However, the rate of expansion of the UV-irradiated sperm nuclei and the size of male pronuclei were reduced apparently. Unlike the female pronucleus, the male pronucleus derived from sperm genome inactivated by UV irradiation did not form chromosomes, but became a dense chromatin body (DCB). At mitotic anaphase, DCB did not participate in the karyokinesis of the first cleavage as evidenced by chromosomal nondisjunction, demonstrating the effectiveness of using UV irradiation to induce gynogenetic scallop embryos.

  18. Comparative Proteomic Identification of Mature and Immature Sperm in the Catfish Cranoglanis bouderius

    PubMed Central

    Xie, Shaolin; Wang, Chao; Lv, Zijun; Zou, Jixing

    2016-01-01

    To understand the molecular responses of mature and immature sperm in the catfish Cranoglanis bouderius, we used the iTRAQ proteomics approach to perform proteomic profiling of spermatogenesis in C. bouderius. As a result, 1,941 proteins were identified, including 361 differentially expressed proteins, 157 upregulated proteins and 204 downregulated proteins in mature sperm relative to immature sperm. All of the identified proteins were categorized into seven types of subcellular localizations and three molecular functions and were found to be involved in nine biological processes. All of the differential proteins were involved in 235 different pathways. Moreover, we found that the tricarboxylic acid (TCA) pathway played an important role in the energy metabolism of sperm and that the EABB pathway was involved in the mechanism of spermatogenesis. Our study is the first to use the iTRAQ-based proteomic approach to analyze the catfish sperm proteome, and the results we obtained using this approach are valuable for understanding the molecular mechanisms of fish spermatogenesis. PMID:26964044

  19. Comparative Proteomic Identification of Mature and Immature Sperm in the Catfish Cranoglanis bouderius.

    PubMed

    Chen, Jintao; Zhou, Aiguo; Xie, Shaolin; Wang, Chao; Lv, Zijun; Zou, Jixing

    2016-01-01

    To understand the molecular responses of mature and immature sperm in the catfish Cranoglanis bouderius, we used the iTRAQ proteomics approach to perform proteomic profiling of spermatogenesis in C. bouderius. As a result, 1,941 proteins were identified, including 361 differentially expressed proteins, 157 upregulated proteins and 204 downregulated proteins in mature sperm relative to immature sperm. All of the identified proteins were categorized into seven types of subcellular localizations and three molecular functions and were found to be involved in nine biological processes. All of the differential proteins were involved in 235 different pathways. Moreover, we found that the tricarboxylic acid (TCA) pathway played an important role in the energy metabolism of sperm and that the EABB pathway was involved in the mechanism of spermatogenesis. Our study is the first to use the iTRAQ-based proteomic approach to analyze the catfish sperm proteome, and the results we obtained using this approach are valuable for understanding the molecular mechanisms of fish spermatogenesis.

  20. Ability of Catalonian donkey sperm to penetrate zona pellucida-free bovine oocytes matured in vitro.

    PubMed

    Taberner, E; Morató, R; Mogas, T; Miró, J

    2010-04-01

    An experiment was designed to study the interaction between fresh/frozen-thawed donkey spermatozoa and zona pellucida (ZP)-free bovine oocytes in an attempt to develop a model for assessing cryopreserved Catalonian donkey sperm function. Semen from five donkeys was collected using an artificial vagina. Sperm motility and viability were immediately assessed and the semen sample cryopreserved. Sperm viability and motility were then reassessed immediately after thawing. The motion characteristics of the fresh and frozen-thawed spermatozoa were determined using a computer-assisted sperm analysis system. In vitro-matured cow oocytes were inseminated with different percent live donkey sperm (high (>60%) or low (<40%) viability donkey sperm). After 18h of co-incubation, the oocytes were fixed, stained with 4',6-diamidino-2-phenylindole (DAPI) and examined for sperm penetration, the number of penetrated spermatozoa per oocyte, and male pronucleus formation. Frozen-thawed spermatozoa from high viability semen showed significantly lower VCL, VAP and mean ALH values than did high viability fresh spermatozoa. In contrast, frozen-thawed spermatozoa of low viability had significantly higher velocity values than fresh spermatozoa of low viability. A significant positive correlation (P<0.01) was detected between percentage fertilization and viability (r=0.84), and between percentage fertilization and certain CASA parameters (VAP, r=0.56; VCL, r=0.61 and mean ALH, r=0.68). Fresh or frozen-thawed high viability spermatozoa penetrated 90.1% and 85.4% of bovine oocytes respectively. Lower rates of penetration were observed for fresh and frozen-thawed low viability spermatozoa (34% and 22.5% respectively). The donkey spermatozoa were able to fuse with the oolema and even to decondense and form the male pronucleus (85-94%). Larger numbers of penetrated spermatozoa per oocyte were recorded when high viability sperm samples were used, whether fresh (3.02 vs. 1.12 for low viability sperm

  1. Methyl-parathion decreases sperm function and fertilization capacity after targeting spermatocytes and maturing spermatozoa

    SciTech Connect

    Pina-Guzman, Belem; Sanchez-Gutierrez, M.; Marchetti, Francesco; Hernandez-Ochoa, I.; Solis-Heredia, M.J .; Quintanilla-Vega, B.

    2009-05-03

    Paternal germline exposure to organophosphorous pesticides (OP) has been associated with reproductive failures and adverse effects in the offspring. Methyl parathion (Me-Pa), a worldwide-used OP, has reproductive adverse effects and is genotoxic to sperm. Oxidative damage has been involved in the genotoxic and reproductive effects of OP. The purpose of this study was to determine the effects of Me-Pa on spermatozoa function and ability to fertilize. Male mice were exposed to Me-Pa (20 mg/kg bw, i.p.) and spermatozoa from epididymis-vas deferens were collected at 7 or 28 days post-treatment (dpt) to assess the effects on maturing spermatozoa and spermatocytes, respectively. DNA damage was evaluated by nick translation (NT-positive cells) and SCSA (percentDFI); lipoperoxidation (LPO) by malondialdehyde production; sperm function by spontaneous- and induced-acrosome reactions (AR); mitochondrial membrane potential (MMP) by using the JC-1 flurochrome; and, fertilization ability by an in vitro assay and in vivo mating. Results showed alterations in DNA integrity (percentDFI and NT-positive cells) at 7 and 28 dpt, in addition to decreased sperm quality and a decrease in induced-AR; reduced MMP and LPO was observed only at 7 dpt. We found negative correlations between LPO and all sperm alterations. Altered sperm functional parameters were associated with reduced fertilization rates at both times, evaluated either in vitro or in vivo. These results show that Me-Pa exposure of maturing spermatozoa and spermatocytes affects many sperm functional parameters that result in a decreased fertilizing capacity. Oxidative stress seems to be a likely mechanism ofthe detrimental effects of Me-Pa in male germ cells.

  2. Methyl-parathion decreases sperm function and fertilization capacity after targeting spermatocytes and maturing spermatozoa.

    PubMed

    Piña-Guzmán, B; Sánchez-Gutiérrez, M; Marchetti, F; Hernández-Ochoa, I; Solís-Heredia, M J; Quintanilla-Vega, B

    2009-07-15

    Paternal germline exposure to organophosphorous pesticides (OP) has been associated with reproductive failures and adverse effects in the offspring. Methyl-parathion (Me-Pa), a worldwide-used OP, has reproductive adverse effects and is genotoxic to sperm, possibly via oxidative damage. This study investigated the stages of spermatogenesis susceptible to be targeted by Me-Pa exposure that impact on spermatozoa function and their ability to fertilize. Male mice were exposed to Me-Pa (20 mg/kg bw, i.p.) and spermatozoa from epididymis-vas deferens were collected at 7 or 28 days post-treatment (dpt) to assess the effects on maturing spermatozoa and spermatocytes, respectively. Spermatozoa were examined for DNA damage by nick translation (NT-positive cells) and SCSA (%DFI), lipoperoxidation (LPO) by malondialdehyde production, sperm function by spontaneous- and induced-acrosome reactions (AR), mitochondrial membrane potential (MMP) by using the JC-1 fluorochrome, and fertilization ability by an in vitro assay and in vivo mating. Alterations on DNA integrity (%DFI and NT-positive cells) in spermatozoa collected at 7 and 28 dpt, and decreases in sperm quality and induced-AR were observed; reduced MMP and LPO were observed at 7 dpt only. Negative correlations between LPO and sperm alterations were found. Altered sperm functional parameters evaluated either in vitro or in vivo were associated with reduced fertilization rates at both times. These results show that Me-Pa exposure of maturing spermatozoa and spermatocytes affects many sperm functional parameters that result in a decreased fertilizing capacity. Oxidative stress seems to be a likely mechanism of the detrimental effects of Me-Pa exposure in male germ cells.

  3. Lipid Rafts: Keys to Sperm Maturation, Fertilization, and Early Embryogenesis

    PubMed Central

    Kawano, Natsuko; Yoshida, Kaoru; Miyado, Kenji; Yoshida, Manabu

    2011-01-01

    Cell membranes are composed of many different lipids and protein receptors, which are important for regulating intracellular functions and cell signaling. To orchestrate these activities, the cell membrane is compartmentalized into microdomains that are stably or transiently formed. These compartments are called “lipid rafts”. In gamete cells that lack gene transcription, distribution of lipids and proteins on these lipid rafts is focused during changes in their structure and functions such as starting flagella movement and membrane fusion. In this paper, we describe the role of lipid rafts in gamete maturation, fertilization, and early embryogenesis. PMID:21490798

  4. Systematic identification and characterization of long non-coding RNAs in mouse mature sperm

    PubMed Central

    Zhang, Xiaoning; Gao, Fengxin; Fu, Jianbo; Zhang, Peng; Wang, Yuqing; Zeng, Xuhui

    2017-01-01

    Increasing studies have shown that mature spermatozoa contain many transcripts including mRNAs and miRNAs. However, the expression profile of long non-coding RNAs (lncRNAs) in mammalian sperm has not been systematically investigated. Here, we used highly purified RNA to investigate lncRNA expression profiles in mouse mature sperm by stranded-specific RNA-seq. We identified 20,907 known and 4,088 novel lncRNAs transcripts, and the existence of intact lncRNAs was confirmed by RT-PCR and fluorescence in situ hybridization on two representative lncRNAs. Compared to round spermatids, 1,794 upregulated and 165 downregulated lncRNAs and 4,435 upregulated and 3,920 downregulated mRNAs were identified in sperm. Based on the “Cis and Trans” RNA-RNA interaction principle, we found 14,259 targeted coding genes of differently expressed lncRNAs. In terms of Gene ontology (GO) analysis, differentially expressed lncRNAs targeted genes mainly related to nucleic acid metabolic, protein modification, chromatin and histone modification, heterocycle compound metabolic, sperm function, spermatogenesis and other processes. In contrast, differentially expressed transcripts of mRNAs were highly enriched for protein metabolic process and RNA metabolic, spermatogenesis, sperm motility, cell cycle, chromatin organization, heterocycle and aromatic compound metabolic processes. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the differentially expressed lncRNAs were involved in RNA transport, mRNA surveillance pathway, PI3K-Akt signaling pathway, AMPK signaling pathway, protein processing in endoplasmic reticulum. Metabolic pathways, mRNA surveillance pathway, AMPK signaling pathway, cell cycle, RNA transport splicesome and endocytosis incorporated with the differentially expressed mRNA. Furthermore, many lncRNAs were specifically expressed in testis/sperm, and 880 lncRNAs were conserved between human and mouse. In summary, this study provides a preliminary

  5. Epididymosomes: a heterogeneous population of microvesicles with multiple functions in sperm maturation and storage.

    PubMed

    Sullivan, Robert

    2015-01-01

    Extracellular microvesicles present in the epididymal fluid have been named epididymosomes. Many epididymosome-associated proteins are transferred to spermatozoa during their maturation in the excurrent duct. Epididymosomes are heterogeneous, with their size varying between 50 and 250 nm. Two distinct population of epididymosomes characterized by different protein compositions and diameters have been isolated from the bovine epididymal fluid using different centrifugation protocols. One subpopulation of epididymosomes was characterized by CD9 and other tetraspanin partners. Transfer of proteins from these epididymosomes to maturing spermatozoa in co-incubation experiments was inhibited by antibodies against tetraspanin proteins. This suggests that this subpopulation of epididymosomes is involved in the acquisition of proteins involved in maturation by spermatozoa in the epididymis. The other population of epididymosomes was characterized by ELSPBP1 (epididymal sperm binding protein 1), known for its affinity for the phospholipid choline group. Flow cytometric analyses showed that ELSPBP1-positive epididymosomes only interacted with dying or dead epididymal spermatozoa in a Zn 2 + -dependent manner. BLVRA (biliverdin reductase) was identified as a partner of ELSPBP1. This enzyme reduces biliverdin to bilirubin: two molecules with powerful anti-oxidant properties. We hypothesize that BLVRA is involved in an ROS-scavenging mechanism protecting live epididymal spermatozoa against detrimental molecules (ROS) released by dying cells. Therefore, it appears that there are at least two epididymosome population with distinct functions: targeting specific proteins to transiting spermatozoa by tetraspanin-mediated membrane fusion, and protection of epididymal spermatozoa against ROS released from dying cells. Further work is needed to understand functions of epididymosomes in epididymal physiology and sperm maturation and storage.

  6. Testicular descent, sperm maturation and capacitation. Lessons from our most distant relatives, the monotremes.

    PubMed

    Ecroyd, Heath; Nixon, Brett; Dacheux, Jean-Louis; Jones, Russell C

    2009-01-01

    The present review examines whether monotremes may help to resolve three questions relating to sperm production in mammals: why the testes descend into a scrotum in most mammals, why spermatozoa are infertile when they leave the testes and require a period of maturation in the specific milieu provided by the epididymides, and why ejaculated spermatozoa cannot immediately fertilise an ovum until they undergo capacitation within the female reproductive tract. Comparisons of monotremes with other mammals indicate that there is a need for considerable work on monotremes. It is hypothesised that testicular descent should be related to epididymal differentiation. Spermatozoa and ova from both groups share many of the proteins that are thought to be involved in gamete interaction, and although epididymal sperm maturation is significant it is probably less complex in monotremes than in other mammals. However, the monotreme epididymis is unique in forming spermatozoa into bundles of 100 with greatly enhanced motility compared with individual spermatozoa. Bundle formation involves a highly organised interaction with epididymal proteins, and the bundles persist during incubation in vitro, except in specialised medium, in which spermatozoa separate after 2-3 h incubation. It is suggested that this represents an early form of capacitation.

  7. Effect of low oxygen tension atmosphere and maturation media supplementation on nuclear maturation, cortical granules migration and sperm penetration in swine in vitro fertilization.

    PubMed

    Marques, M G; de Barros, F R O; Goissis, M D; Cavalcanti, P V; Viana, C H C; Assumpção, M E O D; Visintin, J A

    2012-06-01

    The aim of this study was to evaluate the efficiency of low oxygen tension (5% CO(2) , 5% O(2) and 90% N(2) ) on in vitro oocyte maturation using defined media (0.1% polyvinyl alcohol - PVA) or 10% porcine follicular fluid (PFF)-supplemented media. To achieve this goal, oocytes were evaluated regarding cortical granules (GCs) migration, nuclear maturation and sperm penetration. Oocytes were in vitro matured under different conditions: 5% or 20% O(2) atmosphere and 0.1% PVA- or 10% PFF-supplemented media and evaluated at 0 and 44 h of maturation. To evaluate the migration of CGs and nuclear maturation, by confocal microscopy, oocytes were incubated with 100 μg of FITC-PNA/ml and 10 μg/ml of propidium iodide. To address sperm penetration, after maturation, in vitro fertilization for 6 h and in vitro culture for 18 h, zygotes were incubated with 10 mg/ml Hoechst 33342. Pronuclei and polar bodies were quantified using an epifluorescence microscope. Atmosphere conditions did not affect the CGs migration, but media supplementation did. Oocytes matured in 10% PFF media had a higher percentage of CGs in the oocyte periphery than oocytes matured in PVA-supplemented media. However, this fact did not have effect on in vitro sperm penetration levels. No effect of atmosphere conditions and media supplementation was observed on the rates of metaphase II oocytes. Therefore, the use of low oxygen tension in association with PVA maturation media does not improve the in vitro maturation system of porcine oocytes, because its use did not improve nuclear maturation, CGs migration and zygotes monospermic rates.

  8. Effects of dietary energy on sexual maturation and sperm production in Holstein bulls.

    PubMed

    Harstine, B R; Maquivar, M; Helser, L A; Utt, M D; Premanandan, C; DeJarnette, J M; Day, M L

    2015-06-01

    In prepubertal bulls and heifers of dairy and beef breeds, puberty can be induced to occur earlier than typical with targeted high-energy diets due to precocious activation of the endocrine mechanisms that regulate puberty. Precocious activation of puberty in bulls intended for use in the AI industry has the potential to hasten and perhaps increase sperm production. It was hypothesized that feeding bulls a high-energy diet beginning at 8 wk of age would advance the prepubertal rise in LH and lead to advanced testicular maturation and age at puberty. From 58 to 230 ± 0.3 d of age, Holstein bulls received either a high-energy diet (HE;n = 9; targeted ADG 1.5 kg/d) or a control diet (CONT;n = 10; targeted ADG 0.75 kg/d). Thereafter, all bulls were fed a similar diet. The HE treatment increased LH secretion at 125 d of age, testosterone concentrations from 181 to 210 d, and scrotal circumference (SC) from 146 to 360 d of age relative to the CONT treatment. Beginning at 241 ± 5 d of age, semen collection (artificial vagina) was attempted every 14 d in bulls from the HE (n = 8) and CONT (n = 7) treatment until each bull attained puberty (ejaculate containing 50 × 10 spermatozoa with 10% motility). To assess semen production as mature bulls, semen was collected thrice weekly beginning at 541 ± 5 d of age until slaughter at 569 ± 5 d of age. After slaughter, epididymal and testicular measurements were collected and testicular tissue was fixed to determine seminiferous tubule diameter. Age at puberty did not differ between treatments (310 ± 35 d). Although testis and epididymal weight and testis volume were greater (P < 0.05) in the HE than the CONT treatment, sperm production of mature bulls did not differ between treatments. Diameter of seminiferous tubules also did not differ between treatments. We conclude that the HE advanced aspects of sexual maturation and increased testes size, but this was not reflected in hastened puberty or sperm production in the present

  9. Estimation of the Optimal Timing of Fertilization for Embryo Development of In Vitro-Matured Bovine Oocytes Based on the Times of Nuclear Maturation and Sperm Penetration

    PubMed Central

    KOYAMA, Keisuke; KANG, Sung-Sik; HUANG, Weiping; YANAGAWA, Yojiro; TAKAHASHI, Yoshiyuki; NAGANO, Masashi

    2014-01-01

    ABSTRACT The objective of this research was to estimate the optimal timing for fertilization to achieve proper embryonic development of in vitro-matured bovine oocytes. First, cumulus-oocyte complexes were subjected to in vitro maturation (IVM) for 14–22 hr. The timing when 50% of oocytes reached metaphase II stage was estimated to be 17.5 hr after IVM start. Next, using oocytes subjected to IVM for 12–30 hr, sperm penetration was examined after 4–18 hr of in vitro fertilization (IVF). A significant negative correlation between IVM duration and the timing when 50% of oocytes were penetrated by sperm after IVF start was observed (P<0.01). Finally, oocytes subjected to 12–30 hr of IVM were inseminated and cultured for 6 days to examine embryonic development. In the group with 22 hr of IVM, the percentages of cleaved embryos and blastocysts were the highest values in all groups. According to the regression equation describing the time from nuclear maturation to sperm penetration (x) and the percentage of blastocysts (y) (y=7.23x − 0.297x2, P<0.01), the blastocyst rate peaked when sperm penetration occurred at 12.2 hr after achieving nuclear maturation. In conclusion, under the present IVM/IVF conditions, it was estimated that oocytes acquired their highest developmental competence at about 30 hr after IVM start, and thus, the optimal IVM duration was calculated to be about 21 hr. PMID:24430663

  10. Estimation of the optimal timing of fertilization for embryo development of in vitro-matured bovine oocytes based on the times of nuclear maturation and sperm penetration.

    PubMed

    Koyama, Keisuke; Kang, Sung-Sik; Huang, Weiping; Yanagawa, Yojiro; Takahashi, Yoshiyuki; Nagano, Masashi

    2014-05-01

    The objective of this research was to estimate the optimal timing for fertilization to achieve proper embryonic development of in vitro-matured bovine oocytes. First, cumulus-oocyte complexes were subjected to in vitro maturation (IVM) for 14-22 hr. The timing when 50% of oocytes reached metaphase II stage was estimated to be 17.5 hr after IVM start. Next, using oocytes subjected to IVM for 12-30 hr, sperm penetration was examined after 4-18 hr of in vitro fertilization (IVF). A significant negative correlation between IVM duration and the timing when 50% of oocytes were penetrated by sperm after IVF start was observed (P<0.01). Finally, oocytes subjected to 12-30 hr of IVM were inseminated and cultured for 6 days to examine embryonic development. In the group with 22 hr of IVM, the percentages of cleaved embryos and blastocysts were the highest values in all groups. According to the regression equation describing the time from nuclear maturation to sperm penetration (x) and the percentage of blastocysts (y) (y=7.23x - 0.297x(2), P<0.01), the blastocyst rate peaked when sperm penetration occurred at 12.2 hr after achieving nuclear maturation. In conclusion, under the present IVM/IVF conditions, it was estimated that oocytes acquired their highest developmental competence at about 30 hr after IVM start, and thus, the optimal IVM duration was calculated to be about 21 hr.

  11. Can bovine in vitro-matured oocytes selectively process X- or Y-sorted sperm differentially?

    PubMed

    Bermejo-Alvarez, P; Rizos, D; Rath, D; Lonergan, P; Gutiérrez-Adán, A

    2008-10-01

    It has been reported that the mammalian female could have a preconceptual influence on the sex of her offspring, and it has been hypothesized that this influence could go some way toward accounting for the reported lower fertility following insemination with sex-sorted sperm. To test whether in vitro matured oocytes are able to select X- or Y-bearing spermatozoa following in vitro fertilization (IVF), we fertilized in vitro 1788 oocytes with X-sorted semen, Y-sorted semen, a mix of X- and Y-sorted semen, and unsorted semen from the same bull, and cultured until Day 9. Fertility was assessed by recording cleavage rate at 48 h postinsemination (hpi) and blastocyst development until Day 9. Embryos were sexed at the two- to four-cell stage and the blastocyst stage. The proportion of zygotes cleaving at 48 hpi was not different between X- and Y-sorted groups and the mix of X- and Y-sorted semen group; however, all were significantly lower than the unsorted group (P < 0.001). Blastocyst yield on Day 6 was significantly higher (P < or = 0.01) in the control group compared with the rest of the groups. Cumulative blastocyst yields on Days 7, 8, and 9 were also significantly higher (P < or = 0.01) in the unsorted group compared with the sorted groups. The proportion of female and male two- to four-cell embryos obtained following IVF with X- and Y-sorted sperm was 88% and 89%, respectively and the sex ratio at the two- to four-cell stage was not different following IVF with unsorted or sorted/recombined sperm (56.9% males vs. 57% males, respectively). At the blastocyst stage, similar percentages were obtained. In conclusion, the differences in cleavage and blastocyst development using sorted versus unsorted sperm are not due to the oocyte preferentially selecting sperm of one sex over another, but are more likely due to spermatic damage caused by the sorting procedure.

  12. The Drosophila chromosomal protein Mst77F is processed to generate an essential component of mature sperm chromatin

    PubMed Central

    2016-01-01

    In most animals, the bulk of sperm DNA is packaged with sperm nuclear basic proteins (SNBPs), a diverse group of highly basic chromosomal proteins notably comprising mammalian protamines. The replacement of histones with SNBPs during spermiogenesis allows sperm DNA to reach an extreme level of compaction, but little is known about how SNBPs actually function in vivo. Mst77F is a Drosophila SNBP with unique DNA condensation properties in vitro, but its role during spermiogenesis remains unclear. Here, we show that Mst77F is required for the compaction of sperm DNA and the production of mature sperm, through its cooperation with protamine-like proteins Mst35Ba/b. We demonstrate that Mst77F is incorporated in spermatid chromatin as a precursor protein, which is subsequently processed through the proteolysis of its N-terminus. The cleavage of Mst77F is very similar to the processing of protamine P2 during human spermiogenesis and notably leaves the cysteine residues in the mature protein intact, suggesting that they participate in the formation of disulfide cross-links. Despite the rapid evolution of SNBPs, sperm chromatin condensation thus involves remarkably convergent mechanisms in distantly related animals. PMID:27810970

  13. Bovine in vitro fertilization: in vitro oocyte maturation and sperm capacitation with heparin.

    PubMed

    Parrish, John J

    2014-01-01

    As a result of research in the 1980s on in vitro maturation, sperm capacitation, and in vitro fertilization, the bovine is now one of the important models for development. Further, the current production of bovine embryos in vitro rivals that of in vivo embryo production for commercial applications. Researchers of today may be unaware of why decisions were made in the procedures. This review addresses the state of the art at the time of the work by Parrish et al. (Bovine in vitro fertilization with frozen thawed semen. Theriogenology 1986;25:591-600), and how later work would explain success or failure of competing procedures. Important was the use of frozen semen and heparin capacitation, because this allowed future researchers/practitioners to change sperm numbers and capacitation conditions to adjust for variations among bulls. The large numbers of citation of the original work stand the testament of time in the repeatability and success of the procedures. The work was done within the environment of the N.L. First laboratory and the unique interactions with a large number of talented graduate students, postdoctoral researchers, and technicians.

  14. Evidence for the involvement of zinc in the association of CRISP1 with rat sperm during epididymal maturation.

    PubMed

    Maldera, Julieta A; Vasen, Gustavo; Ernesto, Juan I; Weigel-Muñoz, Mariana; Cohen, Débora J; Cuasnicu, Patricia S

    2011-09-01

    Rat epididymal protein CRISP1 (cysteine-rich secretory protein 1) associates with sperm during maturation and participates in fertilization. Evidence indicates the existence of two populations of CRISP1 in sperm: one loosely bound and released during capacitation, and one strongly bound that remains after this process. However, the mechanisms underlying CRISP1 binding to sperm remain mostly unknown. Considering the high concentrations of Zn(2+) present in the epididymis, we investigated the potential involvement of this cation in the association of CRISP1 with sperm. Caput sperm were coincubated with epididymal fluid in the presence or absence of Zn(2+), and binding of CRISP1 to sperm was examined by Western blot analysis. An increase in CRISP1 was detected in sperm exposed to Zn(2+), but not if the cation was added with ethylenediaminetetra-acetic acid (EDTA). The same results were obtained using purified CRISP1. Association of CRISP1 with sperm was dependent on epididymal fluid and Zn(2+) concentrations and incubation time. Treatment with NaCl (0.6 M) removed the in vitro-bound CRISP1, indicating that it corresponds to the loosely bound population. Flow cytometry of caput sperm exposed to biotinylated CRISP1/avidin-fluorescein isothiocyanate revealed that only the cells incubated with Zn(2+) exhibited an increase in fluorescence. When these sperm were examined by epifluorescence microscopy, a clear staining in the tail, accompanied by a weaker labeling in the head, was observed. Detection of changes in the tryptophan fluorescence emission spectra of CRISP1 when exposed to Zn(2+) supported a direct interaction between CRISP1 and Zn(2+). Incubation of either cauda epididymal fluid or purified CRISP1 with Zn(2+), followed by native-PAGE and Western blot analysis, revealed the presence of high-molecular-weight CRISP1 complexes not detected in fluids treated with EDTA. Altogether, these results support the involvement of CRISP1-Zn(2+) complexes in the association of

  15. Delivery following intracytoplasmic injection of mature sperm cells recovered by testicular fine needle aspiration in a case of hypergonadotropic azoospermia due to maturation arrest.

    PubMed

    Lewin, A; Weiss, D B; Friedler, S; Ben-Shachar, I; Porat-Katz, A; Meirow, D; Schenker, J G; Safran, A

    1996-04-01

    This is the first reported delivery following intracytoplasmic sperm injection (ICSI) of mature live testicular sperm cells collected in a case of hypergonadotrophic azoospermia with maturation arrest. The 30 year old couple presented with primary infertility of 11 years duration, the man being submitted in childhood to five orchidopexy operations for the treatment of cryptorchism. He had elevated serum follicle stimulating hormone (FSH; 18.8 IU/I), an atrophic left testis and a normal sized right testis, the biopsy of which diagnosed maturation arrest and focal scarring. The couple refused donor insemination for religious reasons and the only option was an attempt at testicular sperm collection. Multiple testicular and epididymal fine needle aspirations were performed, using an aspiration handle loaded with 20 ml syringe and 21-23 gauge butterfly needles. The mature spermatozoa recovered were used to inseminate the oocytes by ICSI. Prior to this procedure, the patient's wife underwent ovulation induction using a long protocol of mid-luteal gonadotrophin-releasing hormone analogue/human menopausal gonadotrophin (GnRHa/HMG). At oocyte retrieval, ten oocytes were recovered. Eight live sperm cells were recovered from the aspirates of the right testis. Following ICSI into four metaphase II and two metaphase I oocytes, one mature oocyte was fertilized, cleaved and was transferred to the uterus 48 h after oocyte retrieval. The patient conceived and delivered a 3300 g boy at term. In conclusion, our results demonstrate that this novel approach should be considered in cases with hypergonadotrophic azoospermia due to testicular failure. Further experience is needed to establish the exact criteria for its use.

  16. Sperm Proteome: What Is on the Horizon?

    PubMed

    Mohanty, Gayatri; Swain, Nirlipta; Samanta, Luna

    2015-06-01

    As the mammalian spermatozoa transcends from the testis to the end of the epididymal tubule, the functionally incompetent spermatozoa acquires its fertilizing capability. Molecular changes in the spermatozoa at the posttesticular level concern qualitative and quantitative modifications of proteins along with their sugar moieties and membranous lipids mostly associated with motility, egg binding, and penetration processes. Proteomic studies have identified numerous sperm-specific proteins, and recent reports have provided a further understanding of their function with respect to male fertility. High-throughput techniques such as mass spectrometry have shown drastic potential for the identification and study of sperm proteins. In fact, compelling evidence has provided that proteins are critically important in cellular remodeling event and that aberrant expression is associated with pronounced defects in sperm function. This review highlights the posttesticular functional transformation in the epididymis and female reproductive tract with due emphasis on proteomics.

  17. Influence of macrophage migration inhibitory factor (MIF) on the zinc content and redox state of protein-bound sulphydryl groups in rat sperm: indications for a new role of MIF in sperm maturation.

    PubMed

    Eickhoff, Regina; Baldauf, Christina; Koyro, Hans-Werner; Wennemuth, Gunther; Suga, Yasushi; Seitz, Jürgen; Henkel, Ralf; Meinhardt, Andreas

    2004-08-01

    The function of macrophage migration inhibitory factor (MIF) in sperm maturation was studied by investigating its role in the biochemical maturation of the outer dense fibres. Rat sperm obtained from the caput and cauda epididymis were stimulated overnight with either recombinant MIF or MIF-containing vesicles originating from epididymal fluid at various concentrations. The zinc content of both the sperm and the medium was determined by means of atomic absorption spectrometry. Incubation in both recombinant MIF and vesicular MIF resulted in a statistically significant decrease of the zinc content in stimulated caput sperm of approximately 50%. In parallel, the conditioned media showed a clear increase in the concentration of this trace metal. The effect of MIF was less marked in cauda sperm. In addition, we demonstrated a statistically significant increase of detectable free thiol groups in the sperm mid- and principle piece in isolated rat sperm after stimulation with MIF at concentrations of 25 and 50 ng/ml. Our data suggest that MIF plays an important role in the maturation process of rat sperm during epididymal transit by inducing the elimination of zinc and affecting the amount of free sulphydryl groups in the sperm flagella.

  18. The presence of centrioles and centrosomes in ovarian mature cystic teratoma cells suggests human parthenotes developed in vitro can differentiate into mature cells without a sperm centriole

    SciTech Connect

    Lee, Bo Yon; Shim, Sang Woo; Kim, Young Sun; Kim, Seung Bo

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer The sperm centriole is the progenitor of centrosomes in all somatic cells. Black-Right-Pointing-Pointer Centrioles and centrosomes exist in parthenogenetic ovarian teratoma cells. Black-Right-Pointing-Pointer Without a sperm centriole, parthenogenetic oocytes produce centrioles and centrosomes. Black-Right-Pointing-Pointer Parthenogenetic human oocytes can develop and differentiate into mature cells. -- Abstract: In most animals, somatic cell centrosomes are inherited from the centriole of the fertilizing spermatozoa. The oocyte centriole degenerates during oogenesis, and completely disappears in metaphase II. Therefore, the embryos generated by in vitro parthenogenesis are supposed to develop without any centrioles. Exceptional acentriolar and/or acentrosomal developments are possible in mice and in some experimental cells; however, in most animals, the full developmental potential of parthenogenetic cells in vitro and the fate of their centrioles/centrosomes are not clearly understood. To predict the future of in vitro human parthenogenesis, we explored the centrioles/centrosomes in ovarian mature cystic teratoma cells by immunofluorescent staining and transmission electron microscopy. We confirmed the presence of centrioles and centrosomes in these well-known parthenogenetic ovarian tumor cells. Our findings clearly demonstrate that, even without a sperm centriole, parthenotes that develop from activated oocytes can produce their own centrioles/centrosomes, and can even develop into the well-differentiated mature tissue.

  19. Migration of sperm cells during pollen tube elongation in Arabidopsis thaliana: behavior during transport, maturation and upon dissociation of male germ unit associations.

    PubMed

    Ge, Lili; Gou, Xiaoping; Yuan, Tong; Strout, Greg W; Nakashima, Jin; Blancaflor, Elison B; Tian, Hui Qiao; Russell, Scott D

    2011-02-01

    The promoter sequence of sperm-expressed gene, PzIPT isolated from the S(vn) (sperm associated with the vegetative nucleus) of Plumbago zeylanica, was fused to a green fluorescent protein (GFP) reporter sequence and transformed into Arabidopsis thaliana to better visualize the live behavior of angiosperm sperm cells. Angiosperm sperm cells are not independently motile, migrating in a unique cell-within-a-cell configuration within the pollen tube. Sperm cells occur in association with the vegetative nucleus forming a male germ unit (MGU). In Arabidopsis, GFP was expressed equally in both sperm cells and was observed using a spinning disk confocal microscope, which allowed long duration observation of cells without bleaching or visible laser radiation damage. Pollen activation is reflected by conspicuous movement of sperm and pollen cytoplasm. Upon pollen germination, sperm cells enter the forming tube and become oriented, typically with a sperm cytoplasmic projection leading the sperm cells in the MGU, which remains intact throughout normal pollen tube elongation. Maturational changes, including vacuolization, general rounding and entry into G2, were observed during in vitro culture. When MGUs were experimentally disrupted by mild temperature elevation, sperm cells no longer tracked the growth of the tube and separated from the MGU, providing critical direct evidence that the MGU is a functional unit required for sperm transmission.

  20. Hypotonic resistance of boar spermatozoa: sperm subpopulations and relationship with epididymal maturation and fertility.

    PubMed

    Druart, Xavier; Gatti, Jean-Luc; Huet, Sylvie; Dacheux, Jean-Louis; Humblot, Patrice

    2009-02-01

    Hypotonic resistance of boar spermatozoa was investigated by measuring the ratio of live/dead spermatozoa (SYBR-14/propidium iodide) by flow cytometry after hypotonic stress. The survival rate of ejaculated spermatozoa incubated in hypotonic solutions ranging from 3 to 330 mmol/kg followed a sigmoid curve that fitted a simple logistic model. The critical osmolality value (Osm(crit)) at which 50% of spermatozoa died was determined with this model. Hypotonic resistance of spermatozoa increased with temperature between 15 and 39 degrees C and decreased after hydrogen superoxide treatment, but was not modified during 8 days of preservation in Beltsville thawing solution. Hypotonic resistance markedly decreased during epididymal maturation and after ejaculation as Osm(crit) at 15 degrees C was 54.7+/-3.2, 68.5+/-10.6, 116.7+/-2.1 and 194.3+/-3.7 mmol/kg for the caput, corpus, cauda and ejaculated spermatozoa respectively. Hypo-osmotic stress of 100 mmol/kg revealed a sperm subpopulation exhibiting increased hypotonic resistance compared with the whole ejaculate (Osm(crit)=67.8+/-2.1 mmol/kg). Consistent differences were observed between lean and standard breeds (Pietrain versus Large White) and between boars within the same breed. According to data collected by artificial insemination centers during a large-scale field trial, hypotonic resistance of ejaculates was found to be positively correlated with in vivo fertility.

  1. Can the rapid identification of mature spermatozoa during microdissection testicular sperm extraction guide operative planning?

    PubMed

    Alrabeeah, K; Doucet, R; Boulet, E; Phillips, S; Al-Hathal, N; Bissonnette, F; Kadoch, I J; Zini, A

    2015-05-01

    The minimum sperm count and quality that must be identified during microdissection testicular sperm extraction (micro-TESE) to deem the procedure successful remains to be established. We conducted a retrospective study of 81 consecutive men with non-obstructive azoospermia who underwent a primary (first) micro-TESE between March 2007 and October 2013. Final assessment of sperm recovery [reported on the day of (intracytoplasmic sperm injection) ICSI] was recorded as (i) successful (available spermatozoa for ICSI) or (ii) unsuccessful (no spermatozoa for ICSI). The decision to perform a unilateral (with limited or complete microdissection) or bilateral micro-TESE was guided by the intra-operative identification of sperm recovery (≥5 motile or non-motile sperm) from the first testicle. Overall, sperm recovery was successful in 56% (45/81) of the men. A unilateral micro-TESE was performed in 47% (38/81) of the men (based on intra-operative identification of sperm) and in 100% (38/38) of these men, spermatozoa was found on final assessment. In 42% (16/38) of the unilateral cases, a limited microdissection was performed (owing to the rapid intra-operative identification of sperm). The remaining 43 men underwent a bilateral micro-TESE and 16% (7/43) of these men had sperm identified on final assessment. The cumulative ICSI pregnancy rates (per cycle started and per embryo transfer) were 47% (21/45) and 60% (21/35), respectively, with a mean (±SD) of 1.9 ± 1.0 embryos transferred. The data demonstrate that intra-operative assessment of sperm recovery can correctly identify those men that require a unilateral micro-TESE. Moreover, the rapid identification of sperm recovery can allow some men to undergo a limited unilateral micro-TESE and avoid the need for complete testicular microdissection.

  2. Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility

    PubMed Central

    Dudiki, Tejasvi; Kadunganattil, Suraj; Ferrara, John K.; Kline, Douglas W.; Vijayaraghavan, Srinivasan

    2015-01-01

    Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function. PMID:26569399

  3. Birth after human chorionic gonadotropin-primed oocyte in vitro maturation and fertilization with testicular sperm in a normo-ovulatory patient

    PubMed Central

    González-Ortega, Claudia; Piña-Aguilar, Raul Eduardo; Cancino-Villareal, Patricia; Gutiérrez-Gutiérrez, Antonio Martin

    2016-01-01

    In this report, we present a case of in vitro maturation (IVM) with surgical retrieved testicular sperm in a normo-ovulatory female. Human chorionic gonadotropin-primed IVM, testicular biopsy for sperm retrieval and intracytoplasmic sperm injection with fresh sperm were performed. Fourteen cumulus-oocyte complexes were obtained in germinal vesicle or metaphase I stage, eight oocytes reached metaphase II, seven presumptive zygotes were obtained, and three cleavage stages embryos in day 2 were transferred producing a singleton pregnancy. A single healthy newborn was obtained. Our results suggest that IVM may be an alternative for in vitro fertilization in normo-ovulatory women even if surgical retrieval of sperm is needed. Further research is required to depict contributing factors to the success of IVM in indications different from polycystic ovaries syndrome and the role of male gamete. PMID:27803591

  4. Fine structure of acrosome biogenesis and of mature sperm in the bivalve molluscs Glycymeris sp. (Pteriomorphia) and Eurhomalea rufa (Heterodonta)

    NASA Astrophysics Data System (ADS)

    Guerra, Rosa; Sousa, Mário; Torres, Artur; Oliveira, Elsa; Baldaia, Luis

    2003-03-01

    Proacrosomal vesicles form during the pachytene stage, being synthetized by the Golgi complex in Glycymeris sp., and by both the Golgi and the rough endoplasmic reticulum in Eurhomalea rufa. During early spermiogenesis, a single acrosomal vesicle forms and its apex becomes linked to the plasma membrane while it migrates. In Glycymeris sp., the acrosomal vesicle then turns cap-shaped (1.8 μm) and acquires a complex substructure. In E. rufa, proacrosomal vesicles differentiate their contents while still at the premeiotic stage; as the acrosomal vesicle matures and its contents further differentiate, it elongates and becomes longer than the nucleus (3.2 μm), while the subacrosomal space develops a perforatorium. Before condensation, chromatin turns fibrillar in Glycymeris sp., whereas it acquires a cordonal pattern in E. rufa. Accordingly, the sperm nucleus of Glycymeris sp. is conical and elongated (8.3 μm), and that of E. rufa is short and ovoid (1.1 μm). In the midpiece (Glycymeris sp.: 1.1 μm; E. rufa: 0.8 μm), both species have four mitochondria encircling two linked orthogonal (Glycymeris sp.) or orthogonal and tilted (30-40°; E. rufa) centrioles. In comparison with other Arcoida species, sperm of Glycymeris sp. appear distinct due to the presence of an elongated nucleus, a highly differentiated acrosome, and four instead of five mitochondria. The same occurs with E. rufa regarding other Veneracea species, with the acrosome of the mature sperm strongly resembling that of the recent Mytilinae.

  5. In vitro maturation and intracytoplasmic sperm injection of oocytes collected from hormonally stimulated common wombats, Vombatus ursinus.

    PubMed

    West, M; Lacham-Kaplan, O; Cleary, M; Galloway, D; Shaw, J; Trounson, A O; Paris, M C J

    2007-04-01

    Porcine FSH/LH stimulation successfully induced development of multiple large (>or=4mm) antral follicles in 10 of 11 common wombats. A mean of 5.5 metaphase II (MII) oocytes were aspirated from wombats that were stimulated during the follicular phase of the oestrous cycle (n=3) or after pouch young removal (n=3). Three subadults (n=3) and two anoestrus adults did not produce MII oocytes despite pFSH/pLH administration. In vitro maturation of immature oocytes at the time of aspiration doubled the number of MII oocytes that could be collected from pFSH/pLH stimulated wombats. Immature oocytes with cumulus attached, matured more readily to the MII stage than immature oocytes without cumulus. Following intracytoplasmic sperm injection (ICSI), approximately 5% of the oocytes that were MII at the time of collection cleaved. Approximately 5% of those that were matured by in vitro maturation (IVM) formed two polar bodies following ICSI, although they not cleave. Parthenogenesis cannot be excluded. This demonstrates that assisted reproductive technologies may be applicable to the common wombat.

  6. Characterisation of mouse epididymosomes reveals a complex profile of microRNAs and a potential mechanism for modification of the sperm epigenome

    PubMed Central

    Reilly, Jackson N.; McLaughlin, Eileen A.; Stanger, Simone J.; Anderson, Amanda L.; Hutcheon, Kate; Church, Kiralee; Mihalas, Bettina P.; Tyagi, Sonika; Holt, Janet E.; Eamens, Andrew L.; Nixon, Brett

    2016-01-01

    Recent evidence has shown that the sperm epigenome is vulnerable to dynamic modifications arising from a variety of paternal environment exposures and that this legacy can serve as an important determinant of intergenerational inheritance. It has been postulated that such exchange is communicated to maturing spermatozoa via the transfer of small non-protein-coding RNAs (sRNAs) in a mechanism mediated by epididymosomes; small membrane bound vesicles released by the soma of the male reproductive tract (epididymis). Here we confirm that mouse epididymosomes encapsulate an impressive cargo of >350 microRNAs (miRNAs), a developmentally important sRNA class, the majority (~60%) of which are also represented by the miRNA signature of spermatozoa. This includes >50 miRNAs that were found exclusively in epididymal sperm and epididymosomes, but not in the surrounding soma. We also documented substantial changes in the epididymosome miRNA cargo, including significant fold changes in almost half of the miRNAs along the length of the epididymis. Finally, we provide the first direct evidence for the transfer of several prominent miRNA species between mouse epididymosomes and spermatozoa to afford novel insight into a mechanism of intercellular communication by which the sRNA payload of sperm can be selectively modified during their post-testicular maturation. PMID:27549865

  7. Production of lion (Panthera leo) blastocysts after in vitro maturation of oocytes and intracytoplasmic sperm injection.

    PubMed

    Fernandez-Gonzalez, Lorena; Hribal, Romy; Stagegaard, Julia; Zahmel, Jennifer; Jewgenow, Katarina

    2015-04-01

    Assisted reproductive techniques are becoming widely applied to the breeding of endangered species, but establishing reliable protocols for the production of embryos in vitro is challenging because of the scarcity of sample material. In our study, we applied an assisted reproductive technique protocol for IVM and intracytoplasmic sperm injection (ICSI), developed in the domestic cat, to oocytes retrieved from ovaries of four 2-year-old lionesses (Panthera leo) eight hours postmortem. In total, 68 cumulus-oocyte complexes of good quality were randomly distributed and cultured for 32 to 34 hours in two different maturation culture media, consisting of Medium 199 with Earle's salts, 3 mg/mL BSA, 0.1 mg/mL cysteine, 1.4 mg/mL sodium pyruvate, 0.6 mg/mL sodium lactate, 0.15 mg/mL l-glutamine, and 0.055 mg/mL gentamicin. Hormonal supplementation of IVM_1 was 0.02 IU/mL FSH and 0.05 IU/mL LH; IVM_2 consisted of 1.64 IU/mL FSH, 1.06 IU/mL LH, and 1 μg/mL 17ß-estradiol. Differences in hormonal supplementation did not produce significant differences in oocyte maturation rates, which were 39.4% in IVM_1 and 34.3% in IVM_2. Matured oocytes were microinjected with homologous frozen-thawed spermatozoa, and subsequent cleavage rates were 30.8% and 58.3%, respectively. Half of the embryos derived from oocytes matured in IVM_1 developed into blastocysts, whereas only 28.6% of embryos from oocytes matured in IVM_2 reached the blastocyst stage. Morula stages were present from Day 6 onward, and blastocyst stages from Day 9 on, indicating a slower developmental speed in comparison with domestic cats. This is the first report of in vitro-produced blastocysts using ICSI in the lion, and the results report that IVM and ICSI can be successfully performed with cumulus-oocyte complexes retrieved from ovaries after eight hours of shipping, obtaining competent embryos in culture.

  8. Spermatogenesis in the blue swimming crab, Portunus pelagicus, and evidence for histones in mature sperm nuclei.

    PubMed

    Stewart, Michael J; Stewart, Praphaporn; Soonklang, Nantawan; Linthong, Vichai; Hanna, Peter J; Duan, Wei; Sobhon, Prasert

    2010-06-01

    Spermatogenesis in the blue swimming crab, Portunus pelagicus, is described by light and electron microscopy. The testis is composed of anterior (AT) and posterior (PT) lobes, that are partitioned into lobules by connective tissue trabecula, and further divided into zones (germinal, transformation and evacuation), each with various stages of cellular differentiation. The vas deferens is classified into three distinct regions: anterior (AVD), median (MVD), and posterior (PVD), on the presence of spermatophores and two secretions, termed substance I and II. Based on the degree and patterns of heterochromatin, spermatogenesis is classified into 13 stages: two spermatogonia (SgA and SgB), six primary spermatocytes (leptotene, zygotene, pachytene, diplotene, diakinesis, and metaphase), a secondary spermatocyte (SSc), three spermatids (St 1-3), and a mature spermatozoon. Spermatid stages are differentiated by chromatin decondensation and the formation of an acrosomal complex, which is unique to brachyurans. Mature spermatozoa are aflagellated, and have a nuclear projection and a spherical acrosome. AUT-PAGE and Western blots show that, during chromatin decondensation, there is a reduction of most histones, with only small amounts of H2B and H3 remaining in mature spermatozoa.

  9. Porcine embryo production following in vitro fertilization and intracytoplasmic sperm injection from vitrified immature oocytes matured with a granulosa cell co-culture system.

    PubMed

    Casillas, Fahiel; Ducolomb, Yvonne; Lemus, Ana E; Cuello, Cristina; Betancourt, Miguel

    2015-10-01

    This study was designed to evaluate the capacity of vitrified-warmed porcine immature oocytes to mature and to be fertilized using in vitro fertilization or intracytoplasmic sperm injection, and to determine the subsequent embryo development. Immature oocytes were vitrified using ethylene glycol and dimethylsulphoxide as cryoprotectants and the Cryolock method. After warming oocytes were cultured 44 h for maturation. Oocytes were randomly distributed in three treatment groups and subjected to in vitro fertilization (Experiment 1) or intracytoplasmic sperm injection (Experiment 2) procedures. The results indicate that the embryo development was higher in denuded oocytes co-cultured with granulosa cells (NkO-CC group) fertilized by in vitro fertilization or intracytoplasmic sperm injection compared to cumulus-cell oocyte complexes (COCs group), showing no significant differences with control. Vitrified denuded oocytes matured with a co-culture system NkO-CC group, displayed higher cleavage rate and blastocyst production than vitrified COCs group. Blastocysts were successfully obtained after IVF and ICSI procedures; however, the development to the blastocyst stage was better after IVF. These results show that the vitrification-warming media, the employment of a granulosa cell co-culture system and the Cryolock method during vitrification, increased the nuclear and cytoplasmic maturation of vitrified porcine immature oocytes. Further experiments are required to enhance porcine embryo production after vitrification.

  10. Manipulation and in vitro maturation of Xenopus laevis oocytes, followed by intracytoplasmic sperm injection, to study embryonic development.

    PubMed

    Miyamoto, Kei; Simpson, David; Gurdon, John B

    2015-02-09

    Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

  11. Sperm Competition, Sperm Numbers and Sperm Quality in Muroid Rodents

    PubMed Central

    Gómez Montoto, Laura; Magaña, Concepción; Tourmente, Maximiliano; Martín-Coello, Juan; Crespo, Cristina; Luque-Larena, Juan José

    2011-01-01

    developmental pathways underlying processes of sperm formation, maturation, transport in the female reproductive tract, and preparation for fertilization must all evolve in concert. PMID:21464956

  12. Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa.

    PubMed

    Nandi, Pinki; Ghosh, Swatilekha; Jana, Kuladip; Sen, Parimal C

    2012-01-01

    Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.

  13. Alternative splicing, promoter methylation, and functional SNPs of sperm flagella 2 gene in testis and mature spermatozoa of Holstein bulls.

    PubMed

    Guo, F; Yang, B; Ju, Z H; Wang, X G; Qi, C; Zhang, Y; Wang, C F; Liu, H D; Feng, M Y; Chen, Y; Xu, Y X; Zhong, J F; Huang, J M

    2014-02-01

    The sperm flagella 2 (SPEF2) gene is essential for development of normal sperm tail and male fertility. In this study, we characterized first the splice variants, promoter and its methylation, and functional single-nucleotide polymorphisms (SNPs) of the SPEF2 gene in newborn and adult Holstein bulls. Four splice variants were identified in the testes, epididymis, sperm, heart, spleen, lungs, kidneys, and liver tissues through RT-PCR, clone sequencing, and western blot analysis. Immunohistochemistry revealed that the SPEF2 was specifically expressed in the primary spermatocytes, elongated spermatids, and round spermatids in the testes and epididymis. SPEF2-SV1 was differentially expressed in the sperms of high-performance and low-performance adult bulls; SPEF2-SV2 presents the highest expression in testis and epididymis; SPEF2-SV3 was only detected in testis and epididymis. An SNP (c.2851G>T) in exon 20 of SPEF2, located within a putative exonic splice enhancer, potentially produced SPEF2-SV3 and was involved in semen deformity rate and post-thaw cryopreserved sperm motility. The luciferase reporter and bisulfite sequencing analysis suggested that the methylation pattern of the core promoter did not significantly differ between the full-sib bulls that presented hypomethylation in the ejaculated semen and testis. This finding indicates that sperm quality is unrelated to SPEF2 methylation pattern. Our data suggest that alternative splicing, rather than methylation, is involved in the regulation of SPEF2 expression in the testes and sperm and is one of the determinants of sperm motility during bull spermatogenesis. The exonic SNP (c.2851G>T) produces aberrant splice variants, which can be used as a candidate marker for semen traits selection breeding of Holstein bulls.

  14. Cytochemical study of spermiogenesis and mature spermatozoa in the lizard Tropidurus itambere (Reptilia, Squamata).

    PubMed

    Ferreira, Adelina; Dolder, Heidi

    2003-01-01

    The present study was undertaken to detect essential components of spermatozoa by ultrastructural and cytochemical analyses of testis and epididymis of the lizard Tropidurus itambere at different points of its annual reproductive cycle. Cytochemical investigations of spermiogenesis have not been performed so far in Squamata and are scarce for lower vertebrates. Essential components are: 1) polysaccharides, identified by PAS staining, abundantly present in Sertoli cell elongations, acrosomal vesicles and the acrosome of sperm cells; 2) glycoconjugate variations, labeled by different lectins and used to investigate cell modifications during spermiogenesis and found in mature spermatozoa in the female's seminal receptacle; 3) basic proteins, present in large quantities in spermatozoa in the subacrosomal cone, the pericentriolar material, the midpiece dense bodies, the peripheral fibers of the axoneme, and the fibrous sheath of the flagellum; 4) the final reaction product of acid phosphatase activity in several stages of acrosome development, specifically in the clear zone and epinuclear electron-lucent region of spermatozoal acrosomes, as well as in very active lysosomes found during the quiescent period of the reproductive cycle; 5) lipids, abundantly present in the cytoplasm of Leydig cells during the quiescent period. The cytochemical methods show that the ultrastructurally complex acrosome is also biochemically heterogeneous, with specific layers rich in glycoproteins, basic proteins or acid phosphatase. These different components may play a role during sperm penetration into the ovule. Basic proteins are largely responsible for structures surrounding the axoneme to provide resistance to the flagellum. In the quiescent period, acid phosphatase activity is involved in the elimination of superfluous sperm cells, whereas lipids in Leydig cells are used for hormone synthesis which starts at this time point to initiate a new reproductive cycle. Variations in lectin

  15. Attempt at intracytoplasmic sperm injection of in vitro matured oocytes in common minke whales (Balaenoptera acutorostrata) captured during the Kushiro Coast Survey.

    PubMed

    Fukui, Yutaka; Iwayama, Hiroshi; Matsuoka, Taiki; Nagai, Hiroki; Koma, Noriko; Mogoe, Toshihiro; Ishikawa, Hajime; Fujise, Yoshihiro; Hirabayashi, Masumi; Hochi, Shinichi; Kato, Hidehiro; Ohsumi, Seiji

    2007-08-01

    The present study was conducted during the Kushiro Coast Survey in an attempt to produce common minke whale embryos. In Experiment 1, we attempted to determine the appropriate culture duration (30 or 40 h) for in vitro maturation (IVM) of immature oocytes using the Well of the Well method. In Experiment 2, and intracytoplasmic sperm injection (ICSI) was applied to matured oocytes from prepubertal and adult common minke whales after IVM culture (40 or 48 h), and then their embryonic development was assessed. In Experiment 1, the maturation rate of oocytes cultured for 40 h (30.4%) was significantly higher than that of oocytes cultured for 30 h (6.8%; P<0.01). In Experiment 2, a total of 35 and 46 immature oocytes derived from adult (n=2) and prepubertal (n=6) minke whales, respectively, were cultured for 40 or 48 h. The maturation rate in the oocytes from the adult whales (34.2%) tended to be higher than that of the oocytes from the prepubertal whales (19.6%), but there was no significant difference. Following ICSI, 3 out of the 10 inseminated and cultured oocytes from the adult whales cleaved (2-, 8-, and 16-cell stages); all of these oocytes had been matured for 40 in culture. However, these oocytes did not develop to further stages. Only one of the 6 oocytes derived from the prepubertal whales, IVM cultured for 40 h and inseminated, developed to the 4-cell stage. The present results indicate that a 40 h IVM culture produces significantly higher rates of in vitro maturation than a 30 h IVM culture for common minke whale oocytes. Following ICSI, some oocytes cleaved to the 16-cell stage, but no further development was observed.

  16. Low-Dose Urinary Human Chorionic Gonadotropin Is Effective for Oocyte Maturation in In Vitro Fertilization/ Intracytoplasmic Sperm Injection Cycles Independent of Body Mass Index

    PubMed Central

    R. Hoyos, Luis; Khan, Sana; Dai, Jing; Singh, Manvinder; P. Diamond, Michael; E. Puscheck, Elizabeth; O. Awonuga, Awoniyi

    2017-01-01

    Background: Currently, there is no agreement on the optimal urinary derived human chorionic gonadotropin (u-hCG) dose requirement for initiating final oocyte maturation prior to oocyte collection in in vitro fertilization (IVF), but doses that range from 2500- 15000 IU have been used. We intended to determine whether low dose u-hCG was effective for oocyte maturation in IVF/intracytoplasmic sperm injection (ICSI) cycles independent of body mass index (BMI). Materials and Methods: We retrospectively evaluated a cohort of 295 women who underwent their first IVF/ICSI cycles between January 2003 and December 2010 at the Division of Reproductive Endocrinology and Infertility, Wayne State University, Detroit, MI, USA. Treatment cycles were divided into 3 groups based on BMI (kg/ m2): <25 (n=136), 25- <30 (n=84), and ≥30 (n=75) women. Patients received 5000, 10000 or 15000 IU u-hCG for final maturation prior to oocyte collection. The primary outcome was clinical pregnancy rates (CPRs) and secondary outcome was live birth rates (LBRs). Results: Only maternal age negatively impacted (P<0.001) CPR [odds ratio (OR=0.85, confidence interval (CI: 0.79-0.91)] and LBR (OR=0.84, CI: 0.78-0.90). Conclusion: Administration of lower dose u-hCG was effective for oocyte maturation in IVF and did not affect the CPRs and LBRs irrespective of BMI. Women’s BMI need not be taken into consideration in choosing the appropriate dose of u-hCG for final oocyte maturation prior to oocyte collection in IVF. Only maternal age at the time of IVF negatively influenced CPRs and LBRs in this study. PMID:28367299

  17. In vitro maturation, fertilization, embryo development & clinical outcome of human metaphase-I oocytes retrieved from stimulated intracytoplasmic sperm injection cycles

    PubMed Central

    Álvarez, Cristina; García-Garrido, Carmen; Taronger, Roser; de Merlo, Gaspar González

    2013-01-01

    Background & objectives: The major cause of fertilisation failure after ICSI is failure of the oocyte to initiate the biochemical processes necessary for activation. This inability could be ascribed to cytoplasmic immaturity of those gametes even if they had reached nuclear maturity. The activation of a mature oocyte is characterised by release from metaphase II (MII) arrest and extrusion of the second polar body, followed by pro-nuclear formation. The aim of this study was to evaluate the fate of in vitro matured (IVM) metaphase I (MI) oocytes subjected to intracytoplasmic sperm injection (ICSI) at different time intervals after extrusion of the first polar body (1PB) in in vitro fertilization (IVF) cycles. Methods: A total of 8030 oocytes were collected from 1400 ICSI cycles, 5504 MII at the time of cumulus retrieval. Four hundred eight metaphase II (MII) (27.1%) matured to MII after in vitro culture for 2-26 h and 5389 sibling MII in the moment of oocyte denudation were injected. On the other hand, 49 ICSI cycles containing only MI oocytes at retrieval were injected at three different time intervals after reaching the MII. The intervals were as follows: 2-6 h (n=10), 8-11 h (n=4) and 23-26 h (n=10). Fertilization and development potential were evaluated in both studies. Results: Fertilization, embryo cleavage and quality were significantly lower in IVM MI compared to MII at time of denudation. Pregnancy rate was higher in group MII. Pregnancy was achieved in three embryo transfers when ICSI was performed within 2-6 h (group I) and 8-11 h (group II) after PB extrusion. One pregnancy was obtained in group I and a healthy neonate was born. Interpretation & conclusions: Immature oocytes from women whose ovaries have been stimulated could be matured, fertilized by ICSI, cleaved in vitro and to give rise to a live birth. However, the developmental competence of embryos derived from immature oocytes is reduced, compared with sibling in vivo matured oocytes. Further

  18. Protein disulfide isomerase-P5, down-regulated in the final stage of boar epididymal sperm maturation, catalyzes disulfide formation to inhibit protein function in oxidative refolding of reduced denatured lysozyme.

    PubMed

    Akama, Kuniko; Horikoshi, Tomoe; Sugiyama, Atsushi; Nakahata, Satoko; Akitsu, Aoi; Niwa, Nobuyoshi; Intoh, Atsushi; Kakui, Yasutaka; Sugaya, Michiko; Takei, Kazuo; Imaizumi, Noriaki; Sato, Takaya; Matsumoto, Rena; Iwahashi, Hitoshi; Kashiwabara, Shin-ichi; Baba, Tadashi; Nakamura, Megumi; Toda, Tosifusa

    2010-06-01

    In mammalian spermiogenesis, sperm mature during epididymal transit to get fertility. The pig sharing many physiological similarities with humans is considered a promising animal model in medicine. We examined the expression profiles of proteins from boar epididymal caput, corpus, and cauda sperm by two-dimensional gel electrophoresis and peptide mass fingerprinting. Our results indicated that protein disulfide isomerase-P5 (PDI-P5) human homolog was down-regulated from the epididymal corpus to cauda sperm, in contrast to the constant expression of protein disulfide isomerase A3 (PDIA3) human homolog. To examine the functions of PDIA3 and PDI-P5, we cloned and sequenced cDNAs of pig PDIA3 and PDI-P5 protein precursors. Each recombinant pig mature PDIA3 and PDI-P5 expressed in Escherichia coli showed thiol-dependent disulfide reductase activities in insulin turbidity assay. Although PDIA3 showed chaperone activity to promote oxidative refolding of reduced denatured lysozyme, PDI-P5 exhibited anti-chaperone activity to inhibit oxidative refolding of lysozyme at an equimolar ratio. SDS-PAGE and Western blotting analysis suggested that disulfide cross-linked and non-productively folded lysozyme was responsible for the anti-chaperone activity of PDI-P5. These results provide a molecular basis and insights into the physiological roles of PDIA3 and PDI-P5 in sperm maturation and fertilization.

  19. Chromosomal aberrations in in-vitro matured oocytes influence implantation and ongoing pregnancy rates in a mouse model undergoing intracytoplasmic sperm injection.

    PubMed

    Li, Min; Zhao, Hong-Cui; Li, Rong; Yu, Yang; Qiao, Jie

    2014-01-01

    Implantation failure and early pregnancy loss have been reported to be closely related to the quality of mammalian oocytes; however, the pregnant outcome of embryos from in-vitro matured (IVM) oocytes remains unknown. In this study we examined spindle assembly and chromosome segregation during differentiation, and the duration of IVM of mouse oocytes. The resulting implantation and pregnancy outcomes were analyzed to clarify the relationship between the spindle and chromosomes of IVM oocytes and implantation and early pregnancy. Cumulus-enclosed germinal vesicle oocytes were collected and randomly cultured in IVM medium with different IVM durations. One part of IVM oocytes were analyzed the spindle and chromosome morphology by immunofluorescence method, and the other part of them were fertilized by intracytoplasmic sperm injection. The resulting embryos were transferred into pseudo-pregnant female mice, and the post-implantation and full term development was observed. The chromosome aberrations and incorrect spindle assembly seems not affect the early development and blastocyst cell number derived from IVM oocytes, however the development potential of the resulting embryos after implantation were significant decreased with the ratio increasing of chromosome aberrations and incorrect spindle assembly. Accordingly, the full-term development was also decreased. In conclusion, the present study showed the spindle assembly of in vitro-matured oocytes was one of the most important factors that affected the implantation and ongoing pregnancy rates of IVM oocytes, and the improvement by an appropriate duration of maturation in vitro will enhance the post-implantation development potential of the resulting embryos, and decrease implantation failure and early pregnancy loss.

  20. Early sexual maturity in local boars of Northeastern India: age-related changes in testicular growth, epididymal sperm characteristics and peripheral testosterone levels.

    PubMed

    Kumaresan, A; Bujarbaruah, K M; Kadirvel, G; Khargharia, G; Sarma, Rumi G; Goswami, J; Basumatary, Rantu; Palaniappan, Kavitha; Bardoloi, R K

    2011-03-01

    The present study reports the age related changes in the peripheral testosterone levels, testicular and epididymal growth and development and cauda epididymal spermiogram in local pigs of Northeastern India, which attain sexual maturity around 3 months of age. Local boars (n = 20) were castrated at monthly intervals from 2 to 6 months of age (4 boars per month) to study the testicular growth and development and the epididymal spermiogram. Blood samples, collected from local boars (n = 6) at monthly intervals from 2 to 6 months of age, were analyzed for testosterone levels by radioimmunoassay. Compared to Hampshire boars, significantly (P < 0.05) high testosterone levels were observed in the local boars as early as 2 months of age. The mean (± SEM) level of testosterone in the local boars at 2, 3 and 4 months of age was 11.89 ± 1.52, 20.45 ± 1.33 and 20.38 ± 2.0 ngml(-1), respectively. Though there was consistently significant (P < 0.05) difference in the body weight between Hampshire and local pigs, the same was not observed in case of testicular weight except at 3 and 6 months of age. In line with the above observation, the testis:body weight ratio (gram testis per kg body weight) was significantly (P < 0.05) higher in the local boars compared to the Hampshire boars at any time of observation, which ranged from 0.8 to 1.0 in case of Hampshire and from 2.3 to 3.0 in local boars. The sperm concentration in the cauda epididymal fluid of local boars at 2, 3 and 6 months of age was 2255 ± 186.6, 3685 ± 103.8 and 4325 ± 146.2 million/ml, respectively and the sperm motility, viability and total abnormality was 73.3, 75.2 and 6.2%, respectively at 3 months of age. Taken together, the testosterone level, testicular growth and development and epididymal spermiogram indicate the trait of early sexual maturity in the local pigs as compared to Hampshire.

  1. Sperm proteome and reproductive technologies in mammals.

    PubMed

    Li, Chun-Jin; Wang, Dong; Zhou, Xu

    2016-10-01

    Sperm is highly differentiated cell that can be easily obtained and purified. Mature sperm is considered to be transcriptionally and translationally silent and incapable of protein synthesis. Recently, a large number of proteins have been identified in sperm from different species by using the proteomic approaches. Clinically, sperm proteins can be used as markers for male infertility due to different protein profiles identified in sperm from fertile and infertile male animals. Recent evidences have shown that the conditions of sperm preservation in vitro can also change the sperm protein profiles. This paper reviews the recent scientific publications available to address sperm proteome and their relationship with sperm cryopreservation, capacitation, fertilization, and separation of X and Y sperm. Future directions in the application of sperm proteomics to develop or optimize reproductive technologies in mammals are also discussed.

  2. Extracellular vesicles: roles in gamete maturation, fertilization and embryo implantation

    PubMed Central

    Machtinger, Ronit; Laurent, Louise C.; Baccarelli, Andrea A.

    2016-01-01

    BACKGROUND Extracellular vesicles (EVs) are membrane-bound vesicles, found in biofluids, that carry and transfer regulatory molecules, such as microRNAs (miRNAs) and proteins, and may mediate intercellular communication between cells and tissues. EVs have been isolated from a wide variety of biofluids, including plasma, urine, and, relevant to this review, seminal, follicular and uterine luminal fluid. We conducted a systematic search of the literature to review and present the currently available evidence on the possible roles of EVs in follicular growth, resumption of oocyte development and maturation (meiosis), sperm maturation, fertilization and embryo implantation. METHODS MEDLINE, Embase and Web of Science databases were searched using keywords pertaining to EVs, including ‘extracellular vesicles’, ‘microvesicles’, ‘microparticles’ and ‘exosomes’, combined with a range of terms associated with the period of development between fertilization and implantation, including ‘oocyte’, 'sperm’, 'semen’, 'fertilization’, ‘implantation’, ‘embryo’, ‘follicular fluid’, ‘epididymal fluid’ and ‘seminal fluid’. Relevant research articles published in English (both animal and human studies) were reviewed with no restrictions on publication date (i.e. from earliest database dates to July 2015). References from these articles were used to obtain additional articles. RESULTS A total of 1556 records were retrieved from the three databases. After removing duplicates and irrelevant titles, we reviewed the abstracts of 201 articles, which included 92 relevant articles. Both animal and human studies unequivocally identified various types of EVs in seminal, follicular and ULFs. Several studies provided evidence for the roles of EVs in these biofluids. In men, EVs in seminal fluid were linked with post-testicular sperm maturation, including sperm motility acquisition and reduction of oxidative stress. In women, EVs in follicular fluid were

  3. Feline spermatozoa from fresh and cryopreserved testicular tissues have comparable ability to fertilize matured oocytes and sustain the embryo development after intracytoplasmic sperm injection.

    PubMed

    Buarpung, S; Tharasanit, T; Comizzoli, P; Techakumphu, M

    2013-01-01

    Cryopreservation of testicular tissue associated with intracytoplasmic sperm injection (ICSI) is a critical tool that still needs to be explored for preserving the fertility of endangered species. Using the domestic cat as a model for wild felids, the study aimed at determining the effect of different cryoprotectants and freezing techniques (two-step freezing vs. controlled slow freezing) on the sperm quality (membrane and DNA integrity). Then, spermatozoa were extracted from frozen-thawed testicular tissues and used for ICSI to assess early gamete activation or developmental competence in vitro. The percentage of spermatozoa with intact plasma membrane was not different (P > 0.05) among nonfrozen control, glycerol-, and ethylene glycol-frozen tissues (63.2 ± 2%, 58.2 ± 2.6%, 53.3 ± 2.3%, respectively). However, these percentages were significantly lower (P < 0.05) in groups of dimethyl sulfoxide (46.3 ± 3.3%) and 1,2 propanediol (44.3 ± 2.9%) when compared with control. Conventional freezing combined with 5% (vol/vol) glycerol best preserved sperm membrane integrity (55.0 ± 2.7%) when compared with other freezing techniques. The incidence of DNA fragmentation was found to be low (0.2%-1.1%) in all freezing protocols. After ICSI with frozen testicular spermatozoa, male and female gametes were asynchronously activated and the percentages of normal fertilization at 6, 12, and 18 hours were 11.2%, 20.6%, and 22.1%, respectively. Metaphase II-arrested oocytes containing or not a decondensed sperm head were predominantly found after ICSI with frozen testicular spermatozoa. Although two-pronucleus formation could be observed as soon as 6 hours post ICSI (10%), the rate increased dramatically after 12 and 18 hours post ICSI (17.2% and 19.5%, respectively). ICSI using frozen-thawed testicular spermatozoa yielded cleavage (32.7%), morula (6.5%), and blastocyst (4.4%) percentages similar to nonfrozen control (P > 0.05). It is concluded that conventional freezing

  4. Effect of recombinant human follicle-stimulating hormone and luteinizing hormone on in vitro maturation of porcine oocytes evaluated by the subsequent in vitro development of embryos obtained by in vitro fertilization, intracytoplasmic sperm injection, or parthenogenetic activation.

    PubMed

    Silvestre, M A; Alfonso, J; García-Mengual, E; Salvador, I; Duque, C C; Molina, I

    2007-05-01

    The aim of this work was to study the effect of recombinant human (rh) FSH and LH on in vitro maturation of pig oocytes compared with a conventional hormonal supplement based on equine (PMSG) and human chorionic gonadotropins (hCG), as evaluated by the developmental ability of 3 types of pig embryos obtained by in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), or artificial activation (ATA). In Exp. 1, one cumulus-oocyte complex group (A group) was supplemented with rh-FSH and rh-LH (0.1 IU/mL each), and the other group (B group) was supplemented with PMSG and hCG (10 IU/mL each). No differences in nuclear maturation between the A and B groups were observed (68.5 vs. 71.4%, respectively). No differences were detected between hormonal treatments in the rates of cleavage or blastocyst formation of ATA, IVF, and ICSI embryos. Total cell number of the embryos was not significantly different in any experimental group (A: 31.1, 28.5, and 19.8 vs. B: 25.2, 25.5, and 20.6 for ATA, IVF, and ICSI embryos, respectively). In Exp. 2, the effects of different concentrations of rh-FSH and rh-LH (0.5, 0.1, or 0.05 IU/mL) in maturation medium on nuclear maturation and in vitro development of embryos obtained by IVF were studied. No effect of different hormonal concentrations on blastocyst formation rates was observed (8.5, 13.0, and 5.7%, respectively). Blastocyst cell number was not different in any experimental group. In conclusion, the results obtained here permit us to substitute PMSG and hCG with rh-FSH and rh-LH and to produce pig embryos obtained by IVF, ICSI, or ATA.

  5. Association of sperm morphology and the sperm deformity index (SDI) with poly (ADP-ribose) polymerase (PARP) cleavage inhibition.

    PubMed

    Aziz, Nabil; Sharma, Rakesh K; Mahfouz, Reda; Jha, Rajesh; Agarwal, Ashok

    2011-06-30

    Apoptosis was induced in immature and mature sperm in the presence or absence of poly (ADP-ribose) polymerase (PARP) inhibitor. The association of cleaved (cPARP) with sperm morphology was examined using sperm deformity index (SDI) score. The SDI scores are associated with PARP cleavage as an early marker of apoptosis.

  6. Intracellular pH in sperm physiology.

    PubMed

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L; Darszon, Alberto

    2014-08-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca(2+) channel; Slo3, a K(+) channel; the sperm-specific Na(+)/H(+) exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction.

  7. Sugar‐coated sperm: Unraveling the functions of the mammalian sperm glycocalyx

    PubMed Central

    Tecle, Eillen

    2015-01-01

    SUMMARY Mammalian spermatozoa are coated with a thick glycocalyx that is assembled during sperm development, maturation, and upon contact with seminal fluid. The sperm glycocalyx is critical for sperm survival in the female reproductive tract and is modified during capacitation. The complex interplay among the various glycoconjugates generates numerous signaling motifs that may regulate sperm function and, as a result, fertility. Nascent spermatozoa assemble their own glycans while the cells still possess a functional endoplasmic reticulum and Golgi in the seminiferous tubule, but once spermatogenesis is complete, they lose the capacity to produce glycoconjugates de novo. Sperm glycans continue to be modified, during epididymal transit by extracellular glycosidases and glycosyltransferases. Furthermore, epididymal cells secrete glycoconjugates (glycophosphatidylinositol‐anchored glycoproteins and glycolipids) and glycan‐rich microvesicles that can fuse with the maturing sperm membrane. The sperm glycocalyx mediates numerous functions in the female reproductive tract, including the following: inhibition of premature capacitation; passage through the cervical mucus; protection from innate and adaptive female immunity; formation of the sperm reservoir; and masking sperm proteins involved in fertilization. The immense diversity in sperm‐associated glycans within and between species forms a remarkable challenge to our understanding of essential sperm glycan functions. Mol. Reprod. Dev. 82: 635–650, 2015. © 2015 The Authors. Molecular Reproduction and Development published by Wiley Periodicals, Inc. PMID:26061344

  8. A germline-specific isoform of eIF4E (IFE-1) is required for efficient translation of stored mRNAs and maturation of both oocytes and sperm.

    PubMed

    Henderson, Melissa A; Cronland, Elizabeth; Dunkelbarger, Steve; Contreras, Vince; Strome, Susan; Keiper, Brett D

    2009-05-15

    Fertility and embryonic viability are measures of efficient germ cell growth and development. During oogenesis and spermatogenesis, new proteins are required for both mitotic expansion and differentiation. Qualitative and quantitative changes in protein synthesis occur by translational control of mRNAs, mediated in part by eIF4E, which binds the mRNAs 5' cap. IFE-1 is one of five eIF4E isoforms identified in C. elegans. IFE-1 is expressed primarily in the germ line and associates with P granules, large mRNPs that store mRNAs. We isolated a strain that lacks IFE-1 [ife-1(bn127)] and demonstrated that the translation of several maternal mRNAs (pos-1, pal-1, mex-1 and oma-1) was inefficient relative to that in wild-type worms. At 25 degrees C, ife-1(bn127) spermatocytes failed in cytokinesis, prematurely expressed the pro-apoptotic protein CED-4/Apaf-1, and accumulated as multinucleate cells unable to mature to spermatids. A modest defect in oocyte development was also observed. Oocytes progressed normally through mitosis and meiosis, but subsequent production of competent oocytes became limiting, even in the presence of wild-type sperm. Combined gametogenesis defects decreased worm fertility by 80% at 20 degrees C; ife-1 worms were completely sterile at 25 degrees C. Thus, IFE-1 plays independent roles in late oogenesis and spermatogenesis through selective translation of germline-specific mRNAs.

  9. A novel SoxB2 gene is required for maturation of sperm nucleus during spermiogenesis in the Chinese mitten crab, Eriocheir sinensis

    PubMed Central

    Liu, Zhi-Qiang; Jiang, Xue-Hui; Qi, Hai-Yan; Xiong, Liang-Wei; Qiu, Gao-Feng

    2016-01-01

    SRY-related HMG box (Sox) genes are characterized by the presence of a DNA-binding HMG domain and involved in a diverse range of developmental processes. In this study, we identified a novel Sox gene, designated as EsSoxB2-1, from the Chinese mitten crab Eriocheir sinensis. The EsSoxB2-1 encodes a protein of 259 amino acids, sharing the highest identity with the beetle Tribolium castaneum SOX21b. Unlike insect Sox21b, however, EsSoxB2-1 is intronless and exhibits a gonad-specific expression pattern at both mRNA and protein level. Two core promoters in 5′ flanking region were demonstrated to be essential for inducing transcriptional regulatory activity. The transcription of EsSoxB2-1 mRNA begins in spermatogonia stage, while the translation of EsSOXB2-1 protein initiates at spermiogenesis stage. Interestingly, EsSOXB2-1 protein was exclusively localized in the nucleus of spermatid and spermatozoa even at the end of acrosome reaction, and was bound to the uncondensed chromatin in nucleoplasm of mature spermatozoa. Knockdown of EsSoxB2-1 by RNAi leads to abnormal transformation of the nucleus during spermiogenesis. Together, these findings demonstrated the requirement of EsSoxB2-1 for the spermatozoa nucleus maturation and also suggested that EsSoxB2-1 would be delivered into fertilized eggs along with chromatins as a paternal transcription factor for regulating early embryonic development. PMID:27561408

  10. Sperm competition leads to functional adaptations in avian testes to maximize sperm quantity and quality.

    PubMed

    Lüpold, Stefan; Wistuba, Joachim; Damm, Oliver S; Rivers, James W; Birkhead, Tim R

    2011-05-01

    The outcome of sperm competition (i.e. competition for fertilization between ejaculates from different males) is primarily determined by the relative number and quality of rival sperm. Therefore, the testes are under strong selection to maximize both sperm number and quality, which are likely to result in trade-offs in the process of spermatogenesis (e.g. between the rate of spermatogenesis and sperm length or sperm energetics). Comparative studies have shown positive associations between the level of sperm competition and both relative testis size and the proportion of seminiferous (sperm-producing) tissue within the testes. However, it is unknown how the seminiferous tissue itself or the process of spermatogenesis might evolve in response to sperm competition. Therefore, we quantified the different germ cell types and Sertoli cells (SC) in testes to assess the efficiency of sperm production and its associations with sperm length and mating system across 10 species of New World Blackbirds (Icteridae) that show marked variation in sperm length and sperm competition level. We found that species under strong sperm competition generate more round spermatids (RS)/spermatogonium and have SC that support a greater number of germ cells, both of which are likely to increase the maximum sperm output. However, fewer of the RS appeared to elongate to mature spermatozoa in these species, which might be the result of selection for discarding spermatids with undesirable characteristics as they develop. Our results suggest that, in addition to overall size and gross morphology, testes have also evolved functional adaptations to maximize sperm quantity and quality.

  11. Virtual azoospermia and cryptozoospermia--fresh/frozen testicular or ejaculate sperm for better IVF outcome?

    PubMed

    Hauser, Ron; Bibi, Guy; Yogev, Leah; Carmon, Ariella; Azem, Foad; Botchan, Amnon; Yavetz, Haim; Klieman, Sandra E; Lehavi, Ofer; Amit, Ami; Ben-Yosef, Dalit

    2011-01-01

    Men diagnosed as having azoospermia occasionally have a few mature sperm cells in other ejaculates. Other men may have constant, yet very low quality and quantity of sperm cells in their ejaculates, resulting in poor intracytoplasmic sperm injection (ICSI) outcome. It has not been conclusively established which source of sperm cells is preferable for ICSI when both ejaculate and testicular (fresh or frozen) sperm cells are available. It is also unclear whether there is any advantage of fresh over frozen sperm if testicular sperm is to be used. We used ejaculate, testicular (fresh or frozen) sperm cells, or both for ICSI in 13 couples. Five of these couples initially underwent ICSI by testicular sperm extraction, because the males had total azoospermia, and in later cycles with ejaculate sperm cells. Ejaculate sperm cells were initially used for ICSI in the other 8 patients, and later with testicular sperm cells. The fertilization rate was significantly higher when fresh or frozen-thawed testicular sperm cells were used than when ejaculated sperm cells were used. Likewise, the quality of the embryos from testicular (fresh and frozen) sperm was higher than from ejaculated sperm (65.3% vs 53.2%, respectively, P < .05). The use of fresh testicular sperm yielded better implantation rates than both frozen testicular sperm and ejaculate. Therefore, fresh testicular sperm should be considered first for ICSI in patients with virtual azoospermia or cryptozoospermia because of their superior fertility.

  12. Prudent sperm use by leaf-cutter ant queens

    PubMed Central

    den Boer, Susanne P. A.; Baer, Boris; Dreier, Stephanie; Aron, Serge; Nash, David R.; Boomsma, Jacobus J.

    2009-01-01

    In many species, females store sperm between copulation and egg fertilization, but the consequences of sperm storage and patterns of sperm use for female life history and reproductive success have not been investigated in great detail. In hymenopteran insect societies (ants, bees, wasps), reproduction is usually monopolized by one or relatively few queens, who mate only during a brief period early in life and store sperm for later use. The queens of some ants are particularly long-lived and have the potential to produce millions of offspring during their life. To do so, queens store many sperm cells, and this sperm must remain viable throughout the years of storage. Queens should also be under strong selection to use stored sperm prudently when fertilizing eggs. We used the leaf-cutter ant Atta colombica to investigate the dynamics of sperm use during egg fertilization. We show that queens are able to fertilize close to 100 per cent of the eggs and that the average sperm use per egg is very low, but increases with queen age. The robustness of stored sperm was found to decrease with years of storage, signifying that senescence affects sperm either directly or indirectly via the declining glandular secretions or deteriorating sperm-storage organs. We evaluate our findings with a heuristic model, which suggests that the average queen has sperm for almost 9 years of normal colony development. We discuss the extent to which leaf-cutter ant queens have been able to optimize their sperm expenditure and infer that our observed averages of sperm number, sperm robustness and sperm use are consistent with sperm depletion being a significant cause of mortality of mature colonies of Atta leaf-cutter ants. PMID:19710057

  13. Prudent sperm use by leaf-cutter ant queens.

    PubMed

    den Boer, Susanne P A; Baer, Boris; Dreier, Stephanie; Aron, Serge; Nash, David R; Boomsma, Jacobus J

    2009-11-22

    In many species, females store sperm between copulation and egg fertilization, but the consequences of sperm storage and patterns of sperm use for female life history and reproductive success have not been investigated in great detail. In hymenopteran insect societies (ants, bees, wasps), reproduction is usually monopolized by one or relatively few queens, who mate only during a brief period early in life and store sperm for later use. The queens of some ants are particularly long-lived and have the potential to produce millions of offspring during their life. To do so, queens store many sperm cells, and this sperm must remain viable throughout the years of storage. Queens should also be under strong selection to use stored sperm prudently when fertilizing eggs. We used the leaf-cutter ant Atta colombica to investigate the dynamics of sperm use during egg fertilization. We show that queens are able to fertilize close to 100 per cent of the eggs and that the average sperm use per egg is very low, but increases with queen age. The robustness of stored sperm was found to decrease with years of storage, signifying that senescence affects sperm either directly or indirectly via the declining glandular secretions or deteriorating sperm-storage organs. We evaluate our findings with a heuristic model, which suggests that the average queen has sperm for almost 9 years of normal colony development. We discuss the extent to which leaf-cutter ant queens have been able to optimize their sperm expenditure and infer that our observed averages of sperm number, sperm robustness and sperm use are consistent with sperm depletion being a significant cause of mortality of mature colonies of Atta leaf-cutter ants.

  14. A comparison of ejaculated and testicular spermatozoa aneuploidy rates in patients with high sperm DNA damage.

    PubMed

    Moskovtsev, Sergey I; Alladin, Naazish; Lo, Kirk C; Jarvi, Keith; Mullen, J Brendan M; Librach, Clifford L

    2012-06-01

    Testicular spermatozoa are utilized to achieve pregnancy in couples with severe male factor infertility. Several studies suggest that aneuploidy rates in spermatozoa are elevated at the testicular level in infertile patients compared to ejaculates of normal controls. However, essential data regarding aneuploidy rates between ejaculated and testicular spermatozoa in the same individuals is lacking. The purpose of our study was to compare aneuploidy rates at the testicular and post-testicular level from the same patients with persistently high sperm DNA damage. Ejaculates and testicular biopsies were obtained from eight patients with persistently high DNA damage (>30%). Both ejaculated and testicular samples were analyzed for sperm DNA damage and sperm aneuploidy for chromosomes 13, 18, 21, X, and Y. In addition, semen samples from ten normozoospermic men presenting for fertility evaluation served as a control group. A strong correlation between the alteration of spermatogenesis and chromatin deterioration was observed in our study. In the same individuals, testicular samples showed a significantly lower DNA damage compared to ejaculated spermatozoa (14.9% ± 5.0 vs. 40.6% ± 14.8, P<0.05), but significantly higher aneuploidy rates for the five analyzed chromosomes (12.41% ± 3.7 vs. 5.77% ± 1.2, P<0.05). While testicular spermatozoa appear favourable for ICSI in terms of lower DNA damage, this potential advantage could be offset by the higher aneuploidy rates in testicular spermatozoa.

  15. Early Versus Late Maturation Arrest: Reproductive Outcomes of Testicular Failure

    PubMed Central

    Weedin, John W.; Bennett, Richard C.; Fenig, David M.; Lamb, Dolores J.; Lipshultz, Larry I.

    2013-01-01

    Purpose There is a paucity of data characterizing infertile men with maturation arrest. We hypothesized that men with early stage maturation arrest could be clinically distinguished from men with late maturation arrest and would have worse reproductive outcomes. Materials and Methods We retrospectively reviewed the records of all patients with nonobstructive azoospermia and cryptozoospermia who underwent testis mapping and sperm extraction from 2002 to 2009 and for whom histopathological findings were available. Patients had uniform maturation arrest if multiple biopsies revealed maturation arrest at the spermatogonia/spermatocyte (early maturation arrest) or the spermatid (late maturation arrest) stage. Clinical parameters and pregnancy outcomes of in vitro fertilization/intracytoplasmic sperm injection were examined. Statistical analysis consisted of univariate and multivariate analysis. Results Uniform maturation arrest was identified in 49 of 219 men (22.3%) undergoing testicular sperm extraction. On multivariate analysis men with maturation arrest had significantly larger testes (p = 0.01), decreased follicle-stimulating hormone (p = 0.05) and more detectable genetic abnormalities (p = 0.01) than men with other histopathological conditions. Men with late maturation arrest had decreased follicle-stimulating hormone (p = 0.02), increased testosterone (p = 0.03) and a higher sperm retrieval rate at testicular sperm extraction (p = 0.01) than men with early maturation arrest. Predictors of successful sperm retrieval were larger testes, cryptozoospermia, late maturation arrest and hypospermatogenesis (each p ≤0.05). Pregnancy outcomes for men with maturation arrest were not significantly different from those for men with other histopathological conditions. Conclusions Maturation arrest is a common, diverse histopathological subtype of severe male infertility. Compared to men with late maturation arrest those with early maturation arrest have increased follicle

  16. Acrosomo-nuclear syndrome in canine sperm.

    PubMed

    Hrudka, F

    1983-01-01

    An acrosomo-nuclear syndrome in sperm of an infertile semicryptorchid dog is described. Based on an EM study of developing and mature sperm the syndrome is defined by simultaneous occurrence of these symptoms: 1) intranuclear inclusions of acrosomal origin, 2) maldifferentiated apical segment of acrosome, 3) intraacrosomal inclusions of Sertoli cell origin, 4) characteristic change of nuclear shape and 5) retained cytoplasmic droplet. The cause of the syndrome and possibility of a transfer of somatic factors are discussed.

  17. The Rhesus Macaque (Macaca mulatta) Sperm Proteome*

    PubMed Central

    Skerget, Sheri; Rosenow, Matthew; Polpitiya, Ashoka; Petritis, Konstantinos; Dorus, Steve; Karr, Timothy L.

    2013-01-01

    Mass spectrometry based proteomics has facilitated sperm composition studies in several mammalian species but no studies have been undertaken in non-human primate species. Here we report the analysis of the 1247 proteins that comprise the Rhesus macaque (Macaca mulatta) sperm proteome (termed the MacSP). Comparative analysis with previously characterized mouse and human sperm proteomes reveals substantial levels of orthology (47% and 40% respectively) and widespread overlap of functional categories based on Gene Ontology analyses. Approximately 10% of macaque sperm genes (113/1247) are significantly under-expressed in the testis as compared with other tissues, which may reflect proteins specifically acquired during epididymal maturation. Phylogenetic and genomic analyses of three MacSP ADAMs (A-Disintegrin and Metalloprotease proteins), ADAM18-, 20- and 21-like, provides empirical support for sperm genes functioning in non-human primate taxa which have been subsequently lost in the lineages leading to humans. The MacSP contains proteasome proteins of the 20S core subunit, the 19S proteasome activator complex and an alternate proteasome activator PA200, raising the possibility that proteasome activity is present in mature sperm. Robust empirical characterization of the Rhesus sperm proteome should greatly expand the possibility for targeted molecular studies of spermatogenesis and fertilization in a commonly used model species for human infertility. PMID:23816990

  18. Sperm cells manipulation employing dielectrophoresis.

    PubMed

    Rosales-Cruzaley, E; Cota-Elizondo, P A; Sánchez, D; Lapizco-Encinas, Blanca H

    2013-10-01

    Infertility studies are an important growing field, where new methods for the manipulation, enrichment and selection of sperm cells are required. Microfluidic techniques offer attractive advantages such as requirement of low sample volume and short processing times in the range of second or minutes. Presented here is the application of insulator-based dielectrophoresis (iDEP) for the enrichment and separation of mature and spermatogenic cells by employing a microchannel with cylindrical insulating structures with DC electric potentials in the range of 200-1500 V. The results demonstrated that iDEP has the potential to concentrate sperm cells and distinguish between mature and spermatogenic cells by exploiting the differences in shape which lead to differences in electric polarization. Viability assessments revealed that a significant percentage of the cells are viable after the dielectrophoretic treatment, opening the possibility for iDEP to be developed as a tool in infertility studies.

  19. Management and Breeding Soundness of Mature Bulls.

    PubMed

    Palmer, Colin W

    2016-07-01

    Mature bulls must be fed a balanced ration, vaccinated appropriately, and undergo a breeding soundness evaluation to ensure they meet what is required of a short, but intense breeding season. To be classified as a satisfactory potential breeder, minimum standards for physical soundness, scrotal circumference, sperm motility, and sperm morphology must be achieved using an accepted bull-breeding soundness evaluation format. Sperm production requires approximately 70 days. Heat and stress are the most common insults to spermatogenesis, causing an increase in morphologic abnormalities with obesity-associated scrotal fat accumulation being the most frequent cause of elevated testicular temperature in mature bulls.

  20. Etiologies of sperm oxidative stress

    PubMed Central

    Sabeti, Parvin; Pourmasumi, Soheila; Rahiminia, Tahereh; Akyash, Fatemeh; Talebi, Ali Reza

    2016-01-01

    Sperm is particularly susceptible to reactive oxygen species (ROS) during critical phases of spermiogenesis. However, the level of seminal ROS is restricted by seminal antioxidants which have beneficial effects on sperm parameters and developmental potentials. Mitochondria and sperm plasma membrane are two major sites of ROS generation in sperm cells. Besides, leukocytes including polymer phonuclear (PMN) leukocytes and macrophages produce broad category of molecules including oxygen free radicals, non-radical species and reactive nitrogen species. Physiological role of ROS increase the intracellular cAMP which then activate protein kinase in male reproductive system. This indicates that spermatozoa need small amounts of ROS to acquire the ability of nuclear maturation regulation and condensation to fertilize the oocyte. There is a long list of intrinsic and extrinsic factors which can induce oxidative stress to interact with lipids, proteins and DNA molecules. As a result, we have lipid peroxidation, DNA fragmentation, axonemal damage, denaturation of the enzymes, over generation of superoxide in the mitochondria, lower antioxidant activity and finally abnormal spermatogenesis. If oxidative stress is considered as one of the main cause of DNA damage in the germ cells, then there should be good reason for antioxidant therapy in these conditions. PMID:27351024

  1. Intracellular pH in Sperm Physiology

    PubMed Central

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L.; Darszon, Alberto

    2014-01-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca2+ channel; Slo3, a K+ channel; the sperm-specific Na+/H+ exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction. PMID:24887564

  2. Accurate sperm morphology assessment predicts sperm function.

    PubMed

    Abu Hassan Abu, D; Franken, D R; Hoffman, B; Henkel, R

    2012-05-01

    Sperm morphology has been associated with in vitro as well as in vivo fertilisation. The study aimed to evaluate the possible relation between the percentage of spermatozoa with normal morphology and the following sperm functional assays: (i) zona-induced acrosome reaction (ZIAR); (ii) DNA integrity; (iii) chromatin condensation; (iv) sperm apoptosis; and (v) fertilisation rates. Regression analysis was employed to calculate the association between morphology and different functional tests. Normal sperm morphology correlated significantly with the percentages of live acrosome-reacted spermatozoa in the ZIAR (r = 0.518; P < 0.0001; n = 92), DNA integrity (r = -0.515; P = 0.0018; n = 34), CMA(3) -positive spermatozoa (r = -0.745; P < 0.0001; n = 92), sperm apoptosis (r = -0.395; P = 0.0206; n = 34) and necrosis (r = -0.545; P = 0.0009; n = 34). Negative correlations existed between for the acrosome reaction, and DNA integrity, while negative associations were recorded with the percentages of CMA(3) -positive spermatozoa, apoptotic and necrotic spermatozoa. Sperm morphology is related to sperm dysfunction such as poor chromatin condensation, acrosome reaction and DNA integrity. Negative and significant correlations existed between normal sperm morphology and chromatin condensation, the percentage of spermatozoa with abnormal DNA and spermatozoa with apoptotic activity. The authors do not regard sperm morphology as the only test for the diagnosis of male fertility, but sperm morphology can serve as a valuable indicator of underlying dysfunction.

  3. Comparative Transcriptomics of Arabidopsis thaliana Sperm Cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In flowering plants the two sperm cells are embedded within the cytoplasm of the growing pollen tube and as such are passively transported to the embryo sac, wherein double fertilization occurs upon their release. Understanding the mechanisms and conditions by which male gametes mature and take part...

  4. Protective effect of hyaluronic acid on cryopreserved boar sperm.

    PubMed

    Qian, Li; Yu, Sijiu; Zhou, Yan

    2016-06-01

    This study aimed to evaluate the effects of supplementing freezing and thawing media with hyaluronic acid (HA) on the quality parameters of frozen-thawed boar spermatozoa. Boar semen samples were collected from seven mature Yorkshire boars once a week using the gloved hand technique; these samples were frozen-thawed in the extender with added HA. Boar sperm was cryopreserved in the extender with HA added at concentrations of 0 (used as control), 4, 6, 8, 8 and 12mg/L, and their effects on the quality of frozen-thawed boar sperm were evaluated. HA addition to the extender significantly improved sperm motility, sperm membrane integrity, mitochondrial activity, acrosomal integrity, superoxide dismutase and catalase activity, but decreased sperm malondialdehyde level (p<0.05). Therefore, HA could be a promising cryoprotectant for boar sperm.

  5. Sperm head phenotype and male fertility in ram semen.

    PubMed

    Maroto-Morales, A; Ramón, M; García-Álvarez, O; Montoro, V; Soler, A J; Fernández-Santos, M R; Roldan, E R S; Pérez-Guzmán, M D; Garde, J J

    2015-12-01

    Although there is ample evidence for the effects of sperm head shape on sperm function, its impact on fertility has not been explored in detail at the intraspecific level in mammals. Here, we assess the relationship between sperm head shape and male fertility in a large-scale study in Manchega sheep (Ovis aries), which have not undergone any selection for fertility. Semen was collected from 83 mature rams, and before insemination, head shapes were measured for five parameters: area, perimeter, length, width, and p2a (perimeter(2)/2×π×area) using a computer-assisted sperm morphometric analysis. In addition, a cluster analysis using sperm head length and p2a factor was performed to determine sperm subpopulations (SPs) structure. Our results show the existence of four sperm SPs, which present different sperm head phenotype: SP1 (large and round), SP2 (short and elongated), SP3 (shortest and round), and SP4 (large and the most elongated). No relationships were found between males' fertility rates and average values of sperm head dimensions. However, differences in fertility rates between rams were strongly associated to the proportion of spermatozoa in an ejaculate SP with short and elongated heads (P < 0.001). These findings show how the heterogeneity in sperm head shape of the ejaculate has an effect on reproductive success, and highlight the important role of modulation of the ejaculate at the intraspecific level.

  6. Not All Sperm Are Equal: Functional Mitochondria Characterize a Subpopulation of Human Sperm with Better Fertilization Potential

    PubMed Central

    Sousa, Ana Paula; Amaral, Alexandra; Baptista, Marta; Tavares, Renata; Caballero Campo, Pedro; Caballero Peregrín, Pedro; Freitas, Albertina; Paiva, Artur; Almeida-Santos, Teresa; Ramalho-Santos, João

    2011-01-01

    Human sperm samples are very heterogeneous and include a low amount of truly functional gametes. Distinct strategies have been developed to characterize and isolate this specific subpopulation. In this study we have used fluorescence microscopy and fluorescence-activated cell sorting to determine if mitochondrial function, as assessed using mitochondrial-sensitive probes, could be employed as a criterion to obtain more functional sperm from a given ejaculate. We first determined that mitochondrial activity correlated with the quality of distinct human samples, from healthy donors to patients with decreased semen quality. Furthermore, using fluorescence-activated cell sorting to separate sperm with active and inactive mitochondria we found that this was also true within samples. Indeed, sperm with active mitochondria defined a more functional subpopulation, which contained more capacitated and acrosome intact cells, sperm with lower chromatin damage, and, crucially, sperm more able to decondense and participate in early development using both chemical induction and injection into mature bovine oocytes. Furthermore, cell sorting using mitochondrial activity produced a more functional sperm subpopulation than classic swim-up, both in terms of improvement in a variety of functional sperm parameters and in statistical significance. In conclusion, whatever the true biological role of sperm mitochondria in fertilization, mitochondrial activity is a clear hallmark of human sperm functionality. PMID:21448461

  7. Kinesin-1 Prevents Capture of the Oocyte Meiotic Spindle by the Sperm Aster

    PubMed Central

    McNally, Karen L.P.; Fabritius, Amy S.; Ellefson, Marina L.; Flynn, Jonathan R.; Milan, Jennifer A.; McNally, Francis J.

    2012-01-01

    Centrioles are lost during oogenesis and inherited from the sperm at fertilization. In the zygote, the centrioles recruit pericentriolar proteins from the egg to form a mature centrosome that nucleates a sperm aster. The sperm aster then captures the female pronucleus to join the maternal and paternal genomes. Because fertilization occurs before completion of female meiosis, some mechanism must prevent capture of the meiotic spindle by the sperm aster. Here we show that in wild-type Caenorhabditis elegans zygotes, maternal pericentriolar proteins are not recruited to the sperm centrioles until after completion of meiosis. Depletion of kinesin-1 heavy chain or its binding partner resulted in premature centrosome maturation during meiosis and growth of a sperm aster that could capture the oocyte meiotic spindle. Kinesin prevents recruitment of pericentriolar proteins by coating the sperm DNA and centrioles and thus prevents triploidy by a non-motor mechanism. PMID:22465668

  8. Flow cytometry of sperm

    SciTech Connect

    Gledhill, B.L.

    1987-09-21

    This brief paper summarizes automated flow cytometric determination of sperm morphology and flow cytometry/sorting of sperm with application to sex preselection. In the latter context, mention is made of results of karyotypic determination of sex chromosome ratios in albumin-processed human sperm. 23 refs., 1 fig., 1 tab.

  9. Calcium signaling and the MAPK cascade are required for sperm activation in Caenorhabditis elegans.

    PubMed

    Liu, Zhiyu; Wang, Bin; He, Ruijun; Zhao, Yanmei; Miao, Long

    2014-02-01

    In nematode, sperm activation (or spermiogenesis), a process in which the symmetric and non-motile spermatids transform into polarized and crawling spermatozoa, is critical for sperm cells to acquire fertilizing competence. SPE-8 dependent and SPE-8 independent pathways function redundantly during sperm activation in both males and hermaphrodites of Caenorhabditis elegans. However, the downstream signaling for both pathways remains unclear. Here we show that calcium signaling and the MAPK cascade are required for both SPE-8 dependent and SPE-8 independent sperm activation, implying that both pathways share common downstream signaling components during sperm activation. We demonstrate that activation of the MAPK cascade is sufficient to activate spermatids derived from either wild-type or spe-8 group mutant males and that activation of the MAPK cascade bypasses the requirement of calcium signal to induce sperm activation, indicating that the MAPK cascade functions downstream of or parallel with the calcium signaling during sperm activation. Interestingly, the persistent activation of MAPK in activated spermatozoa inhibits Major Sperm Protein (MSP)-based cytoskeleton dynamics. We demonstrate that MAPK plays dual roles in promoting pseudopod extension during sperm activation but also blocking the MSP-based, amoeboid motility of the spermatozoa. Thus, though nematode sperm are crawling cells, morphologically distinct from flagellated sperm, and the molecular machinery for motility of amoeboid and flagellated sperm is different, both types of sperm might utilize conserved signaling pathways to modulate sperm maturation.

  10. Comparative transcriptomics of Arabidopsis sperm cells.

    PubMed

    Borges, Filipe; Gomes, Gabriela; Gardner, Rui; Moreno, Nuno; McCormick, Sheila; Feijó, José A; Becker, Jörg D

    2008-10-01

    In flowering plants, the two sperm cells are embedded within the cytoplasm of the growing pollen tube and as such are passively transported to the embryo sac, wherein double fertilization occurs upon their release. Understanding the mechanisms and conditions by which male gametes mature and take part in fertilization are crucial goals in the study of plant reproduction. Studies of gene expression in male gametes of maize (Zea mays) and Plumbago and in lily (Lilium longiflorum) generative cells already showed that the previously held view of transcriptionally inert male gametes was not true, but genome-wide studies were lacking. Analyses in the model plant Arabidopsis (Arabidopsis thaliana) were hindered, because no method to isolate sperm cells was available. Here, we used fluorescence-activated cell sorting to isolate sperm cells from Arabidopsis, allowing GeneChip analysis of their transcriptome at a genome-wide level. Comparative analysis of the sperm cell transcriptome with those of representative sporophytic tissues and of pollen showed that sperm has a distinct and diverse transcriptional profile. Functional classifications of genes with enriched expression in sperm cells showed that DNA repair, ubiquitin-mediated proteolysis, and cell cycle progression are overrepresented Gene Ontology categories. Moreover, analysis of the small RNA and DNA methylation pathways suggests that distinct mechanisms might be involved in regulating the epigenetic state of the paternal genome. We identified numerous candidate genes whose involvement in sperm cell development and fertilization can now be directly tested in Arabidopsis. These results provide a roadmap to decipher the role of sperm-expressed proteins.

  11. Sperm is epigenetically programmed to regulate gene transcription in embryos

    PubMed Central

    Teperek, Marta; Simeone, Angela; Gaggioli, Vincent; Miyamoto, Kei; Allen, George E.; Erkek, Serap; Kwon, Taejoon; Marcotte, Edward M.; Zegerman, Philip; Bradshaw, Charles R.; Peters, Antoine H.F.M.; Gurdon, John B.; Jullien, Jerome

    2016-01-01

    For a long time, it has been assumed that the only role of sperm at fertilization is to introduce the male genome into the egg. Recently, ideas have emerged that the epigenetic state of the sperm nucleus could influence transcription in the embryo. However, conflicting reports have challenged the existence of epigenetic marks on sperm genes, and there are no functional tests supporting the role of sperm epigenetic marking on embryonic gene expression. Here, we show that sperm is epigenetically programmed to regulate embryonic gene expression. By comparing the development of sperm- and spermatid-derived frog embryos, we show that the programming of sperm for successful development relates to its ability to regulate transcription of a set of developmentally important genes. During spermatid maturation into sperm, these genes lose H3K4me2/3 and retain H3K27me3 marks. Experimental removal of these epigenetic marks at fertilization de-regulates gene expression in the resulting embryos in a paternal chromatin-dependent manner. This demonstrates that epigenetic instructions delivered by the sperm at fertilization are required for correct regulation of gene expression in the future embryos. The epigenetic mechanisms of developmental programming revealed here are likely to relate to the mechanisms involved in transgenerational transmission of acquired traits. Understanding how parental experience can influence development of the progeny has broad potential for improving human health. PMID:27034506

  12. The regulation of spermatogenesis and sperm function in nematodes

    PubMed Central

    Ellis, Ronald E.; Stanfield, Gillian M.

    2014-01-01

    In the nematode C. elegans, both males and self-fertile hermaphrodites produce sperm. As a result, researchers have been able to use a broad range of genetic and genomic techniques to dissect all aspects of sperm development and function. Their results show that the early stages of spermatogenesis are controlled by transcriptional and translational processes, but later stages are dominated by protein kinases and phosphatases. Once spermatids are produced, they participate in many interactions with other cells — signals from the somatic gonad determine when sperm activate and begin to crawl, signals from the female reproductive tissues guide the sperm, and signals from sperm stimulate oocytes to mature and be ovulated. The sperm also show strong competitive interactions with other sperm and oocytes. Some of the molecules that mediate these processes have conserved functions in animal sperm, others are conserved proteins that have been adapted for new roles in nematode sperm, and some are novel proteins that provide insights into evolutionary change. The advent of new techniques should keep this system on the cutting edge of research in cellular and reproductive biology. PMID:24718317

  13. Formation of primary sperm conjugates in a haplogyne spider (Caponiidae, Araneae) with remarks on the evolution of sperm conjugation in spiders.

    PubMed

    Lipke, Elisabeth; Michalik, Peter

    2012-11-01

    Sperm conjugation, where two or more sperm are physically united, is a rare but widespread pheno-menon across the animal kingdom. One group well known for its different types of sperm conjugation are spiders. Particularly, haplogyne spiders show a high diversity of sperm traits. Besides individual cleistospermia, primary (synspermia) and secondary (coenospermia, "spermatophore") sperm conjugation occurs. However, the evolution of sperm conjugates and sperm is not understood in this group. Here, we look at how sperm are transferred in Caponiidae (Haplogynae) in pursuit of additional information about the evolution of sperm transfer forms in spiders. Additionally, we investigated the male reproductive system and spermatozoa using light- and transmission electron-microscopy and provide a 3D reconstruction of individual as of well as conjugated spermatozoa. Mature spermatozoa are characterized by an extremely elongated, helical nucleus resulting in the longest spider sperm known to date. At the end of spermiogenesis, synspermia are formed by complete fusion of four spermatids. Thus, synspermia might have evolved early within ecribellate Haplogynae. The fused sperm cells are surrounded by a prominent vesicular area. The function of the vesicular area remains still unknown but might be correlated with the capacitation process inside the female. Further phylogenetic and functional implications of the spermatozoa and sperm conjugation are discussed.

  14. Increased aneuploidy rate in sperm with fragmented DNA as determined by the sperm chromatin dispersion (SCD) test and FISH analysis.

    PubMed

    Muriel, Lourdes; Goyanes, Vicente; Segrelles, Enrique; Gosálvez, Jaime; Alvarez, Juan G; Fernández, José Luis

    2007-01-01

    Previous studies suggest that sperm DNA fragmentation may be associated with aneuploidy. However, currently available tests have not made it possible to simultaneously perform DNA fragmentation and chromosomal analyses on the same sperm cell. The recently introduced sperm chromatin dispersion (SCD) test allows users to determine this relationship. Semen samples from 16 males, including 4 fertile donors, 7 normozoospermic, 3 teratozoospermic, 1 asthenozoospermic, and 1 oligoasthenoteratozoospermic, were processed for DNA fragmentation analysis by the SCD test using the Halosperm kit. Three-color fluorescence in situ hybridization (FISH) was performed on SCD-processed slides to determine aneuploidy for chromosomes X, Y, and 18. Spermatozoa with DNA fragmentation showed a 4.4 +/- 1.9-fold increase in diploidy rate and a 5.9 +/- 3.5-fold increase in disomy rate compared to spermatozoa without DNA fragmentation. The overall aneuploidy rate was 4.6 +/- 2.0-fold higher in sperm with fragmented DNA (Wilcoxon rank test: P < .001 in the 3 comparisons). A higher frequency of DNA fragmentation was found in sperm cells containing sex chromosome aneuploidies originated in both first and second meiotic divisions. The observed increase in aneuploidy rate in sperm with fragmented DNA may suggest that the occurrence of aneuploidy during sperm maturation may lead to sperm DNA fragmentation as part of a genomic screening mechanism developed to genetically inactivate sperm with a defective genomic makeup.

  15. Valosin-containing protein/p97 interacts with sperm-activating and sperm-attracting factor (SAAF) in the ascidian egg and modulates sperm-attracting activity.

    PubMed

    Kondoh, Eri; Konno, Aru; Inaba, Kazuo; Oishi, Tohru; Murata, Michio; Yoshida, Manabu

    2008-10-01

    Sperm chemotaxis toward an egg is observed in many animals, and the control of sperm-attracting activity is thought to play an important role in ensuring fertilization. However, the mechanism underlying the release of a sperm attractant from an egg is still obscure. In this study, we examined the systems involved in the release of sperm-activating and sperm-attracting factor (SAAF), which is the sperm attractant of the ascidian Ciona intestinalis. Here, we show that the egg acquires sperm-attracting activity after germinal vesicle breakdown. Further, since the cytoplasmic extracts of immature oocytes exhibit no sperm-attracting activity, the SAAF in oocytes may be activated after germinal vesicle breakdown. We found 13 SAAF-binding proteins in an egg plasma membrane extract and identified five proteins by proteomic analysis: valosin-containing protein (VCP)/p97, proteasome alpha 2 subunit, MGC97756 protein, proteasome subunit Y, and beta-tubulin. In particular, the interaction between VCP/p97 and SAAF was confirmed by a pull-down assay. VCP/p97 is initially localized in the germinal vesicle, and during oocyte maturation, it shifts to the endoplasmic reticulum in the cortical regions. Thus, VCP/p97 is a potential modulator of SAAF release from the egg.

  16. Metabolic rate limits the effect of sperm competition on mammalian spermatogenesis.

    PubMed

    delBarco-Trillo, Javier; Tourmente, Maximiliano; Roldan, Eduardo R S

    2013-01-01

    Sperm competition leads to increased sperm production in many taxa. This response may result from increases in testes size, changes in testicular architecture or changes in the kinetics of spermatogenesis, but the impact of each one of these processes on sperm production has not been studied in an integrated manner. Furthermore, such response may be limited in species with low mass-specific metabolic rate (MSMR), i.e., large-bodied species, because they cannot process energy and resources efficiently enough both at the organismic and cellular levels. Here we compare 99 mammalian species and show that higher levels of sperm competition correlated with a) higher proportions of seminiferous tubules, b) shorter seminiferous epithelium cycle lengths (SECL) which reduce the time required to produce sperm, and c) higher efficiencies of Sertoli cells (involved in sperm maturation). These responses to sperm competition, in turn, result in higher daily sperm production, more sperm stored in the epididymides, and more sperm in the ejaculate. However, the two processes that require processing resources at faster rates (SECL and efficiency of Sertoli cells) only respond to sperm competition in species with high MSMR. Thus, increases in sperm production with intense sperm competition occur via a complex network of mechanisms, but some are constrained by MSMR.

  17. Mammalian sperm morphometry.

    PubMed Central

    Gage, M J

    1998-01-01

    Understanding the adaptive significance of sperm form and function has been a challenge to biologists because sperm are highly specialized cells operating at a microscopic level in a complex environment. A fruitful course of investigation has been to use the comparative approach. This comparative study attempts to address some fundamental questions of the evolution of mammalian sperm morphometry. Data on sperm morphometry for 445 mammalian species were collated from published sources. I use contemporary phylogenetic analysis to control for the inherent non-independence of species and explore relationships between the morphometric dimensions of the three essential spermatozoal components: head, mid-piece and flagellum. Energy for flagellar action is metabolized by the mitochondrial-dense mid-piece and these combine to propel the sperm head, carrying the male haplotype, to the ovum. I therefore search for evolutionary associations between sperm morphometry and body mass, karyotype and the duration of oestrus. In contrast to previous findings, there is no inverse correlation between body weight and sperm length. Sperm mid-piece and flagellum lengths are positively associated with both head length and area, and the slopes of these relationships are discussed. Flagellum length is positively associated with mid-piece length but, in contrast to previous research and after phylogenetic control, I find no relationship between flagellum length and the volume of the mitochondrial sheath. Sperm head dimensions are not related to either genome mass or chromosome number, and there are no relationships between sperm morphometry and the duration of oestrus. PMID:9474794

  18. Assessment of Chromatin Maturity in Human Spermatozoa: Useful Aniline Blue Assay for Routine Diagnosis of Male Infertility

    PubMed Central

    Chakroun, Nozha; Ben Zarrouk, Soumaya; Sellami, Hanen; Kebaili, Sahbi; Rebai, Tarek; Keskes, Leila

    2013-01-01

    During spermatogenesis, sperm chromatin undergoes structural changes and results in a high condensation. This nuclear compaction would be useful as a predictor of sperm fertilization capacity and pregnancy outcome. We purpose to evaluate firstly the relationship among chromatin maturity assessed by aniline blue staining (AB) and the semen parameters in infertile men. Secondly, we analyzed whether the sperm gradient density centrifugation is effective to select mature spermatozoa. Fifty-one ejaculates were investigated by semen analysis and stained for chromatin condensation with AB to distinguish between unstained mature sperm and stained immature sperm. AB was applied also on 12 ejaculates which proceeded by density gradient centrifugation to compare the rates of immature sperm before and after selection. Neat semen were divided into two groups: G1 (n = 31): immature sperm <20% and G2 (n = 20): immature sperm ≥20%. No significant differences were detected in sperm concentration, motility, and normal morphology between G1 and G2. However, the rates of some morphology abnormalities were higher in G2: head abnormalities (P = 0.01) and microcephalic sperm (P = 0.02). We founded significant correlation between sperm immaturity and acrosome abnormalities (r = 0.292; P = 0.03). Sperm selection has significantly reduced the rates of immature sperm. A better understanding of chromatin structure and its impact on the sperm potential is needed to explore male infertility. PMID:24198830

  19. Genetic sperm defects.

    PubMed

    Chenoweth, Peter J

    2005-08-01

    Genetic sperm defects are specific sperm defects, which have been shown to have a genetic mode of transmission. Such genetic linkage, either direct or indirect, has been associated with a number of sperm defects in different species, with this number increasing with improved diagnostic capabilities. A number of sperm defects, which have proven or suspected genetic modes of transmission are discussed herein, with particular emphasis on cattle. These include: 1. Acrosome defects (knobbed, ruffled and incomplete); 2. Head defects (abnormal condensation, decapitated, round head, rolled head, nuclear crest); 3. Midpiece abnormalities ("Dag" defect, "corkscrew" defect, "pseudo-droplet" defect); 4. Tail defects ("tail stump" defect, primary ciliary dyskinesia).

  20. Binding of Sperm to the Zona Pellucida Mediated by Sperm Carbohydrate-Binding Proteins is not Species-Specific in vitro between Pigs and Cattle

    PubMed Central

    Takahashi, Kazuya; Kikuchi, Kazuhiro; Uchida, Yasuomi; Kanai-Kitayama, Saeko; Suzuki, Reiichiro; Sato, Reiko; Toma, Kazunori; Geshi, Masaya; Akagi, Satoshi; Nakano, Minoru; Yonezawa, Naoto

    2013-01-01

    Carbohydrates are candidates for the basis of species-selective interaction of gametes during mammalian fertilization. In this study, we sought to clarify the roles of sugar residues in the species-selective, sperm–oocyte interaction in pigs and cattle. Acrosome-intact porcine and bovine sperm exhibited their strongest binding affinities for β-Gal and α-Man residues, respectively. Porcine-sperm specificity changed from β-Gal to α-Man after the acrosome reaction, while bovine-sperm specificity did not. Binding of acrosome-intact and acrosome-reacted sperm decreased after trypsinization, indicating that the carbohydrate-binding components are proteins. While immature oocytes bound homologous sperm preferentially to heterologous sperm, oocytes matured in vitro bound similar numbers of homologous and heterologous sperm. Lectin staining revealed the aggregation of α-Man residues on the outer surface of the porcine zona during maturation. In both species, zona-free, mature oocytes bound homologous sperm preferentially to heterologous sperm. The lectin-staining patterns of the zona pellucida and zona-free oocytes coincided with the carbohydrate-binding specificities of acrosome-intact and acrosome-reacted sperm, respectively, supporting the involvement of carbohydrates in gamete recognition in pigs and cattle. These results also indicate that sperm-zona pellucida and sperm–oolemma bindings are not strictly species-specific in pigs and cattle, and further suggest that sperm penetration into the zona and/or fusion with oolemma may be species-specific between pigs and cattle. PMID:24970158

  1. Mouse Sperm Membrane Potential Hyperpolarization Is Necessary and Sufficient to Prepare Sperm for the Acrosome Reaction*

    PubMed Central

    De La Vega-Beltran, Jose Luis; Sánchez-Cárdenas, Claudia; Krapf, Darío; Hernandez-González, Enrique O.; Wertheimer, Eva; Treviño, Claudia L.; Visconti, Pablo E.; Darszon, Alberto

    2012-01-01

    Mammalian sperm are unable to fertilize the egg immediately after ejaculation; they acquire this capacity during migration in the female reproductive tract. This maturational process is called capacitation and in mouse sperm it involves a plasma membrane reorganization, extensive changes in the state of protein phosphorylation, increases in intracellular pH (pHi) and Ca2+ ([Ca2+]i), and the appearance of hyperactivated motility. In addition, mouse sperm capacitation is associated with the hyperpolarization of the cell membrane potential. However, the functional role of this process is not known. In this work, to dissect the role of this membrane potential change, hyperpolarization was induced in noncapacitated sperm using either the ENaC inhibitor amiloride, the CFTR agonist genistein or the K+ ionophore valinomycin. In this experimental setting, other capacitation-associated processes such as activation of a cAMP-dependent pathway and the consequent increase in protein tyrosine phosphorylation were not observed. However, hyperpolarization was sufficient to prepare sperm for the acrosome reaction induced either by depolarization with high K+ or by addition of solubilized zona pellucida (sZP). Moreover, K+ and sZP were also able to increase [Ca2+]i in non-capacitated sperm treated with these hyperpolarizing agents but not in untreated cells. On the other hand, in conditions that support capacitation-associated processes blocking hyperpolarization by adding valinomycin and increasing K+ concentrations inhibited the agonist-induced acrosome reaction as well as the increase in [Ca2+]i. Altogether, these results suggest that sperm hyperpolarization by itself is key to enabling mice sperm to undergo the acrosome reaction. PMID:23095755

  2. Effect of sorafenib on sperm count and sperm motility in male Swiss albino mice

    PubMed Central

    Shetty, Surekha Devadasa; Bairy, Laxminarayana Kurady

    2015-01-01

    The issue of male germ line mutagenesis and the effects on developmental defects in the next generation has become increasingly high profile over recent years. Mutagenic substance affects germinal cells in the testis. Since the cells are undergoing different phases of cell division and maturation, it is an ideal system to study the effect of chemotherapeutic agents. There are lacunae in the literature on the effect of sorafenib on gonadal function. With background, a study was planned to evaluate the effects of sorafenib on sperm count and sperm motility in male Swiss albino mice. Male Swiss albino mice were used for the study. The animals were segregated into control, positive control (PC) and three treatment groups. PC received oral imatinib (100 mg/kg body weight) and treatment groups received 25, 50, and 100 mg/kg body weight of sorafenib orally for 7 consecutive days at intervals of 24 h between two administrations. The control group remained in the home cage for an equal duration of time to match their corresponding treatment groups. The animals were sacrificed at the end of 1st, 2nd, 4th, 5th, 7th, and 10th weeks after the last exposure to drug, respectively. Sperm suspensions were prepared and introduced into a counting chamber. Total sperm count and motility were recorded. There was a significant decrease in sperm count and sperm motility by sorafenib which was comparable with the effect of PC imatinib. Sorafenib adversely affects sperm count and sperm motility which are reversible after discontinuation of treatment. PMID:26605157

  3. Reliable single sperm cryopreservation in Cell Sleepers for azoospermia management.

    PubMed

    Coetzee, K; Ozgur, K; Berkkanoglu, M; Bulut, H; Isikli, A

    2016-03-01

    Conventional sperm freezing methods perform best when freezing sperm samples containing at least hundreds of spermatozoa. In this severe male factor infertility case series, we examined the reproductive outcomes in 12 intracytoplasmic sperm injection cases where spermatozoa used were frozen in Cell Sleepers. Cell Sleepers are novel devices in which individual spermatozoa can be frozen in microdroplets. The case series included five men with obstructive azoospermia, six with nonobstructive azoospermia and one with cryptozoospermia, in whom microscopic sperm retrievals from testicular sperm extraction (TESE), micro-TESE extracts and a centrifugation procedure resulted in less than 50 spermatozoa. A total of 304 microscopically retrieved spermatozoa were frozen in 20 Cell Sleepers using a rapid manual cryopreservation method. A total of 179 mature oocytes were injected with recovered thawed spermatozoa, resulting in a fertilisation rate of 65.9% (118 of 179), with no total fertilisation failures. In 10 cases, an embryo transfer was performed, three on day 3 and seven on day 5, resulting in a per cycle pregnancy rate of 58.3% (seven of 12). Four of the pregnancies have progressed past 20 gestation weeks. The recovery and use of spermatozoa that were frozen in Cell Sleepers was uncomplicated and effective and eliminated the need to perform any microscopic sperm retrieval procedures on the day of oocyte collection. Modification of the routine sperm cryopreservation methodology to include the use of Cell Sleepers increases the range of sperm samples that can be effectively cryopreserved, to include men with severe male factor fertility.

  4. Processes involved in assisted reproduction technologies significantly increase sperm DNA fragmentation and phosphatidylserine translocation.

    PubMed

    Balasuriya, A; Serhal, P; Doshi, A; Harper, J C

    2014-03-01

    Sperm preparation techniques in assisted reproduction technologies (ART) are potential generators of exogenous stresses that cause additional DNA damage. DNA fragmentation tests, such as the sperm chromatin structure assay, involve freezing sperm samples in the absence of cryoprotectant. Thermal, oxidative stress (OS) and freezing are detrimental to sperm DNA fragmentation and phosphatidylserine (PS) translocation. The primary aim of this study was to subject mature sperm to environmental insults that normally occur during ART. We tested the hypotheses that OS, thermal stress and freeze-thawing caused sperm nuclear and membrane damage and that a positive correlation exists between PS translocation and DNA fragmentation. Sperm DNA integrity deteriorates in semen samples from men with advancing age and a sperm concentration of <15 m ml(-1) . The significant increase in sperm DNA fragmentation at 37 °C after merely 1 h is important clinically as semen liquefaction and short-term sperm storage in an ART cycle involve incubating samples at this temperature. Freezing without a cryoprotectant significantly increases the level of sperm nuclear damage, so it is important not to freeze neat semen prior to DNA fragmentation testing. This study highlights the importance of minimising the production of exogenous stresses during sperm preparation in ART.

  5. Sperm Patch-Clamp

    PubMed Central

    Lishko, Polina; Clapham, David E.; Navarro, Betsy; Kirichok, Yuriy

    2014-01-01

    Sperm intracellular pH and calcium concentration ([Ca2+]i) are two central factors that control sperm activity within the female reproductive tract. As such, the ion channels of the sperm plasma membrane that alter intracellular sperm [Ca2+] and pH play important roles in sperm physiology and the process of fertilization. Indeed, sperm ion channels regulate sperm motility, control sperm chemotaxis toward the egg in some species, and may trigger the acrosome reaction. Until recently, our understanding of these important molecules was rudimentary due to the inability to patch-clamp spermatozoa and directly record the activity of these ion channels under voltage clamp. Recently, we overcame this technical barrier and developed a method for reproducible application of the patch-clamp technique to mouse and human spermatozoa. This chapter covers important aspects of application of the patch-clamp technique to spermatozoa, such as selection of the electrophysiological equipment, isolation of spermatozoa for patch-clamp experiments, formation of the gigaohm seal with spermatozoa, and transition into the whole-cell mode of recording. We also discuss potential pitfalls in application of the patch-clamp technique to flagellar ion channels. PMID:23522465

  6. The small RNA content of human sperm reveals pseudogene-derived piRNAs complementary to protein-coding genes

    PubMed Central

    Pantano, Lorena; Jodar, Meritxell; Bak, Mads; Ballescà, Josep Lluís; Tommerup, Niels; Oliva, Rafael; Vavouri, Tanya

    2015-01-01

    At the end of mammalian sperm development, sperm cells expel most of their cytoplasm and dispose of the majority of their RNA. Yet, hundreds of RNA molecules remain in mature sperm. The biological significance of the vast majority of these molecules is unclear. To better understand the processes that generate sperm small RNAs and what roles they may have, we sequenced and characterized the small RNA content of sperm samples from two human fertile individuals. We detected 182 microRNAs, some of which are highly abundant. The most abundant microRNA in sperm is miR-1246 with predicted targets among sperm-specific genes. The most abundant class of small noncoding RNAs in sperm are PIWI-interacting RNAs (piRNAs). Surprisingly, we found that human sperm cells contain piRNAs processed from pseudogenes. Clusters of piRNAs from human testes contain pseudogenes transcribed in the antisense strand and processed into small RNAs. Several human protein-coding genes contain antisense predicted targets of pseudogene-derived piRNAs in the male germline and these piRNAs are still found in mature sperm. Our study provides the most extensive data set and annotation of human sperm small RNAs to date and is a resource for further functional studies on the roles of sperm small RNAs. In addition, we propose that some of the pseudogene-derived human piRNAs may regulate expression of their parent gene in the male germline. PMID:25904136

  7. The sperm epigenome and potential implications for the developing embryo.

    PubMed

    Jenkins, Timothy G; Carrell, Douglas T

    2012-06-01

    Recent work in the field of male fertility has yielded significant increases in our understanding of the sperm epigenome and its potential role in embryonic development. These new findings have enabled a broad classification of a normal epigenetic state in the male gamete and have provided insight into the possible etiologies of some idiopathic male infertility cases. Histone retention and modification, protamine incorporation into the chromatin, DNA methylation, and spermatozoal RNA transcripts appear to play important roles in the epigenetic state of mature sperm. These epigenetic factors may reveal a historical record of spermatogenesis, portend future functions in embryogenesis, and help to elucidate mechanism of pluripotency. In contrast to the once held dogma regarding the importance of the paternal epigenome, the unique epigenetic landscape in sperm appears to serve more than the gamete itself and is likely influential in the developing embryo. In fact, growing evidence suggests that mature sperm provide appropriate epigenetic marks that drive specific genes toward activation and contribute to the pluripotent state of the embryonic cells. Although not definitive, the current literature provides evidence for the role of the sperm epigenome in the embryo. Future work must be focused on the characterization of epigenetic abnormalities commonly found in individuals with compromised fertility to further establish this role. Additionally, studies should target the effects of environment and aging on the sperm epigenetic program and subsequent fertility loss to determine the etiology of aberrant epigenetic profiles.

  8. How nematode sperm crawl.

    PubMed

    Bottino, Dean; Mogilner, Alexander; Roberts, Tom; Stewart, Murray; Oster, George

    2002-01-15

    Sperm of the nematode, Ascaris suum, crawl using lamellipodial protrusion, adhesion and retraction, a process analogous to the amoeboid motility of other eukaryotic cells. However, rather than employing an actin cytoskeleton to generate locomotion, nematode sperm use the major sperm protein (MSP). Moreover, nematode sperm lack detectable molecular motors or the battery of actin-binding proteins that characterize actin-based motility. The Ascaris system provides a simple 'stripped down' version of a crawling cell in which to examine the basic mechanism of cell locomotion independently of other cellular functions that involve the cytoskeleton. Here we present a mechanochemical analysis of crawling in Ascaris sperm. We construct a finite element model wherein (a) localized filament polymerization and bundling generate the force for lamellipodial extension and (b) energy stored in the gel formed from the filament bundles at the leading edge is subsequently used to produce the contraction that pulls the rear of the cell forward. The model reproduces the major features of crawling sperm and provides a framework in which amoeboid cell motility can be analyzed. Although the model refers primarily to the locomotion of nematode sperm, it has important implications for the mechanics of actin-based cell motility.

  9. Sperm and spermatids contain different proteins and bind distinct egg factors.

    PubMed

    Teperek, Marta; Miyamoto, Kei; Simeone, Angela; Feret, Renata; Deery, Michael J; Gurdon, John B; Jullien, Jerome

    2014-09-19

    Spermatozoa are more efficient at supporting normal embryonic development than spermatids, their immature, immediate precursors. This suggests that the sperm acquires the ability to support embryonic development during spermiogenesis (spermatid to sperm maturation). Here, using Xenopus laevis as a model organism, we performed 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry analysis of differentially expressed proteins between sperm and spermatids in order to identify factors that could be responsible for the efficiency of the sperm to support embryonic development. Furthermore, benefiting from the availability of egg extracts in Xenopus, we also tested whether the chromatin of sperm could attract different egg factors compared to the chromatin of spermatids. Our analysis identified: (1) several proteins which were present exclusively in sperm; but not in spermatid nuclei and (2) numerous egg proteins binding to the sperm (but not to the spermatid chromatin) after incubation in egg extracts. Amongst these factors we identified many chromatin-associated proteins and transcriptional repressors. Presence of transcriptional repressors binding specifically to sperm chromatin could suggest its preparation for the early embryonic cell cycles, during which no transcription is observed and suggests that sperm chromatin has a unique protein composition, which facilitates the recruitment of egg chromatin remodelling factors. It is therefore likely that the acquisition of these sperm-specific factors during spermiogenesis makes the sperm chromatin suitable to interact with the maternal factors and, as a consequence, to support efficient embryonic development.

  10. The Effect of Ambient Air Pollution on Sperm Quality

    PubMed Central

    Hansen, Craig; Luben, Thomas J.; Sacks, Jason D.; Olshan, Andrew; Jeffay, Susan; Strader, Lillian; Perreault, Sally D.

    2010-01-01

    Background Research has suggested an association with ambient air pollution and sperm quality. Objectives We investigated the effect of exposure to ozone (O3) and particulate matter < 2.5 μm in aerodynamic diameter (PM2.5) on sperm quality. Methods We reexamined a previous cohort study of water disinfection by-products to evaluate sperm quality in 228 presumed fertile men with different air pollution profiles. Outcomes included sperm concentration, total sperm per ejaculate (count), and morphology, as well as DNA integrity and chromatin maturity. Exposures to O3 and PM2.5 were evaluated for the 90–day period before sampling. We used multivariable linear regression, which included different levels of adjustment (i.e., without and with season and temperature) to assess the relationship between exposure to air pollutants during key periods of sperm development and adverse sperm outcomes. Results Sperm concentration and count were not associated with exposure to PM2.5, but there was evidence of an association (but not statistically significant) with O3 concentration and decreased sperm concentration and count. Additionally, a significant increase in the percentage of sperm cells with cytoplasmic drop [β = 2.64; 95% confidence interval (CI), 0.21–5.06] and abnormal head (β = 0.47; 95% CI, 0.03–0.92) was associated with PM2.5 concentration in the base model. However, these associations, along with all other sperm outcomes, were not significantly associated with either pollutant after controlling for season and temperature. Overall, although we found both protective and adverse effects, there was generally no consistent pattern of increased abnormal sperm quality with elevated exposure to O3 or PM2.5. Conclusions Exposures to O3 or PM2.5 at levels below the current National Ambient Air Quality Standards were not associated with statistically significant decrements in sperm outcomes in this cohort of fertile men. However, some results suggested effects on sperm

  11. Sperm number trumps sperm size in mammalian ejaculate evolution

    PubMed Central

    Fitzpatrick, John L.

    2015-01-01

    Postcopulatory sexual selection is widely accepted to underlie the extraordinary diversification of sperm morphology. However, why does it favour longer sperm in some taxa but shorter in others? Two recent hypotheses addressing this discrepancy offered contradictory explanations. Under the sperm dilution hypothesis, selection via sperm density in the female reproductive tract favours more but smaller sperm in large, but the reverse in small, species. Conversely, the metabolic constraint hypothesis maintains that ejaculates respond positively to selection in small endothermic animals with high metabolic rates, whereas low metabolic rates constrain their evolution in large species. Here, we resolve this debate by capitalizing on the substantial variation in mammalian body size and reproductive physiology. Evolutionary responses shifted from sperm length to number with increasing mammalian body size, thus supporting the sperm dilution hypothesis. Our findings demonstrate that body-size-mediated trade-offs between sperm size and number can explain the extreme diversification in sperm phenotypes. PMID:26582027

  12. Direct visualization of sperm competition and sperm storage in Drosophila.

    PubMed

    Civetta, A

    Drosophila females engage in multiple matings [1] [2] [3] [4] even though they can store sperm in specialized organs for most of their life [5]. The existence of sperm competition in Drosophila has been inferred from the proportion of progeny sired by the second male in double-mating experiments [6] [7] [8]. Investigators have used this approach to quantify genetic variation underlying sperm competition [8] [9] [10], to elucidate its genetic basis [11], to identify the dependence of different male competitive ability on the genotype of the females with which they mate [12] and to discern the potential role of sperm competition in species isolation [13] [14]. This approach assumes that the sperm from two males stored in a female compete to fertilize the eggs. The mechanism by which sperm competition is accomplished is still unknown, however. Here, fluorescence microscopy, cytometry, and differently labeled sperm were used to analyze the fate of sperm inside the female's sperm storage organs, to quantify sperm competition, and to assess how closely paternity success corresponds to the appearance and location of the sperm. The results show that the first male's sperm is retained for a shortened period if the female remates, and that the second males that sire more progeny either induce females to store and use more of their sperm or strongly displace resident sperm.

  13. Intracytoplasmic Sperm Injection (ICSI)

    MedlinePlus

    ... to the inside of the egg (cytoplasm), where fertilization takes place. Sometimes the sperm cannot penetrate the ... ICSI) can be done along with in vitro fertilization (IVF) to help fertilize the egg. During ICSI, ...

  14. Tuning sperm chemotaxis.

    PubMed

    Guerrero, Adán; Wood, Christopher D; Nishigaki, Takuya; Carneiro, Jorge; Darszon, Alberto

    2010-10-01

    Sperm chemotaxis is a long-term puzzle and most of our knowledge comes from studying marine animals that are external fertilizers. Sperm are attracted by diffusible chemical factors (chemoattractants) released from the egg which redirect their swimming paths towards their source. This redirection is driven by increases in flagellar curvature that correlate with transient flagellar Ca(2+) increases. Recent experimental and modelling results provide insights into the signal flow underlying the translation of an external chemical gradient into an intracellular molecular and motor response. A fundamental element of sea-urchin sperm chemotaxis lies in the ability of these cells to suppress Ca(2+)-mediated increases in flagellar curvature while experiencing an increasing chemoattractant gradient. The article considers this new evidence and summarizes the known underlying cellular mechanisms and behavioural strategies that sperm use to locate and fertilize the oocyte.

  15. Low Sperm Count

    MedlinePlus

    ... unknown, it might be related to abnormal testicular temperature regulation. Varicoceles result in reduced quality of the ... can be permanently reduced. Overheating the testicles. Elevated temperatures impair sperm production and function. Although studies are ...

  16. Sperm studies in anesthesiologists

    SciTech Connect

    Wyrobek, A.J.; Brodsky, J.; Gordon, l.; Moore, D.H., II; Watchmaker, G.; Cohen, E.N.

    1981-11-01

    Semen samples were collected from 46 anesthesiologists each of whom had worked a minimum of one year in hospital operating rooms ventilated with modern gas-scavenging devices. Samples collected from 26 beginning residents in anesthesiology served as controls. Concentrations of sperm and percentage of sperm having abnormal head shapes were determined for each sample. No significant differences were found between anesthesiologists and beginning residents. Limiting the analyses to men having no confounding factors (varicocele, recent illness, medications, heavy smoking, frequent sauna use) did not change the results. The sperm concentration and morphology in 13 men did not change signficantly after one year of exposure to anesthetic gases. However, the group of men who had one or more confounding factors (excluding exposure to anesthetic gases) showed significantly higher percentages of sperm abnormalities than did the group of men without such factors. These results suggest that limited exposure to anesthetic gases does not significantly affect sperm production as judged by changes in sperm concentration and morphology. These data are reassuring, but since the hospitals surveyed used modern gas-scavenging devices, men who are occupationally exposed to anesthetic gases without this protection should be studied for fuller assessment of the possible human spermatotoxic effects.

  17. Sperm ultrastructure, morphometry, and abnormal morphology in American black bears (Ursus americanus).

    PubMed

    Brito, L F C; Sertich, P L; Stull, G B; Rives, W; Knobbe, M

    2010-11-01

    The objective of this study was to describe sperm ultrastructure, morphometry, and abnormal morphology in American black bears. Electroejaculation was successful in 53.8% (7/13) of the attempts, but urine contamination was common. Epididymal sperm samples were also obtained from five bears. Sperm had a paddle-like head shape and the ultrastructure was similar to that of most other mammals. The most striking particularity of black bear sperm ultrastructure was a tightening of the nucleus in the equatorial region. Although the differences were not significant in all bears, the overall decrease in sperm nucleus dimensions during transport from the caput epididymis to the cauda suggested increasing compaction of the nucleus during maturation. For ejaculated sperm, nucleus length, width, and base width were 4.9, 3.7, and 1.8 μm, respectively, whereas sperm head length, width, and base width were 6.6, 4.8, and 2.3 μm, and midpiece, tail (including midpiece), and total sperm lengths were 9.8, 68.8, and 75.3 μm. Evaluation of sperm cytoplasmic droplets in the epididymis revealed that proximal droplets start migrating toward a distal position in the caput epididymis and that the process was mostly completed by the time sperm reached the cauda epididymis. The proportion of morphologically normal sperm in the ejaculate was 35.6%; the most prevalent sperm defects were distal cytoplasmic droplets and bent/coiled tails. The morphology of abnormal sperm and the underlying ultrastructural defects were similar to that in other large domestic animals thus suggesting similar underlying pathogenesis of specific sperm defects and similar effects on fertility.

  18. Provisional bilateral symmetry in Xenopus eggs is established during maturation

    NASA Technical Reports Server (NTRS)

    Brown, E. E.; Margelot, K. M.; Danilchik, M. V.

    1994-01-01

    Dorsal-ventral patterning in the Xenopus egg becomes established midway through the first cell cycle during a 30 degree rotation of the subcortical yolk mass relative to the egg cortex. This 'rotation of symmetrisation' is microtubule dependent, and its direction is thought to be cued by the usually eccentric sperm centrosome. The fact that parthenogenetically activated eggs also undergo a directed rotation, despite the absence of a sperm centrosome, suggests that an endogenous asymmetry in the unfertilised egg supports the directed polymerisation of microtubules in the vegetal cortex, in the way that an eccentric sperm centrosome would in fertilised eggs. Consistent with this idea, we noticed that the maturation spot is usually located an average of more than 15 degrees from the geometric centre of the pigmented animal hemisphere. In parthenogenetically activated eggs, this eccentric maturation spot can be used to predict the direction of rotation. Although in most fertilised eggs the yolk mass rotates toward the sperm entry point (SEP) meridian, occasionally this relationship is perturbed significantly; in such eggs, the maturation spot is never on the same side of the egg as the SEP. In oocytes tilted 90 degrees from upright during maturation in vitro, the maturation spot developed 15 degrees or more from the centre of the pigmented hemisphere, always displaced towards the point on the equator that was up during maturation. This experimentally demonstrated lability is consistent with an off-axis oocyte orientation during oogenesis determining its eccentric maturation spot position, and, in turn, its endogenous rotational bias.

  19. Quantitative analysis of radiation-induced changes in sperm morphology

    SciTech Connect

    Young, I.T.; Gledhill, B.L.; Lake, S.; Wyrobek, A.J.

    1982-09-01

    When developing spermatogenic cells are exposed to radiation, chemical carcinogens or mutagens, the transformation in the morphology of the mature sperm can be used to determine the severity of the exposure. In this study five groups of mice with three mice per group received testicular doses of X irradiation at dosage levels ranging from 0 rad to 120 rad. A random sample of 100 mature sperm per mouse was analyzed five weeks later for the quantitative morphologic transformation as a function of dosage level. The cells were stained with gallocyanin chrome alum (GCA) so that only the DNA in the sperm head was visible. The ACUity quantitative microscopy system at Lawrence Livermore National Laboratory was used to scan the sperm at a sampling density of 16 points per linear micrometer and with 256 brightness levels per point. The contour of each cell was extracted using conventional thresholding techniques on the high-contrast images. For each contour a variety of shape features was then computed to characterize the morphology of that cell. Using the control group and the distribution of their shape features to establish the variability of a normal sperm population, the 95% limits on normal morphology were established. Using only four shape features, a doubling dose of approximately 39 rad was determined. That is, at 39 rad exposure the percentage of abnormal cells was twice that occurring in the control population. This compared to a doubling dose of approximately 70 rad obtained from a concurrent visual procedure.

  20. Sperm Motility in Flow

    NASA Astrophysics Data System (ADS)

    Guasto, Jeffrey; Juarez, Gabriel; Stocker, Roman

    2012-11-01

    A wide variety of plants and animals reproduce sexually by releasing motile sperm that seek out a conspecific egg, for example in the reproductive tract for mammals or in the water column for externally fertilizing organisms. Sperm are aided in their quest by chemical cues, but must also contend with hydrodynamic forces, resulting from laminar flows in reproductive tracts or turbulence in aquatic habitats. To understand how velocity gradients affect motility, we subjected swimming sperm to a range of highly-controlled straining flows using a cross-flow microfluidic device. The motion of the cell body and flagellum were captured through high-speed video microscopy. The effects of flow on swimming are twofold. For moderate velocity gradients, flow simply advects and reorients cells, quenching their ability to cross streamlines. For high velocity gradients, fluid stresses hinder the internal bending of the flagellum, directly inhibiting motility. The transition between the two regimes is governed by the Sperm number, which compares the external viscous stresses with the internal elastic stresses. Ultimately, unraveling the role of flow in sperm motility will lead to a better understanding of population dynamics among aquatic organisms and infertility problems in humans.

  1. Sperm-egg interaction.

    PubMed

    Evans, Janice P

    2012-01-01

    A crucial step of fertilization is the sperm-egg interaction that allows the two gametes to fuse and create the zygote. In the mouse, CD9 on the egg and IZUMO1 on the sperm stand out as critical players, as Cd9(-/-) and Izumo1(-/-) mice are healthy but infertile or severely subfertile due to defective sperm-egg interaction. Moreover, work on several nonmammalian organisms has identified some of the most intriguing candidates implicated in sperm-egg interaction. Understanding of gamete membrane interactions is advancing through characterization of in vivo and in vitro fertilization phenotypes, including insights from less robust phenotypes that highlight potential supporting (albeit not absolutely essential) players. An emerging theme is that there are varied roles for gamete molecules that participate in sperm-egg interactions. Such roles include not only functioning as fusogens, or as adhesion molecules for the opposite gamete, but also functioning through interactions in cis with other proteins to regulate membrane order and functionality.

  2. Healthy Sperm: Improving Your Fertility

    MedlinePlus

    Healthy Lifestyle Getting pregnant Healthy sperm aren't always a given. Understand how lifestyle factors can affect your ... as a laptop, might enhance sperm quality. Adopting healthy lifestyle practices to promote your fertility — and avoiding things ...

  3. An update on post-ejaculatory remodeling of the sperm surface before mammalian fertilization.

    PubMed

    Gadella, B M; Boerke, A

    2016-01-01

    The fusion of a sperm with an oocyte to form new life is a highly regulated event. The activation-also termed capacitation-of the sperm cell is one of the key preparative steps required for this process. Ejaculated sperm has to make a journey through the female uterus and oviduct before it can approach the oocyte. The oocyte at that moment also has become prepared to facilitate monospermic fertilization and block immediately thereafter the chance for polyspermic fertilization. Interestingly, ejaculated sperm is not properly capacitated and consequently is not yet able to fertilize the oocyte. During the capacitation process, the formation of competent lipid-protein domains on the sperm head enables sperm-cumulus and zona pellucida interactions. This sperm binding allows the onset for a cascade reaction ultimately resulting in oocyte-sperm fusion. Many different lipids and proteins from the sperm surface are involved in this process. Sperm surface processing already starts when sperm are liberated from the seminiferous tubules and is followed by epididymal maturation where the sperm cell surface is modified and loaded with proteins to ensure it is prepared for its fertilization task. Although cauda epididymal sperm can fertilize the oocyte IVF, they are coated with so-called decapacitation factors during ejaculation. The seminal plasma-induced stabilization of the sperm surface permits the sperm transit through the cervix and uterus but prevents sperm capacitation and thus inhibits fertilization. For IVF purposes, sperm are washed out of seminal plasma and activated to get rid of decapacitation factors. Only after capacitation, the sperm can fertilize the oocyte. In recent years, IVF has become a widely used tool to achieve successful fertilization in both the veterinary field and human medicine. Although IVF procedures are very successful, scientific knowledge is still far from complete when identifying all the molecular players and processes during the first

  4. Sorting of Sperm by Morphology

    NASA Astrophysics Data System (ADS)

    Koh, James; Marcos, Marcos

    2016-11-01

    Many studies have proven that the percentage of morphologically normal sperm is a significant factor in determining the success of assisted reproduction. The velocity of sperm in a microchannel with shear flow subjected to an external field will be explored theoretically. The difference in response between morphologically normal and abnormal sperm will be computed from a statistical approach, to study the feasibility and effectiveness of sorting by an external field to remove abnormal sperm. The full name of this author is Marcos.

  5. A Systematic Analysis of a Deep Mouse Epididymal Sperm Proteome

    SciTech Connect

    Chauvin, Theodore; Xie, Fang; Liu, Tao; Nicora, Carrie D.; Yang, Feng; Camp, David G.; Smith, Richard D.; Roberts, Kenneth P.

    2012-12-21

    Spermatozoa are highly specialized cells that, when mature, are capable of navigating the female reproductive tract and fertilizing an oocyte. The sperm cell is thought to be largely quiescent in terms of transcriptional and translational activity. As a result, once it has left the male reproductive tract, the sperm cell is essentially operating with a static population of proteins. It is therefore theoretically possible to understand the protein networks contained in a sperm cell and to deduce its cellular function capabilities. To this end we have performed a proteomic analysis of mouse sperm isolated from the cauda epididymis and have confidently identified 2,850 proteins, which is the most comprehensive sperm proteome for any species reported to date. These proteins comprise many complete cellular pathways, including those for energy production via glycolysis, β-oxidation and oxidative phosphorylation, protein folding and transport, and cell signaling systems. This proteome should prove a useful tool for assembly and testing of protein networks important for sperm function.

  6. Characterization of proteins in the bovine epididymal and seminal fluid and proteins attached to epididymal and ejaculated sperm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During final maturation sperm lose their ability to biosynthesize, repair, grow, and divide. They have minimal metabolic function, and thus become completely dependent on their environment. In the epididymis, sperm are stored for a long period of time, but upon ejaculation motility is increased and...

  7. Coupling sperm mediated gene transfer and sperm sorting techniques: a new perspective for swine transgenesis.

    PubMed

    De Cecco, Marco; Spinaci, Marcella; Zannoni, Augusta; Bernardini, Chiara; Seren, Eraldo; Forni, Monica; Bacci, Maria Laura

    2010-09-15

    Flow cytometric separation of X and Y chromosome-bearing spermatozoa has been demonstrated to be effective in pigs, allowing the use of boar sexed semen in in vitro trials. Sperm Mediated Gene Transfer (SMGT) is a widely used and efficient technique for the creation of transgenic animals. The present research intended to prove that it is possible to associate sperm sexing with the SMGT technique in order to speed up the assessment of homozygous lines of transgenic pigs. In the first experiment, the sorting protocol was modified in order to obtain the highest DNA uptake by sorted spermatozoa. In the second experiment, spermatozoa that had undergone only sperm sorting, only SMGT, or both procedures (Sorted-SMGT) were used for in in vitro fertilization of in vitro matured oocytes. In the third experiment, transformed blastocysts of the desired gender (male) were obtained with Sorted-SMGT in an in vitro fertilization trial. The method we developed here allowed us to produce transgenic swine blastocysts of pre-determined gender, giving a positive answer at the aim to couple SMGT and sperm sorting in swine, obtaining fertile spermatozoa able to produce transgenic embryos of pre-determined gender.

  8. Glycocalyx characterisation and glycoprotein expression of Sus domesticus epididymal sperm surface samples.

    PubMed

    Fàbrega, Anna; Puigmulé, Marta; Dacheux, Jean-Louis; Bonet, Sergi; Pinart, Elisabeth

    2012-01-01

    The sperm surface is covered with a dense coating of carbohydrate-rich molecules. Many of these molecules are involved in the acquisition of fertilising ability. In the present study, eight lectins (i.e. Arachis hypogae (peanut) agglutinin (PNA), Lens culimaris (lentil) agglutinin-A (LCA), Pisum sativum (pea) agglutin (PSA), Triticum vulgari (wheat) germ agglutinin (WGA), Helix pomatia agglutinin (HPA), Phaseolus vulgaris (red kidney bean) leucoagglutinin (PHA-L), Glycine max (soybean) agglutinin (SBA) and Ulex europaeus agglutinin I (UEA-I)) were investigated to identify changes in the nature and localisation of glycoproteins in boar spermatozoa migrating along the epididymal duct. Complementary procedures included measurement of global lectin binding over the surface of the viable sperm population by flow cytometry, analysis of lectin localisation on the membrane of individual spermatozoa using fluorescence microscopy and the electrophoretic characterisation of the major sperm surface glycoprotein receptors involved in lectin binding. A significant increase was found in sperm galactose, glucose/mannose and N-acetyl-d-glucosamine residues distally in the epididymis. Moreover, the sperm head, cytoplasmic droplet and midpiece were recognised by most of the lectins tested, whereas only HPA and WGA bound to the principal piece and end piece of the sperm tail. Fourteen sperm surface proteins were observed with different patterns of lectin expression between epididymal regions. The sperm glycocalyx modifications observed in the present study provide an insight into the molecular modifications associated with epididymal maturation, which may be correlated with the degree of maturation of ejaculated spermatozoa.

  9. Sperm nuclei glutathione peroxidases and their occurrence in animal species with cysteine-containing protamines.

    PubMed

    Bertelsmann, Holger; Kuehbacher, Markus; Weseloh, Gundolf; Kyriakopoulos, Antonios; Behne, Dietrich

    2007-10-01

    The selenoenzyme sperm nuclei glutathione peroxidase (snGPx), also called the nuclear form of phospholipid hydroperoxide glutathione peroxidase (n-PHGPx), was found to be involved in the stabilization of condensed sperm chromatin, most likely by thiol to disulfide oxidation of the cysteine residues of the mammalian protamines, small nuclear basic proteins in the nuclei of sperm cells. By applying Acidic Urea-PAGE in combination with SDS-PAGE, snGPx with an apparent molecular mass of 34 kDa and a 24-kDa protein were purified from rat sperm nuclei. The 24-kDa protein was identified by means of mass spectrometry as a truncated form of snGPx produced by cleavage at the N-terminal end. After defined processing of spermatozoa and detergent treatment of the sperm nuclei fraction, snGPx and its truncated form were shown to be the only selenoproteins present in mature mammalian sperm nuclei. Both forms were found in mature rat and horse sperm nuclei but in man only snGPx was detected. In trout and chicken, species with sperm cells which likewise undergo chromatin condensation but do not contain cysteine in their protamines, the snGPx proteins were missing. This can be taken as an indirect proof of the function of snGPx to act as protamine cysteine thiol peroxidase in the mammalian species with cysteine-containing protamines.

  10. Aneuploidy detection in human sperm nuclei using PRINS technique

    SciTech Connect

    Girardet, A.; Coignet, L.; Andreo, B.

    1996-08-23

    Rapid and specific identification of chromosomes can be attained in situ using the PRimed IN Situ (PRINS) labelling technique. We have adapted this technique to mature human sperm in combination with a protocol for simultaneous decondensation and denaturation of sperm nuclei. This strategy allowed us to obtain double labelling of human spermatozoa in a <2-hr reaction. In the present study, we report the estimates of disomy for chromosomes 3, 7, 10, 11, and 17 on 64,642 spermatozoa from 2 normal males. The incidences of disomy ranged from 0.28-0.34%. There were no significant interindividual or interchromosomal differences in disomy rates. 27 refs., 1 fig., 3 tabs.

  11. Individual adjustment of sperm expenditure accords with sperm competition theory.

    PubMed

    Pilastro, Andrea; Scaggiante, Marta; Rasotto, Maria B

    2002-07-23

    Sperm competition theory predicts that males should strategically allocate their sperm reserves according to the level of sperm competition, defined as the probability that the sperm of two males compete for fertilizing a given set of ova. Substantial evidence from numerous animal taxa suggests that, at the individual level, sperm expenditure increases when the risk of sperm competition is greater. In contrast, according to the "intensity model" of sperm competition [Parker, G. A., Ball, M. A., Stockley, P. & Gage, M. J. G. (1996) Proc. R. Soc. London Ser. B 263, 1291-1297], when more than two ejaculates compete during a given mating event, sperm expenditure should decrease as the number of competing males increases. Empirical evidence supporting this prediction, however, is still lacking. Here we measured sperm expenditure in two gobiid fishes, the grass (Zosterisessor ophiocephalus) and black goby (Gobius niger), in which up to six sneakers can congregate around the nest of territorial males and release their sperm when females spawn. We show that, in accordance with theory, sneaker males of both species release fewer sperm as the number of competitors increases.

  12. Dietary fatty acids influence sperm quality and function.

    PubMed

    Ferramosca, A; Moscatelli, N; Di Giacomo, M; Zara, V

    2017-03-23

    Recently, obesity has been linked to male infertility. In animal models the administration of a high-fat diet caused a reduction in sperm quality, by impairing gamete energy metabolism. The aim of this study was to investigate a possible effect of dietary fatty acids supplementation in the modulation of sperm energy metabolism and, in turn, in the improvement of sperm quality in rats fed a high-fat diet. Sexually mature male Sprague-Dawley rats were divided into four groups and fed for 4 weeks a standard diet (control group), a high-fat diet (enriched in 35% of fat and 15% sucrose), a high-fat diet supplemented with 2.5% olive oil (a source of monounsaturated fatty acids) or a high-fat diet supplemented with 2.5% krill oil (a source of n-3 polyunsaturated fatty acids). Liver and adipose tissue weight, plasma glucose, insulin and lipid concentrations were determined. Activities of enzymes involved in sperm energetic metabolism were evaluated by spectrophotometric assays. Sperm mitochondrial respiratory efficiency was also assayed. The obtained results suggest that olive oil partially counteracts the negative effects of a high-fat diet on sperm quality, by increasing gamete motility, by reducing oxidative stress and slightly improving mitochondrial respiration efficiency. On the other hand, krill oil determines an increase in sperm concentration and motility, an increase in the activities of lactate dehydrogenase, Krebs cycle enzymes and respiratory chain complexes; a parallel increase in the cellular levels of ATP and a reduction in oxidative damage were also observed. These results suggest that dietary fatty acids are able to positively influence sperm quality and function.

  13. Embryo development of porcine oocytes after injection with miniature pig sperm and their extracts.

    PubMed

    Matsuura, Daizou; Maeda, Teruo

    2009-12-01

    This study examined embryo development of porcine oocytes after microinjection of sperm extracts (SE) in porcine intracytoplasmic sperm injection (ICSI). SE was prepared from miniature pig sperm by a nonionic surfactant, and various concentrations (0.02, 0.04 and 0.08 mg/mL) of SE were injected into the matured oocytes with a first polar body. In the pronuclear stage, the rate of oocytes with two pronuclei and a second polar body (21.4%) in the sperm and SE (0.04 mg/mL) injection group was significantly higher (P < 0.05) compared to other groups. The rate of 2-4-cell stage in sperm and SE (0.04 mg/mL) injection group was 38.1%, and it was significantly higher than that in the sperm injection group (22.9%). The rate of blastocyst stage in sperm and SE (0.04 mg/mL) injection group was 21.4%, the value was significantly higher than those in SE (0.08 mg/mL) injection group (0%), sperm injection group (5.7%), and sperm and SE (0.08 mg/mL) injection group (2.6%). These results suggest that SE induces activation of porcine oocytes and their further embryonic development, and that SE is effective for porcine ICSI.

  14. Motility, ATP levels and metabolic enzyme activity of sperm from bluegill (Lepomis macrochirus).

    PubMed

    Burness, Gary; Moyes, Christopher D; Montgomerie, Robert

    2005-01-01

    Male bluegill displays one of two life history tactics. Some males (termed "parentals") delay reproduction until ca. 7 years of age, at which time they build nests and actively courts females. Others mature precociously (sneakers) and obtain fertilizations by cuckolding parental males. In the current study, we studied the relations among sperm motility, ATP levels, and metabolic enzyme activity in parental and sneaker bluegill. In both reproductive tactics, sperm swimming speed and ATP levels declined in parallel over the first 60 s of motility. Although sneaker sperm initially had higher ATP levels than parental sperm, by approximately 30 s postactivation, no differences existed between tactics. No differences were noted between tactics in swimming speed, percent motility, or the activities of key metabolic enzymes, although sperm from parentals had a higher ratio of creatine phosphokinase (CPK) to citrate synthase (CS). In both tactics, with increasing CPK and CS activity, sperm ATP levels increased at 20 s postactivation, suggesting that capacities for phosphocreatine hydrolysis and aerobic metabolism may influence interindividual variation in rates of ATP depletion. Nonetheless, there was no relation between sperm ATP levels and either swimming speed or percent of sperm that were motile. This suggests that interindividual variation in ATP levels may not be the primary determinant of variation in sperm swimming performance in bluegill.

  15. Advancing age increases sperm chromatin damage and impairs fertility in peroxiredoxin 6 null mice

    PubMed Central

    Ozkosem, Burak; Feinstein, Sheldon I.; Fisher, Aron B.; O’Flaherty, Cristian

    2015-01-01

    Due to socioeconomic factors, more couples are choosing to delay conception than ever. Increasing average maternal and paternal age in developed countries over the past 40 years has raised the question of how aging affects reproductive success of males and females. Since oxidative stress in the male reproductive tract increases with age, we investigated the impact of advanced paternal age on the integrity of sperm nucleus and reproductive success of males by using a Prdx6−/− mouse model. We compared sperm motility, cytoplasmic droplet retention sperm chromatin quality and reproductive outcomes of young (2-month-old), adult (8-month-old), and old (20-month-old) Prdx6−/− males with their age-matched wild type (WT) controls. Absence of PRDX6 caused age-dependent impairment of sperm motility and sperm maturation and increased sperm DNA fragmentation and oxidation as well as decreased sperm DNA compaction and protamination. Litter size, total number of litters and total number of pups per male were significantly lower in Prdx6−/− males compared to WT controls. These abnormal reproductive outcomes were severely affected by age in Prdx6−/− males. In conclusion, the advanced paternal age affects sperm chromatin integrity and fertility more severely in the absence of PRDX6, suggesting a protective role of PRDX6 in age-associated decline in the sperm quality and fertility in mice. PMID:25796034

  16. Recovery and cryopreservation of epididymal sperm from agouti (Dasiprocta aguti) using powdered coconut water (ACP-109c) and Tris extenders.

    PubMed

    Silva, M A; Peixoto, G C X; Santos, E A A; Castelo, T S; Oliveira, M F; Silva, A R

    2011-10-01

    The objective was to compare the use of powdered coconut water (ACP-109c; ACP Biotecnologia, Fortaleza, CE, Brazil) and Tris extenders for recovery and cryopreservation of epididymal sperm from agouti. The caudae epididymus and proximal ductus deferens from 10 sexually mature agoutis were subjected to retrograde washing using ACP-109c (ACP Biotecnologia) or Tris. Epididymal sperm were evaluated for motility, vigor, sperm viability, membrane integrity, and morphology. Samples were centrifuged, and extended in the same diluents plus egg yolk (20%) and glycerol (6%), frozen in liquid nitrogen, and subsequently thawed at 37°C for 1 min, followed by re-evaluation of sperm characteristics. The two extenders were similarly efficient for epididymal recovery, with regard to the number and quality of sperm recovered. However, for both extenders, sperm quality decreased (P < 0.05) after centrifugation and dilution. After sperm cryopreservation and thawing, there were (mean ± SEM) 26.5 ± 2.6% motile sperm with 2.6 ± 0.2 vigor in the ACP-109c (ACP Biotecnologia) group, which was significantly better than 9.7 ± 2.6% motile sperm with 1.2 ± 0.3 vigor in Tris. In conclusion, agouti epididymal sperm were successfully recovered using either ACP-109c (ACP Biotecnologia) or Tris extenders; however, ACP-109c (ACP Biotecnologia) was a significantly better extender for processing and cryopreserving these sperm.

  17. Proteomics and the genetics of sperm chromatin condensation

    PubMed Central

    Oliva, Rafael; Castillo, Judit

    2011-01-01

    Spermatogenesis involves extremely marked cellular, genetic and chromatin changes resulting in the generation of the highly specialized sperm cell. Proteomics allows the identification of the proteins that compose the spermatogenic cells and the study of their function. The recent developments in mass spectrometry (MS) have markedly increased the throughput to identify and to study the sperm proteins. Catalogs of thousands of testis and spermatozoan proteins in human and different model species are becoming available, setting up the basis for subsequent research, diagnostic applications and possibly the future development of specific treatments. The present review intends to summarize the key genetic and chromatin changes at the different stages of spermatogenesis and in the mature sperm cell and to comment on the presently available proteomic studies. PMID:21042303

  18. Isolation and characterization of germ line DNA from mouse sperm.

    PubMed Central

    Shiurba, R; Nandi, S

    1979-01-01

    Mouse germ line DNA was isolated from sperm by a physicochemical procedure that preferentially destroys contaminating somatic cell DNA. The use of reducing conditions and chelating agents in combination with phenol permitted extraction of molecular weight DNA from mature sperm nuclei with approximately 80% efficiency. Less than 0.1% somatic cell DNA contamination remained in sperm DNA prepared by this method. Germ line DNA was characterized by determination of its ultraviolet absorbance spectrum, buoyant density in cesium chloride, and melting profile on a hydroxyapatite column. Contamination by mitochondrial DNA was assessed by cesium chloride/ethidium bromide gradient centrifugation. The significance of the mouse germ line DNA isolation procedure is discussed with respect to the possible genetic transmission of mammary tumor virus and leukemia virus, the origin of antibody diversity, and the origin of testicular teratomas. PMID:291053

  19. Evaluation of cholesterol- treated dromedary camel sperm function by heterologous IVF and AI.

    PubMed

    Crichton, Elizabeth G; Malo, Clara; Pukazhenthi, Budhan S; Nagy, Peter; Skidmore, Julian A

    2016-11-01

    Cholesterol (cholesterol-loaded cyclodextrins: CLC) treatment of dromedary camel sperm prior to freezing enhances cryosurvival. The present study first validated the efficacy of a heterologous zona-free goat oocyte assay (n=115 oocytes) to evaluate camel sperm function in vitro (Experiment 1: n=6 bulls), then examined the effects of CLC treatment (1.5mg/mL CLC; CLC+) versus no treatment (0 CLC) of fresh (Experiment 2: n=4 bulls) and frozen-thawed (Experiment 3: n=5 bulls) camel sperm to penetrate, de-condense and form pro-nuclei in in vitro-matured goat oocytes. Finally, the ability of fresh 0 CLC and CLC+ sperm to fertilize in vivo was studied by artificially inseminating super-ovulated females (n=7-9 per treatment) and examining embryo production (Experiment 4: n=4-5 bulls/treatment). Camel spermatozoa penetrated (60%) and formed pro-nuclei (33%) in goat oocytes demonstrating the utility of this heterologous system for assessing sperm function in vitro. For fresh spermatozoa, 0 CLC-treated sperm performed better than their CLC+ counterparts for all parameters measured (P<0.05). In contrast, cryopreservation resulted in a sharp decline in sperm-oocyte interaction in 0 CLC aliquots but remained unaltered in CLC+ aliquots demonstrating a protective effect of cholesterol treatment. There was no difference between treatments in the in vitro fertilizing ability of frozen-thawed sperm or in the numbers of embryos retrieved following AI with fresh 0 CLC or CLC+ sperm. We conclude that although CLC treatment of dromedary camel sperm improves sperm motility it fails to confer an advantage to them in terms of improved in vitro sperm-oocyte interaction or in vivo fertilization under the conditions tested.

  20. TRY-5 Is a Sperm-Activating Protease in Caenorhabditis elegans Seminal Fluid

    PubMed Central

    Smith, Joseph R.; Stanfield, Gillian M.

    2011-01-01

    Seminal fluid proteins have been shown to play important roles in male reproductive success, but the mechanisms for this regulation remain largely unknown. In Caenorhabditis elegans, sperm differentiate from immature spermatids into mature, motile spermatozoa during a process termed sperm activation. For C. elegans males, sperm activation occurs during insemination of the hermaphrodite and is thought to be mediated by seminal fluid, but the molecular nature of this activity has not been previously identified. Here we show that TRY-5 is a seminal fluid protease that is required in C. elegans for male-mediated sperm activation. We observed that TRY-5::GFP is expressed in the male somatic gonad and is transferred along with sperm to hermaphrodites during mating. In the absence of TRY-5, male seminal fluid loses its potency to transactivate hermaphrodite sperm. However, TRY-5 is not required for either hermaphrodite or male fertility, suggesting that hermaphrodite sperm are normally activated by a distinct hermaphrodite-specific activator to which male sperm are also competent to respond. Within males, TRY-5::GFP localization within the seminal vesicle is antagonized by the protease inhibitor SWM-1. Together, these data suggest that TRY-5 functions as an extracellular activator of C. elegans sperm. The presence of TRY-5 within the seminal fluid couples the timing of sperm activation to that of transfer of sperm into the hermaphrodite uterus, where motility must be rapidly acquired. Our results provide insight into how C. elegans has adopted sex-specific regulation of sperm motility to accommodate its male-hermaphrodite mode of reproduction. PMID:22125495

  1. TRY-5 is a sperm-activating protease in Caenorhabditis elegans seminal fluid.

    PubMed

    Smith, Joseph R; Stanfield, Gillian M

    2011-11-01

    Seminal fluid proteins have been shown to play important roles in male reproductive success, but the mechanisms for this regulation remain largely unknown. In Caenorhabditis elegans, sperm differentiate from immature spermatids into mature, motile spermatozoa during a process termed sperm activation. For C. elegans males, sperm activation occurs during insemination of the hermaphrodite and is thought to be mediated by seminal fluid, but the molecular nature of this activity has not been previously identified. Here we show that TRY-5 is a seminal fluid protease that is required in C. elegans for male-mediated sperm activation. We observed that TRY-5::GFP is expressed in the male somatic gonad and is transferred along with sperm to hermaphrodites during mating. In the absence of TRY-5, male seminal fluid loses its potency to transactivate hermaphrodite sperm. However, TRY-5 is not required for either hermaphrodite or male fertility, suggesting that hermaphrodite sperm are normally activated by a distinct hermaphrodite-specific activator to which male sperm are also competent to respond. Within males, TRY-5::GFP localization within the seminal vesicle is antagonized by the protease inhibitor SWM-1. Together, these data suggest that TRY-5 functions as an extracellular activator of C. elegans sperm. The presence of TRY-5 within the seminal fluid couples the timing of sperm activation to that of transfer of sperm into the hermaphrodite uterus, where motility must be rapidly acquired. Our results provide insight into how C. elegans has adopted sex-specific regulation of sperm motility to accommodate its male-hermaphrodite mode of reproduction.

  2. GLUTs and mammalian sperm metabolism.

    PubMed

    Bucci, Diego; Rodriguez-Gil, Juan Enrique; Vallorani, Claudia; Spinaci, Marcella; Galeati, Giovanna; Tamanini, Carlo

    2011-01-01

    Mammalian cells use glucides as a substrate that can be catabolized through glycolitic pathways or oxidative phosphorylation, used as a source of reducing potential, or used for anabolic aims. An important role in supplying cells with energy is played by different membrane proteins that can actively (sodium-dependent glucose transporters) or passively (glucose transporters; GLUT) transport hexoses through the lipidic bilayer. In particular, GLUTs are a family of 13 proteins that facilitate the transport of sugars and have a peculiar distribution in different tissues as well as a particular affinity for substrates. These proteins are also present in mature sperm cells, which, in fact, need carriers for uptake energetic sources that are important for maintaining cell basic activity as well as specific functions, such as motility and fertilization ability. Likewise, several GLUTs have been studied in various mammalian species (man, bull, rat, mouse, boar, dog, stallion, and donkey) to point out both their actual presence or absence and their localization on plasma membrane. The aim of this work is to give an overall picture of the studies available on GLUTs in mammalian spermatozoa at this moment, pointing out the species peculiarity, the possible role of these proteins, and the potential future research on this item.

  3. Unconventional endocannabinoid signaling governs sperm activation via the sex hormone progesterone.

    PubMed

    Miller, Melissa R; Mannowetz, Nadja; Iavarone, Anthony T; Safavi, Rojin; Gracheva, Elena O; Smith, James F; Hill, Rose Z; Bautista, Diana M; Kirichok, Yuriy; Lishko, Polina V

    2016-04-29

    Steroids regulate cell proliferation, tissue development, and cell signaling via two pathways: a nuclear receptor mechanism and genome-independent signaling. Sperm activation, egg maturation, and steroid-induced anesthesia are executed via the latter pathway, the key components of which remain unknown. Here, we present characterization of the human sperm progesterone receptor that is conveyed by the orphan enzyme α/β hydrolase domain-containing protein 2 (ABHD2). We show that ABHD2 is highly expressed in spermatozoa, binds progesterone, and acts as a progesterone-dependent lipid hydrolase by depleting the endocannabinoid 2-arachidonoylglycerol (2AG) from plasma membrane. The 2AG inhibits the sperm calcium channel (CatSper), and its removal leads to calcium influx via CatSper and ensures sperm activation. This study reveals that progesterone-activated endocannabinoid depletion by ABHD2 is a general mechanism by which progesterone exerts its genome-independent action and primes sperm for fertilization.

  4. A model for the control of DNA integrity by the sperm nuclear matrix

    PubMed Central

    Gawecka, Joanna E; Ribas-Maynou, Jordi; Benet, Jordi; Ward, W Steven

    2015-01-01

    The highly condensed chromatin of mammalian spermatozoa is usually considered to be biologically inert before fertilization. However, we have demonstrated that even in this compacted state, sperm chromatin is subject to degradation at open configurations associated with the nuclear matrix through a process we have termed sperm chromatin fragmentation (SCF). This suggests that a mechanism exists to monitor the health of spermatozoa during transit through the male reproductive tract and to destroy the genome of defective sperm cells. The site of DNA damage in SCF, the matrix attachment sites, are the same that we hypothesize initiate DNA synthesis in the zygote. When sperm that have damaged DNA are injected into the oocyte, the newly created zygote responds by delaying DNA synthesis in the male pronucleus and, if the damage is severe enough, arresting the embryo's development. Here we present a model for paternal DNA regulation by the nuclear matrix that begins during sperm maturation and continues through early embryonic development. PMID:25926613

  5. A Review of New Technologies that may Become Useful for in vitro Production of Boar Sperm.

    PubMed

    Gadella, B M; Ferraz, M A

    2015-07-01

    Making sperm cells outside the original testicular environment in a culture dish has been considered for a long time as impossible due to the very complicated process of spermatogenesis and sperm maturation, which altogether, encompasses a 2-month period. However, new approaches in complex three-dimensional co-cell cultures, micro-perfusion and micro-fluidics technologies, new knowledge in the functioning, culturing and differentiation of spermatogonial stem cells (SSC) and their precursor cells have revolutionized this field. Furthermore, the use of better molecular markers as well as stimulatory factors has led to successful in vitro culture of stem cells either derived from germ line stem cells, from induced pluripotent stem cells (iPSC) or from embryonic stem cells (ESC). These stem cells when placed into small seminiferous tubule fragments are able to become SSC. The SSC beyond self-renewal can also be induced into haploid sperm-like cells under in vitro conditions. In mouse, this in vitro produced sperm can be injected into a mature oocyte and allow post-fertilization development into an early embryo in vitro. After transferring such obtained embryos into the uterus of a recipient mouse, they can further develop into healthy offspring. Recently, a similar approach has been performed with combining selected cells from testicular cell suspensions followed by a complete in vitro culture of seminiferous cords producing sperm-like cells. However, most of the techniques developed are laborious, time-consuming and have low efficiency, placing questionable that it will become useful used for setting up an efficient in vitro sperm production system for the boar. The benefits and drawbacks as well as the likeliness of in vitro pig sperm production to become applied in assisted technologies for swine reproduction are critically discussed. In this contribution, also the process of sperm production in the testis and sperm maturation is reviewed.

  6. Comparative evidence for the evolution of sperm swimming speed by sperm competition and female sperm storage duration in passerine birds.

    PubMed

    Kleven, Oddmund; Fossøy, Frode; Laskemoen, Terje; Robertson, Raleigh J; Rudolfsen, Geir; Lifjeld, Jan T

    2009-09-01

    Sperm swimming speed is an important determinant of male fertility and sperm competitiveness. Despite its fundamental biological importance, the underlying evolutionary processes affecting this male reproductive trait are poorly understood. Using a comparative approach in a phylogenetic framework, we tested the predictions that sperm swim faster with (1) increased risk of sperm competition, (2) shorter duration of female sperm storage, and (3) increased sperm length. We recorded sperm swimming speed in 42 North American and European free-living passerine bird species, representing 35 genera and 16 families. We found that sperm swimming speed was positively related to the frequency of extrapair paternity (a proxy for the risk of sperm competition) and negatively associated with clutch size (a proxy for the duration of female sperm storage). Sperm swimming speed was unrelated to sperm length, although sperm length also increased with the frequency of extrapair paternity. These results suggest that sperm swimming speed and sperm length are not closely associated traits and evolve independently in response to sperm competition in passerine birds. Our findings emphasize the significance of both sperm competition and female sperm storage duration as evolutionary forces driving sperm swimming speed.

  7. Evaluation of antioxidant effects of crocin on sperm quality in cyclophosphamide treated adult mice

    PubMed Central

    Bakhtiary, Zahra; Shahrooz, Rasoul; Ahmadi, Abbas; Zarei, Leila

    2014-01-01

    Cyclophosphamide (CP) is one of the anti-neoplastic drugs. Despite its numerous clinical applications, it has devastating effects on the testicles and declines the sperm quality in treated patients. This study was aimed to investigate the protective effect of crocin in improving the toxicity induced by CP in reproductive system. In this study, 24 male adult mice (6 to 8 weeks) were randomly divided into three groups, control group received normal saline (0.1 mL, IP, daily), the CP group received CP (15 mg kg-1, IP, weekly) and the CP + crocin group received CP along with crocin (200 mg kg-1, IP, daily). After 35 days of treatment, animals were sacrificed. The samples of epididymis in human tubal fluid medium incubated for 30 min in 5% CO2 for flotation of sperm. Sperm were obtained from caudal epididymis using dissecting method. Then, the parameters of sperm quality including sperm count, motility, viability, DNA damage, nuclear maturation, and sperm morphology were evaluated. In CP group, the sperm count, motility, viability, nuclear maturation and sperm morphology were significantly decreased compared to control group (p < 0.05) and in the CP + crocin group all of these parameters significantly increased compared to CP group (p < 0.05). The percentage of sperm with DNA damage in the CP group significantly increased compared to other groups (p < 0.05). The results of this study indicated that the crocin was able to suppress free radicals and enhance the quality of sperm in CP treated animals. PMID:25568721

  8. Turbulence of swarming sperm

    NASA Astrophysics Data System (ADS)

    Creppy, Adama; Praud, Olivier; Druart, Xavier; Kohnke, Philippa L.; Plouraboué, Franck

    2015-09-01

    Collective motion of self-sustained swarming flows has recently provided examples of small-scale turbulence arising where viscous effects are dominant. We report the first observation of universal enstrophy cascade in concentrated swarming sperm consistent with a body of evidence built from various independent measurements. We found a well-defined k-3 power-law decay of a velocity field power spectrum and relative dispersion of small beads consistent with theoretical predictions in 2D turbulence. Concentrated living sperm displays long-range, correlated whirlpool structures of a size that provides an integral scale of turbulence. We propose a consistent explanation for this quasi-2D turbulence based on self-structured laminated flow forced by steric interactions and alignment, a state of active matter that we call "swarming liquid crystal." We develop scaling arguments consistent with this interpretation.

  9. Sperm, Clinics, and Parenthood

    PubMed Central

    2016-01-01

    Abstract In this article I examine a recent approach to regulating assisted reproduction, whereby use of some kind of medical intervention ‘triggers’ laws governing legal parenthood that are more favourable to intending parents and sperm providers. I argue that although perhaps an improvement on the previous legal framework, these laws are problematic for three important reasons. First, they are prone to violating parental rights and unjustly imposing substantial burdens on individuals. Second, they are discriminatory. Third, even if we take a pragmatic approach to the question of parenthood in these cases, these laws fail to properly consider the welfare interests of children. Finally, I conclude by showing that my argument does not entail adopting a laissez‐fair attitude to conception using third‐party sperm. PMID:27523389

  10. Llama oviductal sperm reservoirs: involvement of bulbourethral glands.

    PubMed

    Apichela, S A; Argañaraz, M E; Giuliano, S; Zampini, R; Carretero, I; Miragaya, M; Miceli, D C

    2014-04-01

    The aim of this study was to elucidate the role of llama seminal plasma in the formation of oviductal sperm reservoirs. Female llamas with follicles in the mature phase were mated with a bulbourethral glands-removed male. Females mated with nonbulbourethral glands-removed males were used as control. Oviducts were obtained by surgery 24 h after mating. The uterotubal junction and isthmus were examined by scanning electron microscopy, and mucopolysaccharides were identified by Alcian blue staining. To know the proteins probably involved in sperm reservoir formation, SDS-PAGE of seminal plasma (8% and 18% resolving gel) was made. Spermatozoa only adhered to the oviductal mucosa surface of uterotubal junction of females mated with nonbulbourethral glands-removed males confirming that seminal plasma and, in particular, bulbourethral secretions are related with the oviductal sperm reservoir formation. Histological sections showed sperm in the lumen, immersed in substance, positive for acid mucopolysaccharides. Alcian blue staining of seminal plasma proteins SDS-PAGE showed a band of high molecular weight containing mucopolysaccharides, only present in nonbulbourethral glands-removed males. Bulbourethral glands would secrete at least eight different proteins that most likely participate in the process of sperm storage in the oviduct.

  11. Sperm function test

    PubMed Central

    Talwar, Pankaj; Hayatnagarkar, Suryakant

    2015-01-01

    With absolute normal semen analysis parameters it may not be necessary to shift to specialized tests early but in cases with borderline parameters or with history of fertilization failure in past it becomes necessary to do a battery of tests to evaluate different parameters of spermatozoa. Various sperm function tests are proposed and endorsed by different researchers in addition to the routine evaluation of fertility. These tests detect function of a certain part of spermatozoon and give insight on the events in fertilization of the oocyte. The sperms need to get nutrition from the seminal plasma in the form of fructose and citrate (this can be assessed by fructose qualitative and quantitative estimation, citrate estimation). They should be protected from the bad effects of pus cells and reactive oxygen species (ROS) (leukocyte detection test, ROS estimation). Their number should be in sufficient in terms of (count), structure normal to be able to fertilize eggs (semen morphology). Sperms should have intact and functioning membrane to survive harsh environment of vagina and uterine fluids (vitality and hypo-osmotic swelling test), should have good mitochondrial function to be able to provide energy (mitochondrial activity index test). They should also have satisfactory acrosome function to be able to burrow a hole in zona pellucida (acrosome intactness test, zona penetration test). Finally, they should have properly packed DNA in the nucleus to be able to transfer the male genes (nuclear chromatic decondensation test) to the oocyte during fertilization. PMID:26157295

  12. Cytometry of mammalian sperm

    SciTech Connect

    Gledhill, B.L.

    1983-10-11

    Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. The accessibility of male cells makes them well suited for analytical cytology. We might automate the process of determining sperm morphology but should not do so solely for increased speed. Rather, richer tangible benefits will derive from cytometric evaluation through increased sensitivity, reduced subjectivity, standardization between investigators and laboratories, enhanced archival systems, and the benefits of easily exchanged standardized data. Inroads on the standardization of assays for motility and functional integrity are being made. Flow cytometric analysis of total DNA content of individual sperm is an insensitive means to detect exposure to reproductive toxins because of the small size and low frequency of the DNA content errors. Flow cytometry can be applied to determine the proportions of X- and Y-sperm in semen samples.

  13. Heat Shock Protein 90 Has Roles in Intracellular Calcium Homeostasis, Protein Tyrosine Phosphorylation Regulation, and Progesterone-Responsive Sperm Function in Human Sperm

    PubMed Central

    Chen, Aijun; Jiang, Youfang; Xie, Haifeng; Shi, Qixian; Zhang, Songying; Ni, Ya

    2014-01-01

    Heat shock protein 90 plays critical roles in client protein maturation, signal transduction, protein folding and degradation, and morphological evolution; however, its function in human sperm is not fully understood. Therefore, our objective in this study was to elucidate the mechanism by which heat shock protein 90 exerts its effects on human sperm function. By performing indirect immunofluorescence staining, we found that heat shock protein 90 was localized primarily in the neck, midpiece, and tail regions of human sperm, and that its expression increased with increasing incubation time under capacitation conditions. Geldanamycin, a specific inhibitor of heat shock protein 90, was shown to inhibit this increase in heat shock protein 90 expression in western blotting analyses. Using a multifunctional microplate reader to examine Fluo-3 AM-loaded sperm, we observed for the first time that inhibition of heat shock protein 90 by using geldanamycin significantly decreased intracellular calcium concentrations during capacitation. Moreover, western blot analysis showed that geldanamycin enhanced tyrosine phosphorylation of several proteins, including heat shock protein 90, in a dose-dependent manner. The effects of geldanamycin on human sperm function in the absence or presence of progesterone was evaluated by performing chlortetracycline staining and by using a computer-assisted sperm analyzer. We found that geldanamycin alone did not affect sperm capacitation, hyperactivation, and motility, but did so in the presence of progesterone. Taken together, these data suggest that heat shock protein 90, which increases in expression in human sperm during capacitation, has roles in intracellular calcium homeostasis, protein tyrosine phosphorylation regulation, and progesterone-stimulated sperm function. In this study, we provide new insights into the roles of heat shock protein 90 in sperm function. PMID:25541943

  14. Sperm DNA Fragmentation Index and Hyaluronan Binding Ability in Men from Infertile Couples and Men with Testicular Germ Cell Tumor.

    PubMed

    Marchlewska, Katarzyna; Filipiak, Eliza; Walczak-Jedrzejowska, Renata; Oszukowska, Elzbieta; Sobkiewicz, Slawomir; Wojt, Malgorzata; Chmiel, Jacek; Kula, Krzysztof; Slowikowska-Hilczer, Jolanta

    2016-01-01

    Objective. To investigate sperm DNA fragmentation and sperm functional maturity in men from infertile couples (IC) and men with testicular germ cell tumor (TGCT). Materials and Methods. Semen samples were collected from 312 IC men and 23 men with TGCT before unilateral orchiectomy and oncological treatment. The sperm chromatin dispersion test was performed to determine DNA fragmentation index (DFI) and the ability of sperm to bind with hyaluronan (HA) was assessed. Results. In comparison with the IC men, the men with TGCT had a higher percentage of sperm with fragmented DNA (median 28% versus 21%; p < 0.01) and a lower percentage of HA-bound sperm (24% versus 66%; p < 0.001). Normal results of both analyses were observed in 24% of IC men and 4% of men with TGCT. Negative Spearman's correlations were found between DFI and the percentage of HA-bound sperm in the whole group and in IC subjects and those with TGCT analyzed separately. Conclusions. Approximately 76% of IC men and 96% with TGCT awaiting orchiectomy demonstrated DNA fragmentation and/or sperm immaturity. We therefore recommend sperm banking after unilateral orchiectomy, but before irradiation and chemotherapy; the use of such a deposit appears to be a better strategy to obtain functionally efficient sperms.

  15. Sperm DNA Fragmentation Index and Hyaluronan Binding Ability in Men from Infertile Couples and Men with Testicular Germ Cell Tumor

    PubMed Central

    Filipiak, Eliza; Walczak-Jedrzejowska, Renata; Oszukowska, Elzbieta; Sobkiewicz, Slawomir; Wojt, Malgorzata; Chmiel, Jacek; Kula, Krzysztof; Slowikowska-Hilczer, Jolanta

    2016-01-01

    Objective. To investigate sperm DNA fragmentation and sperm functional maturity in men from infertile couples (IC) and men with testicular germ cell tumor (TGCT). Materials and Methods. Semen samples were collected from 312 IC men and 23 men with TGCT before unilateral orchiectomy and oncological treatment. The sperm chromatin dispersion test was performed to determine DNA fragmentation index (DFI) and the ability of sperm to bind with hyaluronan (HA) was assessed. Results. In comparison with the IC men, the men with TGCT had a higher percentage of sperm with fragmented DNA (median 28% versus 21%; p < 0.01) and a lower percentage of HA-bound sperm (24% versus 66%; p < 0.001). Normal results of both analyses were observed in 24% of IC men and 4% of men with TGCT. Negative Spearman's correlations were found between DFI and the percentage of HA-bound sperm in the whole group and in IC subjects and those with TGCT analyzed separately. Conclusions. Approximately 76% of IC men and 96% with TGCT awaiting orchiectomy demonstrated DNA fragmentation and/or sperm immaturity. We therefore recommend sperm banking after unilateral orchiectomy, but before irradiation and chemotherapy; the use of such a deposit appears to be a better strategy to obtain functionally efficient sperms. PMID:27999814

  16. Simple procedure for sperm cryopreservation in the larvacean tunicate Oikopleura dioica.

    PubMed

    Ouchi, Kazuaki; Nishino, Atsuo; Nishida, Hiroki

    2011-01-01

    The larvacean tunicate Oikopleura dioica is an attractive organism for studies of the development, evolution, and physiology of chordates, showing considerable promise for genetic approaches given its short life cycle of five days. To facilitate future genetic studies, the development of protocols for the maintenance of individual strains is essential. Here we report a simple and practical protocol for the cryopreservation of sperm using liquid nitrogen (-196°C) and dimethyl sulfoxide (DMSO) as a protective agent. The quality of the frozen-thawed sperm was evaluated in terms of fertilizing ability and subsequent development of the fertilized eggs. We examined several parameters to optimize the efficiency of cryopreservation, such as the concentration of DMSO, the method for acclimation of sperm to DMSO before freezing, and for placing sperm in liquid nitrogen, as well as the pH of the seawater used in resuspending the thawed sperm. We confirmed that viable sperm were recovered after preservation for more than one year. In addition, mature animals, and even a subsequent generation, were obtained from eggs fertilized by the cryopreserved sperm. The present procedure seems to be simple and sufficiently practical for maintenance of future established lines of O. dioica using frozen sperm.

  17. Effects of testicular histopathology on sperm retrieval rates and ICSI results in non-obstructive azoospermia.

    PubMed

    Aydin, T; Sofikerim, M; Yucel, B; Karadag, M; Tokat, F

    2015-01-01

    Non-obstructive azoospermia (NOA) is characterised by absence of sperm in the ejaculate. Significant relationship between the pattern of the testis histopathology of NOA and successful sperm retrieval rate is well known. In this study, we assess efficacy of testicular histopathology on sperm retrieval rates and intracytoplasmic sperm injection results after microdissection testicular sperm extraction in cases of non-obstructive azoospermia. It is a retrospective analysis of 111 NOA patients who have histopathological confirmation. According to histopathological findings, the patients were divided into three groups: Sertoli-cell-only syndrome (SCOS), maturation arrest (MA) and hypospermatogenesis. Sperm retrieval rate was significantly higher in hypospermatogenesis group compared with that in SCOS and MA groups. In terms of fertilisation and clinical pregnancy rates, there was no significant difference between the groups. As a result, compared with MA and SCOS, hypospermatogenesis has higher sperm retrieval rates. Our study revealed that once successful sperm retrieval is achieved, fertilisation and clinical pregnancy rates are similar in NOA patients.

  18. Sperm conjugation in mammal reproductive function: Different names for the same phenomenon?

    PubMed

    Monclus, Maria Angeles; Fornes, Miguel Walter

    2016-10-01

    In many mammalian and non-mammalian species, mature sperm interact within the female reproductive tract or inside the epididymal lumen using cohesive forces. This phenomenon, known as "sperm conjugation," is sometimes confused with sperm agglutination, which is the result of the interaction of epididymal or ejaculate spermatozoa upon release into culture medium. In addition to "agglutination," the terms "association," "rouleaux," or "rosettes" are employed interchangeably to describe the conjugation phenomenon, which inevitably causes confusion due to the non-unifying nomenclature. This variety of descriptions is likely due to a poor understanding of the molecular mechanisms involved in such conspicuous cell-cell interaction as well as the different morphologies that result from such interactions among species. Here, we summarize the published data regarding mammalian sperm conjugation, considering the organisms in which sperm interaction was observed; the particular terminology employed; findings regarding the components that enable sperm to adhere; sperm behavior when deposited in the female reproductive tract; and hypotheses formulated to clarify the biological function and, when known, the mechanisms for sperm interaction. We also propose a new classification system for this phenomenon that might clearly unify the criteria used to describe this behavior. Mol. Reprod. Dev. 83: 884-896, 2016 © 2016 Wiley Periodicals, Inc.

  19. Current knowledge on boar sperm metabolism: Comparison with other mammalian species.

    PubMed

    Rodríguez-Gil, Joan E; Bonet, Sergi

    2016-01-01

    A practical consequence of the specific pig reproductive cycle is that the main functional features that distinguish boar spermatozoa cannot be extrapolated to other species. This prevents an overall picture that explains mammalian sperm function from being assumed. Furthermore, the extraordinary complexity of the molecular mechanisms implied in the control and modulation of mature boar sperm functions makes it impossible to provide a complete description of these mechanisms in the limited space of this chapter. Taking this into account, this chapter centers on the description of three highly important specific aspects of boar sperm function. The first aspect is the mechanisms by which boar sperm cells uptake extracellular energy sources. The second aspect is the necessity of mammalian sperm to use other hexoses than glucose as feasible energy sources. The third aspect would be an analysis of the roles that mitochondria could play in the regulation of the overall boar sperm function. As a whole, this revision intends to be an overall picture of regulatory mechanisms involved in the maintenance of proper energy levels of boar sperm and their relationship with the control of the overall boar sperm function.

  20. Giant panda (Ailuropoda melanoleuca) sperm morphometry and function after repeated freezing and thawing.

    PubMed

    Santiago-Moreno, J; Esteso, M C; Pradiee, J; Castaño, C; Toledano-Díaz, A; O'Brien, E; Lopez-Sebastián, A; Martínez-Nevado, E; Delclaux, M; Fernández-Morán, J; Zhihe, Z

    2016-05-01

    This work examines the effects of subsequent cycles of freezing-thawing on giant panda (Ailuropoda melanoleuca) sperm morphometry and function, and assesses whether density-gradient centrifugation (DGC) can increase the number of freezing-thawing cycles this sperm can withstand. A sperm sample was collected by electroejaculation from a mature giant panda and subjected to five freezing-thawing cycles. Although repeated freezing-thawing negatively affected (P < 0.05) sperm motility and membrane integrity, in both nonselected and DCG-selected sperm samples, >60% of the sperm cells in both treatments showed acrosome integrity even after the fifth freezing cycle. In fresh semen, the sperm head length was 4.7 μm, the head width 3.6 μm, area 14.3 μm(2) and perimeter length 14.1 μm. The present results suggest that giant panda sperm trends to be resistant to repeated freezing-thawing, even without DGC selection.

  1. Sperm Morphology Assessment in Captive Neotropical Primates.

    PubMed

    Swanson, W F; Valle, R R; Carvalho, F M; Arakaki, P R; Rodas-Martínez, A Z; Muniz, Japc; García-Herreros, M

    2016-08-01

    The main objective of this study was to evaluate sperm morphology in four neotropical primate species to compare the sperm morphological traits and the sperm morphometric parameters as a basis for establishing normative sperm standards for each species. Data from 80 ejaculates collected from four primate species, Callithrix jacchus, Callimico goeldii, Alouatta caraya and Ateles geoffroyi, were analysed for detection of sperm morphological alterations using subjective World Health Organization (WHO-2010) standards and Sperm Deformity Index (SDI) criteria, objective computer-assisted sperm morphometry analysis (CASMA) and subpopulation sperm determination (SSD) methods. There were multiple differences (p < 0.01) observed among primate species in values obtained from WHO-2010, SDI, CASMA and SSD sperm analysis methods. In addition, multiple significant positive and negative correlations were observed between the sperm morphological traits (SDI, Sperm Deformity Index Head Defects, Sperm Deformity Index Midpiece Defects, Sperm Deformity Index Tail Defects, Normal Sperm, Head Defects, Midpiece Defects and Tail Defects) and the sperm morphometric parameters (SSD, Area (A), Perimeter (P), Length (L), Width (W), Ellipticity, Elongation and Rugosity) (p ≤ 0.046). In conclusion, our findings using different evaluation methods indicate that pronounced sperm morphological variation exists among these four neotropical primate species. Because of the strong relationship observed among morphological and morphometric parameters, these results suggest that application of objective analysis methods could substantially improve the reliability of comparative studies and help to establish valid normative sperm values for neotropical primates.

  2. GLUTATHIONE (GSH) CONCENTRATIONS VARY WITH THE CELL CYCLE IN MATURING HAMSTER OOCYTES, ZYGOTES AND PRE-IMPLANATION EMBRYOS

    EPA Science Inventory

    Abstract
    Glutathione (GSH) is thought to play critical roles in oocyte function including spindle maintenance and provision of reducing power needed to initiate sperm chromatin decondensation. Previous observations that GSH concentrations are higher in mature than immature o...

  3. Sedimentation properties in density gradients correspond with levels of sperm DNA fragmentation, chromatin compaction and binding affinity to hyaluronic acid.

    PubMed

    Torabi, Forough; Binduraihem, Adel; Miller, David

    2017-03-01

    Mature spermatozoa bind hyaluronic acid in the extracellular matrix via hyaladherins. Immature spermatozoa may be unable to interact because they do not express the appropriate hyaladherins on their surface. Fresh human semen samples were fractionated using differential density gradient centrifugation (DDGC) and the ability of these fractions to bind hyaluronic acid was evaluated. The presence of sperm hyaladherins was also assessed. CD44 was located mainly on the acrosome and equatorial segment and became more restricted to the equatorial segment in capacitated spermatozoa. Hyaluronic acid-TRITC (hyaluronic acid conjugated with tetramethylrhodamine isothiocyanante), a generic hyaluronic-acid-binding reagent, labelled the membrane and the neck region, particularly after capacitation. Sperm populations obtained after DDGC or after interaction with hyaluronic acid were assessed for DNA fragmentation and chromatin maturity. Strong relationships between both measures and sperm sedimentation and hyaluronic-acid-binding profiles were revealed. Capacitation enhanced hyaluronic acid binding of both DDGC-pelleted sperm and sperm washed free of seminal fluid. In conclusion, hyaladherins were detected on human sperm and a higher capacity for sperm hyaluronic-acid-binding was shown to correspond with their DDGC sedimentation profiles and with lower levels of DNA fragmentation and better chromatin maturity. Capacitation induced changes in the distribution and presence of hyaladherins may enhance hyaluronic-acid-binding.

  4. Hormone-induced cortical maturation ensures the slow block to polyspermy and does not couple with meiotic maturation in starfish.

    PubMed

    Hirohashi, Noritaka; Harada, Kaori; Chiba, Kazuyoshi

    2008-06-01

    Meiotic progression in starfish oocytes is reinitiated by a maturation-inducing hormone called 1-methyladenine (1-MeAde). In addition to meiotic maturation, 1-MeAde induces cortical maturation in which cortical granules become competent to discharge in response to fusion of a single sperm, which results in the formation of the fertilization envelope. We found that subthreshold concentrations of 1-MeAde induce cortical maturation without germinal vesicle breakdown (GVBD). During cortical maturation, the IP3 sensitivity of calcium stores was increased as well as during meiotic maturation. When oocytes were exposed with 1-MeAde only on a hemisphere of oocytes, the IP3 sensitivity of the cortical region was increased only in the exposed hemisphere, suggesting that signals and components involved in cortical maturation do not readily spread in the cytoplasm. Although a specific inhibitor of phosphatidylinositol-3 kinase, LY294002 blocked both GVBD and cortical maturation, a Cdc2 kinase inhibitor, roscovitine did not block cortical maturation. Inhibition of Akt activation by injecting the competitors for Akt phosphorylation and membrane recruitment also blocked cortical maturation. These results suggest that the signaling pathway leading to Akt activation is common in cortical maturation and meiotic maturation, and Cdc2 activation was not required for cortical maturation.

  5. Harvesting sperm and artificial insemination of mice.

    PubMed

    Duselis, Amanda R; Vrana, Paul B

    2007-01-01

    Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination. Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the female's reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated.Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.

  6. Ion channels, phosphorylation and mammalian sperm capacitation

    PubMed Central

    Visconti, Pablo E; Krapf, Dario; de la Vega-Beltrán, José Luis; Acevedo, Juan José; Darszon, Alberto

    2011-01-01

    Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies. PMID:21540868

  7. Paternal poly (ADP-ribose) metabolism modulates retention of inheritable sperm histones and early embryonic gene expression.

    PubMed

    Ihara, Motomasa; Meyer-Ficca, Mirella L; Leu, N Adrian; Rao, Shilpa; Li, Fan; Gregory, Brian D; Zalenskaya, Irina A; Schultz, Richard M; Meyer, Ralph G

    2014-05-01

    To achieve the extreme nuclear condensation necessary for sperm function, most histones are replaced with protamines during spermiogenesis in mammals. Mature sperm retain only a small fraction of nucleosomes, which are, in part, enriched on gene regulatory sequences, and recent findings suggest that these retained histones provide epigenetic information that regulates expression of a subset of genes involved in embryo development after fertilization. We addressed this tantalizing hypothesis by analyzing two mouse models exhibiting abnormal histone positioning in mature sperm due to impaired poly(ADP-ribose) (PAR) metabolism during spermiogenesis and identified altered sperm histone retention in specific gene loci genome-wide using MNase digestion-based enrichment of mononucleosomal DNA. We then set out to determine the extent to which expression of these genes was altered in embryos generated with these sperm. For control sperm, most genes showed some degree of histone association, unexpectedly suggesting that histone retention in sperm genes is not an all-or-none phenomenon and that a small number of histones may remain associated with genes throughout the genome. The amount of retained histones, however, was altered in many loci when PAR metabolism was impaired. To ascertain whether sperm histone association and embryonic gene expression are linked, the transcriptome of individual 2-cell embryos derived from such sperm was determined using microarrays and RNA sequencing. Strikingly, a moderate but statistically significant portion of the genes that were differentially expressed in these embryos also showed different histone retention in the corresponding gene loci in sperm of their fathers. These findings provide new evidence for the existence of a linkage between sperm histone retention and gene expression in the embryo.

  8. Understanding fertilization through intracytoplasmic sperm injection (ICSI).

    PubMed

    Neri, Queenie V; Lee, Bora; Rosenwaks, Zev; Machaca, Khaled; Palermo, Gianpiero D

    2014-01-01

    Since the establishment of in vitro fertilization, it became evident that almost half of the couples failed to achieve fertilization and this phenomenon was attributed to a male gamete dysfunction. The adoption of assisted fertilization techniques particularly ICSI has been able to alleviate male factor infertility by granting the consistent ability of a viable spermatozoon to activate an oocyte. Single sperm injection, by pinpointing the beginning of fertilization, has been an invaluable tool in clarifying the different aspects of early fertilization and syngamy. However, even with ICSI some couples fail to fertilize due to ooplasmic dysmaturity in relation to the achieved nuclear maturation marked by the extrusion of the first polar body. More uncommon are cases where the spermatozoa partially or completely lack the specific oocyte activating factor. In this work, we review the most relevant aspects of fertilization and its failure through assisted reproductive technologies. Attempts at diagnosing and treating clinical fertilization failure are described.

  9. Understanding fertilization through intracytoplasmic sperm injection (ICSI)

    PubMed Central

    Neri, Queenie V.; Lee, Bora; Rosenwaks, Zev; Machaca, Khaled; Palermo, Gianpiero D.

    2014-01-01

    Summary Since the establishment of in vitro fertilization, it became evident that almost half of the couples failed to achieve fertilization and this phenomenon was attributed to a male gamete dysfunction. The adoption of assisted fertilization techniques particularly ICSI has been able to alleviate male factor infertility by granting the consistent ability of a viable spermatozoon to activate an oocyte. Single sperm injection, by pinpointing the beginning of fertilization, has been an invaluable tool in clarifying the different aspects of early fertilization and syngamy. However, even with ICSI some couples fail to fertilize due to ooplasmic dysmaturity in relation to the achieved nuclear maturation marked by the extrusion of the first polar body. More uncommon are cases where the spermatozoa partially or completely lack the specific oocyte activating factor. In this work, we review the most relevant aspects of fertilization and its failure through assisted reproductive technologies. Attempts at diagnosing and treating clinical fertilization failure are described. PMID:24290744

  10. Sperm ubiquitination in epididymal feline semen.

    PubMed

    Vernocchi, Valentina; Morselli, Maria Giorgia; Varesi, Sara; Nonnis, Simona; Maffioli, Elisa; Negri, Armando; Tedeschi, Gabriella; Luvoni, Gaia Cecilia

    2014-09-01

    Ubiquitin is a 8.5-kDa peptide that tags other proteins for proteasomal degradation. It has been proposed that ubiquitination might be responsible for the elimination of defective spermatozoa during transit through the epididymis in humans and cattle, but its exact biological function in seminal plasma has not yet been clarified. In the domestic cat (Felis catus), the percentage of immature, unviable, and abnormal spermatozoa decreases during the epididymal transit, indicating the existence of a mechanism that removes defective spermatozoa. Magnetic cell separation techniques, based on the use of magnetic beads coated with anti-ubiquitin antibodies, may allow the selective capture of ubiquitinated spermatozoa from semen, thus contributing to the identification of a potential correlation between semen quality and ubiquitination process. Moreover, the selective identification of all the ubiquitinated proteins in different epididymal regions could give a better understanding of the ubiquitin role in feline sperm maturation. The aims of this study were as follows: (1) to verify the possibility of separating ubiquitinated spermatozoa with magnetic ubiquitin beads and identify the morphological and acrosomal differences between whole sample and unbound gametes, (2) to characterize all the ubiquitinated proteins in spermatozoa retrieved in the three epididymal regions by a proteomic approach. The data indicated the presence of ubiquitinated proteins in cat epididymal semen. However, a correlation between abnormal and ubiquitinated spermatozoa has not been found, and ubiquitin cannot be considered as a biomarker of quality of epididymal feline spermatozoa. To the author's knowledge, this is the first identification of all the ubiquitinated proteins of cat spermatozoa collected from different epididymal regions. The proteomic pattern allows a further characterization of cat epididymal semen and represents a contribute to a better understanding of the ubiquitin role in

  11. Chromosomal abnormalities in human sperm

    SciTech Connect

    Martin, R.H.

    1985-01-01

    The ability to analyze human sperm chromosome complements after penetration of zona pellucida-free hamster eggs provides the first opportunity to study the frequency and type of chromosomal abnormalities in human gametes. Two large-scale studies have provided information on normal men. We have studied 1,426 sperm complements from 45 normal men and found an abnormality rate of 8.9%. Brandriff et al. (5) found 8.1% abnormal complements in 909 sperm from 4 men. The distribution of numerical and structural abnormalities was markedly dissimilar in the 2 studies. The frequency of aneuploidy was 5% in our sample and only 1.6% in Brandriff's, perhaps reflecting individual variability among donors. The frequency of 24,YY sperm was low: 0/1,426 and 1/909. This suggests that the estimates of nondisjunction based on fluorescent Y body data (1% to 5%) are not accurate. We have also studied men at increased risk of sperm chromosomal abnormalities. The frequency of chromosomally unbalanced sperm in 6 men heterozygous for structural abnormalities varied dramatically: 77% for t11;22, 32% for t6;14, 19% for t5;18, 13% for t14;21, and 0% for inv 3 and 7. We have also studied 13 cancer patients before and after radiotherapy and demonstrated a significant dose-dependent increase of sperm chromosome abnormalities (numerical and structural) 36 months after radiation treatment.

  12. Heavy ion radiation can promote greater motility and enolase protein expression in ram sperm in in vitro liquid storage.

    PubMed

    He, Yuxuan; Li, Hongyan; He, Jianhua; Zhao, Xingxu

    2014-08-01

    The aim of the study was to determine the effects of heavy ion radiation (HIR) on ram sperm quality during 24h of in vitro liquid storage at 15°C, and identify the most appropriate dose which did not injure, but actually improved sperm quality and confirmed the relationship between highly expressed enolase and ram sperm quality during storage in vitro. Six Dorset ram (Ovis aries) semen pools from five mature and healthy rams were each divided into seven experimental groups with different doses of HIR (0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5Gy) under the same experimental conditions. Sperm motility, viability, ATP content, and the gene and protein expression of enolase were measured at 24h of storage. Irradiated semen which had been stored for 24h, retained not only greater sperm motility, viability, and ATP content, but had greater enolase protein expression. This was evidenced by increased amounts of mRNA for this enzyme and amount of enolase protein as compared with semen from control rams, especially for the 0.1Gy group (P<0.001). These results indicate that HIR can promote enhanced motility and viability during in vitro liquid storage, and the 0.1Gy may be a suitable dose for improving sperm quality. Greater amounts of enolase and ATP content may results from enhanced sperm glycolysis by HIR. HIR enhances sperm glycolysis to generate sufficient ATP for maintaining sperm motility during storage.

  13. An ultrastructural study of spermatogenesis and sperm morula breakdown in Arenicola marina (L.) (Annelida: Polychaeta)

    NASA Astrophysics Data System (ADS)

    Pacey, A. A.; Bentley, M. G.

    1992-06-01

    Spermatogenesis in the lugworm Arenicola marina, in common with other members of Arenicolidae, occurs in the coelomic fluid and results in the formation of discs of mature spermatozoa known as a morulae. Within a morula, individual spermatozoa are connected by a common mass of cytoplasm called the cytophore and therefore make up a syncitium. Immediately prior to spawning, and in response to an endocrine substance known as “Sperm Maturation Factor” (SMF), the structure of the sperm morulae breaks down and free spermatozoa are liberated. These are subsequently spawned from the body cavity. The investigation described here uses transmission electron microscopy to investigate the ultrastructural changes, which accompany spermatogenesis and the breakdown of sperm morulae in response to SMF in vitro. The study demonstrates that the cytophore appears to have a key role both during spermatogenesis and during sperm morula breakdown. The ultrastructure of sperm morulae and of mature spermatozoa is described. The structure of spermatozoa is shown to be primitive with a single flagellum which appears to be coiled at its distal end. The phagocytosis of free spermatozoa by coelomocytes is also described and it is suggested that these may play a role in the resorption of unspawned gametes in vivo.

  14. Yolk protein is expressed in the insect testis and interacts with sperm

    PubMed Central

    Bebas, Piotr; Kotwica, Joanna; Joachimiak, Ewa; Giebultowicz, Jadwiga M

    2008-01-01

    Background Male and female gametes follow diverse developmental pathways dictated by their distinct roles in fertilization. While oocytes of oviparous animals accumulate yolk in the cytoplasm, spermatozoa slough off most of their cytoplasm in the process of individualization. Mammalian spermatozoa released from the testis undergo extensive modifications in the seminal ducts involving a variety of glycoproteins. Ultrastructural studies suggest that glycoproteins are involved in sperm maturation in insects; however, their characterization at the molecular level is lacking. We reported previously that the circadian clock controls sperm release and maturation in several insect species. In the moth, Spodoptera littoralis, the secretion of glycoproteins into the seminal fluid occurs in a daily rhythmic pattern. The purpose of this study was to characterize seminal fluid glycoproteins in this species and elucidate their role in the process of sperm maturation. Results We collected seminal fluid proteins from males before and after daily sperm release. These samples were separated by 2-D gel electrophoresis, and gels were treated with a glycoprotein-detecting probe. We observed a group of abundant glycoproteins in the sample collected after sperm release, which was absent in the sample collected before sperm release. Sequencing of these glycoproteins by mass spectroscopy revealed peptides bearing homology with components of yolk, which is known to accumulate in developing oocytes. This unexpected result was confirmed by Western blotting demonstrating that seminal fluid contains protein immunoreactive to antibody against yolk protein YP2 produced in the follicle cells surrounding developing oocytes. We cloned the fragment of yp2 cDNA from S. littoralis and determined that it is expressed in both ovaries and testes. yp2 mRNA and YP2 protein were detected in the somatic cyst cells enveloping sperm inside the testis. During the period of sperm release, YP2 protein appears in

  15. The localization of protein carboxyl-methylase in sperm tails

    PubMed Central

    1980-01-01

    Protein carboxyl-methylase (PCM), an enzyme known to be involved in exocytotic secretion and chemotaxis, has been studied in rat and rabbit spermatozoa. PCM activity and its substrate methyl acceptor protein(s) (MAP) were demonstrated in the supernate after solubilization of the sperm cell membrane by detergent (Triton X-100). A protein methylesterase that hydrolyzes methyl ester bonds created by PCM was demonstrated in rabbit but not in rat spermatozoa. This enzyme was not solubilized by nonionic detergent. The specific activities of PCM in rat spermatozoa from caput and cauda epididymis were similar and lower than that found in testis. By contrast, MAP substrates were low in testis and increased in parallel with sperm maturation in the epididymis. Multiple MAP were demonstrated in spermatozoa by polyacrylamide gel electrophoresis. The pattern of these proteins was similar in spermatozoa from different portions of the reproductive tract. Fractionation of heads and tails of rat spermatozoa on sucrose gradients indicated that PCM was found exclusively in the tail fraction, whereas MAP was detected both in head and tail fractions. The presence of all the components of the protein carboxyl-methylation system in spermatozoa and the localization of PCM and some of its substrates in the sperm tail are consistent with their involvement in sperm cell motility. PMID:7400214

  16. Chd5 orchestrates chromatin remodeling during sperm development

    PubMed Central

    Li, Wangzhi; Wu, Jie; Kim, Sang-Yong; Zhao, Ming; Hearn, Stephen A.; Zhang, Michael Q.; Meistrich, Marvin L.

    2014-01-01

    One of the most remarkable chromatin remodeling processes occurs during spermiogenesis, the post-meiotic phase of sperm development during which histones are replaced with sperm-specific protamines to repackage the genome into the highly compact chromatin structure of mature sperm. Here we identify Chromodomain helicase DNA binding protein 5 (Chd5) as a master regulator of the histone-to-protamine chromatin remodeling process. Chd5 deficiency leads to defective sperm chromatin compaction and male infertility in mice, mirroring the observation of low CHD5 expression in testes of infertile men. Chd5 orchestrates a cascade of molecular events required for histone removal and replacement, including histone 4 (H4) hyperacetylation, histone variant expression, nucleosome eviction, and DNA damage repair. Chd5 deficiency also perturbs expression of transition proteins (Tnp1/Tnp2) and protamines (Prm1/2). These findings define Chd5 as a multi-faceted mediator of histone-to-protamine replacement and depict the cascade of molecular events underlying chromatin remodeling during this process of extensive chromatin remodeling. PMID:24818823

  17. Sperm competition and sperm cooperation: the potential role of diploid and haploid expression.

    PubMed

    Immler, Simone

    2008-03-01

    Sperm competition is a powerful selective force driving the evolution of sperm shape and function. Recent findings suggest that sperm cooperation is a potential evolutionary response to sperm competition. Sperm cooperation may enhance the performance of the ejaculate increasing a male's chance to outcompete rival males in competition for fertilisation. Whether and how sperm cooperation may evolve is the focal point of this review. The relative importance of haploid and diploid gene expression for the evolution of sperm cooperation and the potential conflict of interest between (i) haploid sperm and diploid male and (ii) among sibling sperm, since sibling sperm only share an average of 50% of their genes in a diploid organism, are discussed. Furthermore, sperm cooperation is defined and the literature for empirical evidence of sperm cooperation is reviewed in light of the author's definitions.

  18. Sperm-agglutinating antibodies and testicular morphology in fifty-nine men with azoospermia or cryptozoospermia.

    PubMed

    Friberg, J; Kjessler, B

    1975-04-01

    The relationship between the state of the germinal epithelium and the type and titer of circulating sperm-agglutinating antibodies has been investigated in a series of 59 azoospermic or occasionally cryptozoospermic men. The patients were grouped according to the condition of the germinal epithelium as observed from testicular biopsy specimens, as well as to type and titer of circulating sperm-agglutinating antibodies investigated by a previously described microagglutination technique. Evidence is presented to suggest that the presence of mature spermatozoa in the testicular structures may be a prerequisite for the spontaneous production of circulating sperm-agglutinating antibodies, at least of the head-to-tail (H-T) agglutinating type. Furthermore, these circulating H-T sperm-agglutinating antibodies, once they are formed, do not seem to interfere adversely with the germinal epithelium of the carrier.

  19. An environmentally relevant organochlorine mixture impairs sperm function and embryo development in the porcine model.

    PubMed

    Campagna, Céline; Guillemette, Christine; Paradis, René; Sirard, Marc-André; Ayotte, Pierre; Bailey, Janice L

    2002-07-01

    We evaluated the effects of an environmentally relevant mixture of more than 15 organochlorines on the development of pig oocytes and sperm during in vitro fertilization (IVF). Oocytes were cocultured with sperm in IVF medium containing increasing concentrations of an organochlorine mixture, similar to that found in women of highly exposed populations. Exposure to the organochlorine mixture diminished oocyte penetration rates and polyspermy in a linear manner. The mixture did not affect rates of cleavage nor development to multicell embryos. However, rates of development to the blastocyst stage were lower at the highest concentration at which oocyte penetration was observed. The same experiment was performed using oocytes that were preexposed during in vitro maturation. This greater exposure to the mixture also reduced penetration in a dose-response manner and affected polyspermy. Frozen-thawed pig sperm were also cultured in IVF medium containing the same organochlorine concentrations. Sperm motility parameters were immediately reduced in a dose-dependent manner by the organochlorines, followed by diminished viability 2 h later. From these results, it appears that reduced sperm quality would account for decreases in fertilization, polyspermy, and blastocyst formation. These results suggest that exposing porcine oocytes and sperm to an environmentally pertinent organochlorine mixture in vitro disrupts the oocyte block to polyspermy, sperm fertility, and further embryonic development, and supports recent concerns that such pollutants harm reproductive health in humans and other species.

  20. Comparison among different cryoprotectants for cryopreservation of epididymal sperm from agouti (Dasyprocta leporina).

    PubMed

    Castelo, T S; Silva, A M; Bezerra, L G P; Costa, C Y M; Lago, A E A; Bezerra, J A B; Campos, L B; Praxedes, E C G; Silva, A R

    2015-12-01

    We verify the effects of different cryoprotectants on the cryopreservation of agouti (Dasyprocta leporina) epididymal sperm. We used 16 pairs of testes-epididymis complexes of sexually mature animals. We immediately evaluated epididymal sperm obtained by retrograde flushing for concentration, motility, vigor, viability, osmotic response, and morphology. Samples were extended in a coconut water extender plus 20% egg yolk, containing glycerol, ethylene glycol, dimethylsulfoxide - DMSO, or dimethylformamide. Finally, samples were stored in 0.25 mL straws, frozen in liquid nitrogen, and thawed after one week, being reevaluated and assessed for membrane integrity using fluorescent probes. The higher values for postthawing sperm motility, vigor, and membrane integrity were achieved by the usage of glycerol, when compared to ethylene glycol and dimethylformamide (P < 0.05); however, no differences were found between glycerol and DMSO (P > 0.05). All cryoprotectants provided a similar effect on the preservation of sperm morphology, osmotic response, and viability (P > 0.05). Therefore, here onwards, there was testing of glycerol and DMSO at 3 and 6% concentrations using the same freezing-thawing protocol reported previously. As the main result, DMSO at 6% concentration provided a decrease in sperm parameters, as well as in the chromatin integrity and in the binding capability of sperm. In conclusion, glycerol 3 or 6% and DMSO 3% can be used as alternative cryoprotectants for agouti epididymal sperm cryopreservation.

  1. An epididymis-specific carboxyl esterase CES5A is required for sperm capacitation and male fertility in the rat

    PubMed Central

    Ru, Yan-Fei; Xue, Hai-Min; Ni, Zi-Mei; Xia, Dong; Zhou, Yu-Chuan; Zhang, Yong-Lian

    2015-01-01

    Despite the fact that the phenomenon of capacitation was discovered over half century ago and much progress has been made in identifying sperm events involved in capacitation, few specific molecules of epididymal origin have been identified as being directly involved in this process in vivo. Previously, our group cloned and characterized a carboxyl esterase gene Ces5a in the rat epididymis. The CES5A protein is mainly expressed in the corpus and cauda epididymidis and secreted into the corresponding lumens. Here, we report the function of CES5A in sperm maturation. By local injection of Lentivirus-mediated siRNA in the CES5A-expressing region of the rat epididymis, Ces5a-knockdown animal models were created. These animals exhibited an inhibited sperm capacitation and a reduction in male fertility. These results suggest that CES5A plays an important role in sperm maturation and male fertility. PMID:25475668

  2. Zinc content of maturing spermatozoa in oestrogen treated rats.

    PubMed

    Srivastava, A; Chowdhury, A R; Setty, B S

    1983-02-01

    Zinc content of spermatozoa collected from the caput and cauda portions of the rat epididymis was determined by atomic absorption spectroscopy. The results showed about 60% reduction in the spermatozoal zinc content by the time they reach the cauda epididymis. This reduction was inhibited in rats receiving micro dose oestrogen which induced 'functional' sterility. It appears that the fall in zinc content of spermatozoa during their transport in the epididymis is related to sperm maturation and that oestrogen treatment interferes with this reduction in sperm zinc content.

  3. Seasonal changes of sperm storage and correlative structures in male and female soft-shelled turtles, Trionyx sinensis.

    PubMed

    Xiangkun, Han; Li, Zhang; Meiying, Li; Huijun, Bao; Nainan, Hei; Qiusheng, Chen

    2008-11-01

    Reproductive ducts of male and female soft-shelled turtles, Trionyx sinensis were examined throughout the year (March, May, September, December) using brightfield and electron microscopes (TEM and SEM), to determine the location and histomorphological characteristics of sperm storage structures as well as their changes at different phases of the seasonal reproductive cycle. Sperm stored in the epididymis were also examined. In the male, spermatogenesis is initiated in spring (May), and then the mature sperm are released in autumn as an episodic event. Spermatogenesis is inactive in winter. However, in this species, the epididymis contains sperm throughout the entire year. Sperm observed in the epididymis are intact and some structures are uniquely different from other reptiles, and is characterized by 35-40 concentric mitochondria with a dense core in the centre. Many glycogen granules are observed in the cytoplasm of the midpiece. However, the epithelial cell type of epididymal duct change in different seasons. The cells are fully developed with a highly secretory activity in September. The materials secreted from the epithelium might have the function as nourishment for the stored sperm. Sperm storage structures in the form of tubules are observed in the wall of the isthmus of the oviduct in hibernating females but are absent in the groups of May and September. These tubules develop either by folding or fusion of the oviductal mucosal folds and are lined by both ciliated and secretory cells. These tubules might provide a microenvironment for the sperm to enable its long-term storage. After being separated 4 months (December-March) from the male, sperm are observed in the tubules of the isthmus of the oviduct. The unique character of the sperm combined with the special sperm storage structures enable the sperm to maintain fertility and activity during their storage.

  4. Relative testis size and sperm morphometry across mammals: no evidence for an association between sperm competition and sperm length.

    PubMed Central

    Gage, Matthew J G; Freckleton, Robert P

    2003-01-01

    Understanding why there is extensive variation in sperm form and function across taxa has been a challenge because sperm are specialized cells operating at a microscopic level in a complex environment. This comparative study collates published data to determine whether the evolution of sperm morphometry (sperm total length and separate component dimensions) is associated with sperm competition (when different males' sperm mix and compete for a female's ova) across 83 mammalian species. We use relative testes mass as an indicator of the intensity of sperm competition across taxa: relative investment into testes is widely accepted to predict the level of sperm competition that a species or population endures. Although we found evidence for positive associations between relative testes mass (controlling for allometry) and sperm morphometry across 83 mammalian species, these relationships were phylogenetically dependent. When we appropriately controlled for phylogenetic association using multiple regression within a phylogenetic framework, there was no relationship between relative testes mass and sperm length across mammals. Furthermore, we found no evidence for associations between relative testes mass and sperm head, mid-piece or flagellar lengths, nor was there a relationship with mid-piece or mitochondrial volumes. Results, therefore, indicate that sperm competition does not select for longer or shorter sperm across mammals, and alternative forces selecting on sperm form and function are discussed. PMID:12769463

  5. Scrotal heat stress effects on sperm viability, sperm DNA integrity, and the offspring sex ratio in mice.

    PubMed

    Pérez-Crespo, M; Pintado, B; Gutiérrez-Adán, A

    2008-01-01

    Evidence exists to suggest detrimental effects of heat stress on male fertility. This study was designed to assess the effects of scrotal heat stress on mature and developing sperm in a mouse model. After receiving shock heat treatment (42 degrees C for 30 min), mature spermatozoa were recovered from the epididymis hours (6) or Days (7, 14, 21, 28, 60) later, to determine the variables: number of spermatozoa, sperm viability, motility and progressive motility, sperm DNA integrity as established by the TUNEL method, embryo implantation rate, and sex ratio of the fetuses conceived using the heat-exposed spermatozoa. Our results indicate that transient mild heat treatment does not affect in the same way the different types of male germ cells. Spermatocytes present within the testis at the time of heat stress resulted into a lower concentration of spermatozoa with reduced viability and low motility. Even though, DNA integrity of spermatozoa resulting from spermatocytes was also compromised by heat stress, the higher degree of DNA damage was found among spermatozoa resulting from spermatids present within the testis at the time of heat stress. At last, heat shock effect on spermatozoa present in the epididymis at the time of thermal stress resulted into a sex ratio distortion. These findings point to a higher sensitivity of spermatocytes to heat exposure and also suggest a different response of X and Y chromosome-bearing spermatozoa to heat stress that warrants further investigation.

  6. 75 FR 81584 - Endangered and Threatened Species; Recovery Plan for the Sperm Whale

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-28

    ... the complex social structure and reproductive behavior of sperm whales have confounded assessments of... of extinction in 100 years) and the global population has at least 1,500 mature, reproductive... extinction that cannot be incorporated into a Population Viability Analysis will be carefully...

  7. Sperm storage and viability in Photinus fireflies.

    PubMed

    Demary, Kristian C

    2005-07-01

    In many species females mate with and store sperm from multiple males, and some female insects have evolved multiple compartments for sperm storage. Sperm storage and sperm viability were investigated in two firefly species, Photinus greeni and P. ignitus, which differ in the morphology of the female reproductive tract. Although the primary spermatheca is similar in both species, P. greeni females have an additional, conspicuous outpocketing within the bursa copulatrix whose potential role in sperm storage was investigated in this study. An assay that distinguishes between live and dead sperm was used to examine sperm viability in male seminal vesicles and sperm storage sites within the female reproductive tract. For both Photinus species, sperm from male seminal vesicles showed significantly higher viability compared to sperm from the primary spermatheca of single mated females. In single mated P. greeni females, sperm taken from the channel outpocketing (secondary spermatheca) showed significantly higher viability compared to sperm from the primary spermatheca. This sperm viability difference was not evident in double mated females. There were no significant differences between P. greeni and P. ignitus females in the viability of sperm from the primary spermatheca. These studies contribute to our understanding of post-mating processes that may influence paternity success, and suggest that sexual conflict over control of fertilizations may occur in multiply mated firefly females.

  8. Morphological diversity of sperm: A mini review

    PubMed Central

    Prakash, Seppan; Prithiviraj, Elumalai; Suresh, Sekar; Lakshmi, Nagella Venkata; Ganesh, Mohanraj Karthik; Anuradha, Murugesan; Ganesh, Lakshmanan; Dinesh, Premavathy

    2014-01-01

    Sperms are highly specialized cells for delivering DNA from male to the ovum. Incredibly, wide degree of diversity in sperm morphology in their basic structures i.e. head, middle piece and tail is found across species. Differences in terms of overall size of the sperm, shape and number of sperm produced are also incredible. One of the key for this variations or diversity in sperm may be associated with female reproductive tract, sperm competition, testicular size and sperm size and number. Establishing a correlation between sperm morphology and factors influencing them is a phenomenal task. In this mini-review these associations and the anatomical and functional adaptations among different from of sperm cells that have evolved to optimize fertilization success are discussed. Nevertheless, explaining these morphological diversities in sperm cells is a challenging question and it seems that evolutionary biologists have only recently engaged in exploring its links and patterns. From the literatures it seems that there is no causal relationship between sperm size and testicular size, however, the accumulated knowledge do indicates evolution of sperm morphology across species has some associations with female reproductive tract, sperm competition and sperm size and number, however interpreting these results for phylogentic correlations should be approached with caution. PMID:24976817

  9. Expression of DNA repair genes in porcine oocytes before and after fertilization by ICSI using freeze-dried sperm.

    PubMed

    Men, Nguyen Thi; Kikuchi, Kazuhiro; Furusawa, Tadashi; Dang-Nguyen, Thanh Quang; Nakai, Michiko; Fukuda, Atsunori; Noguchi, Junko; Kaneko, Hiroyuki; Viet Linh, Nguyen; Xuan Nguyen, Bui; Tajima, Atsushi

    2016-11-01

    Boar sperm freeze-dried with trehalose showed a protective effect against sperm DNA fragmentation. However, normal fertilization and embryonic development were not improved. Damaged sperm may activate maternal DNA repair genes when injected into oocytes. Therefore, we investigated the expression profile of some DNA repair genes in porcine oocytes after intra-cytoplasmic sperm injection. First, the expression levels of MGMT, UDG, XPC, MSH2, XRCC6 and RAD51 genes that are concerned with different types of DNA repair were examined in in vitro mature (IVM) oocytes injected with ejaculated sperm, or freeze-dried sperm with or without trehalose. Quantitative reverse transcription polymerase chain reaction revealed that expression of six DNA repair genes in the oocytes at 4 h after injection did not differ among the four groups. Next, we investigated the gene expression levels of these genes at different stages of maturation. The relative expression levels of UDG and XPC were significantly up-regulated in mature oocytes compared with earlier stages. Furthermore, there was an increased tendency in relative expression of MSH2 and RAD51. These results suggested two possible mechanisms that messenger RNA of DNA repair genes are either accumulated during IVM to be ready for fertilization or increased expression levels of DNA repair genes in oocytes caused by suboptimal IVM conditions.

  10. Sperm-Egg Interaction: Evidence for Boar Sperm Plasma Membrane Receptors for Porcine Zona Pellucida

    NASA Astrophysics Data System (ADS)

    Peterson, Rudolph N.; Russell, Lonnie; Bundman, Donna; Freund, Matthew

    1980-01-01

    Freshly ejaculated, noncapacitated boar sperm bind rapidly and in large numbers to pig egg zona pellucida in vitro. In the present study, the number of sperm bound decreased sharply when sperm motility was lowered by energy poisons or by reducing the temperature. Highly motile sperm from humans, guinea pigs, and rats, added at concentrations ten times higher than control sperm, did not bind to the porcine zona. At the same high concentration, a small number of hamster and bull sperm bound to the zona. Binding of boar sperm to the zona pellucida was blocked almost completely by diluted whole antiserum to sperm plasma membranes and by univalent (Fab) antibody to these membranes. When antibody to sperm plasma membrane was first absorbed with plasma membrane vesicles, sperm binding was not inhibited. These results provide direct evidence for the existence of sperm plasma membrane receptors for the zona pellucida of the pig.

  11. 21 CFR 173.275 - Hydrogenated sperm oil.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... conditions: (a) The sperm oil is derived from rendering the fatty tissue of the sperm whale or is prepared by synthesis of fatty acids and fatty alcohols derived from the sperm whale. The sperm oil obtained...

  12. 21 CFR 173.275 - Hydrogenated sperm oil.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... conditions: (a) The sperm oil is derived from rendering the fatty tissue of the sperm whale or is prepared by synthesis of fatty acids and fatty alcohols derived from the sperm whale. The sperm oil obtained...

  13. 21 CFR 173.275 - Hydrogenated sperm oil.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... conditions: (a) The sperm oil is derived from rendering the fatty tissue of the sperm whale or is prepared by synthesis of fatty acids and fatty alcohols derived from the sperm whale. The sperm oil obtained...

  14. 21 CFR 173.275 - Hydrogenated sperm oil.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... conditions: (a) The sperm oil is derived from rendering the fatty tissue of the sperm whale or is prepared by synthesis of fatty acids and fatty alcohols derived from the sperm whale. The sperm oil obtained...

  15. Using Fresh and Frozen Testicular Sperm Samples in Couples Undergoing ICSI-MicroTESE Treatment

    PubMed Central

    Tavukcuoglu, Safak; AL-Azawi, Tahani; AL-Hasani, Safaa; Khaki, Amir Afshin; Khaki, Arash; Tasdemir, Seval

    2013-01-01

    Background We performed this study to evaluate use of fresh and frozen sperm samples in non-obstructive azoospermia microdissection testicular sperm extraction (micro-TESE-ICSI) treatment. Methods We performed a total of 82 consecutive in vitro fertilization (IVF) cycles at Fertijin IVF Center in Istanbul, Turkey from January 2010 to March 2012. In 43 participants we used fresh sperm and frozen sperm in the remaining 39 cases. We used fresh and frozen thawed micro surgical testicular sperm extraction (micro TESE) sperm for ICSI with metaphase II (MII) oocytes. Results Frozen microTESE sperm was used in 39 cycles, while 43 ICSI cycles were performed using fresh microTESE. Neither the age of male partners (38.33±5.93 and 38.13±8.28) nor that of the female participants (33.16±6.38 and 33.33±6.97) showed significant difference between fresh versus the microTESE and frozen treatment groups, respectively. FSH concentrations were (14.66±13.93 mIU/ml) in fresh TESE group and (17.91±16.29 mIU/ml) in frozen group with no correlations or differences between the two groups. The average number of mature oocytes injected with sperm was 9.23±3.77, versus 9.26±5.26 in cycles using fresh and frozen microTESE sperm, respectively. Fertilization rate was not significantly different in the fresh microTESE (44.79%) than frozen TESE sperm group (46.76%). The average number of transferred embryos was 1.60±0.49 in fresh sperm group and 1.59±0.50 in frozen sperm group. All embryo transfers were performed on day 3. Conclusion Cryopreservation of testicular sperm tissues is more suitable and of great benefite if carried out before ovulation induction and not after, especially in cases with non-obstructive azoospermia. PMID:23926569

  16. Evolution of the vertebrate epididymis.

    PubMed

    Jones, R C

    1998-01-01

    This review examines the structure and function of the extratesticular sperm ducts of vertebrates in terms of their evolutionary development and adaptive significance. The primitive extratesticular duct system of Chondrichthyes is described as an example of the vertebrate archetype. Adaptations of the duct system in higher vertebrates have involved a loss of some structures and specialization of others. The duct system probably evolved as a homeostatic mechanism to facilitate fertilization and some embryological development under conditions protected from the external environment. However, it is argued that the ducts also play an important role in the competition between males to achieve paternity. In vertebrates that practise internal fertilization the ducts are involved in post-testicular maturation and storage of spermatozoa. The biological significance of post-testicular sperm maturation has not been resolved. By contrast, sperm storage is essential in most male vertebrates because of the slow rate of spermatogenesis, particularly in ectotherms. Sperm storage is also important in the competition between males for paternity as it enables a male to mate a 'partner' a number of times during an oestrus in order to reduce the prospect of being cuckolded by another male. The extent of sperm maturation and storage in the epididymis of particular vertebrates depends on the relative roles of the testis and its extragonadal ducts in the competition between males for paternity. These roles depend on a number of factors, including allometric limitations to testis size, metabolic rate and the development of endothermy, and the reproductive strategy of females of the species.

  17. Sperm quality evaluation in Solea senegalensis during the reproductive season at cellular level.

    PubMed

    Beirão, J; Soares, F; Herráez, M P; Dinis, M T; Cabrita, E

    2009-12-01

    Sperm quality seems to be one of the reasons for the reproduction constraints faced by Senegalese sole (Solea senegalensis) aquaculturists. Previous studies in this species indicated that the sperm quality of individuals kept in culture varies throughout the year and that different sperm subpopulations can be identified in ejaculates according to the motility pattern of spermatozoa. Aiming to better understand factors affecting sole sperm quality in captivity, sperm of 11 males was assessed during the reproductive season using different parameters: motility characteristics using CASA analysis; cell plasma membrane resistance to seawater hyperosmolarity; DNA fragmentation with single-cell gel electrophoresis; and early apoptosis, labeled with Annexin-V FITC. Computer-assisted sperm analyses motility data were treated using multivariate analysis to identify the presence of different spermatozoa subpopulations according to their motility pattern. Four distinct sperm subpopulations were obtained: Subpop1, which includes fast linear spermatozoa; Subpop2, made up of fast nonlinear spermatozoa; Subpop3, which includes slow linear spermatozoa; and Subpop4, which contains slow nonlinear spermatozoa. The sperm subpopulation structure varied with time after activation and with male. Low cell resistance to the seawater hyperosmotic conditions was noticed. The Annexin-V assay allowed the identification of an apoptotic population ranging from 6% to 20%. A high percentage of cells (64.1%) showed a DNA fragmentation level below 30%, but these values varied significantly between males. DNA fragmentation appears to be related to cell membrane resistance to hyperosmotic conditions faced by the cells when in contact with seawater. This condition seems to modulate the composition of the motile sperm population and performance after activation. This phenomenon could be related to the spermatozoa maturation process.

  18. Oocyte Activation and Fertilisation: Crucial Contributors from the Sperm and Oocyte.

    PubMed

    Yeste, Marc; Jones, Celine; Amdani, Siti Nornadhirah; Coward, Kevin

    2017-01-01

    This chapter intends to summarise the importance of sperm- and oocyte-derived factors in the processes of sperm-oocyte binding and oocyte activation. First, we describe the initial interaction between sperm and the zona pellucida, with particular regard to acrosome exocytosis. We then describe how sperm and oocyte membranes fuse, with special reference to the discovery of the sperm protein IZUMO1 and its interaction with the oocyte membrane receptor JUNO. We then focus specifically upon oocyte activation, the fundamental process by which the oocyte is alleviated from metaphase II arrest by a sperm-soluble factor. The identity of this sperm factor has been the source of much debate recently, although mounting evidence, from several different laboratories, provides strong support for phospholipase C ζ (PLCζ), a sperm-specific phospholipase. Herein, we discuss the evidence in support of PLCζ and evaluate the potential role of other candidate proteins, such as post-acrosomal WW-binding domain protein (PAWP/WBP2NL). Since the cascade of downstream events triggered by the sperm-borne oocyte activation factor heavily relies upon specialised cellular machinery within the oocyte, we also discuss the critical role of oocyte-borne factors, such as the inositol trisphosphate receptor (IP3R), protein kinase C (PKC), store-operated calcium entry (SOCE) and calcium/calmodulin-dependent protein kinase II (CaMKII), during the process of oocyte activation. In order to place the implications of these various factors and processes into a clinical context, we proceed to describe their potential association with oocyte activation failure and discuss how clinical techniques such as the in vitro maturation of oocytes may affect oocyte activation ability. Finally, we contemplate the role of artificial oocyte activating agents in the clinical rescue of oocyte activation deficiency and discuss options for more endogenous alternatives.

  19. Sperm competition games: the risk model can generate higher sperm allocation to virgin females.

    PubMed

    Ball, M A; Parker, G A

    2007-03-01

    We examine the risk model in sperm competition games for cases where female fertility increases significantly with sperm numbers (sperm limitation). Without sperm competition, sperm allocation increases with sperm limitation. We define 'average risk' as the probability q that females in the population mate twice, and 'perceived risk' as the information males gain about the sperm competition probability with individual females. If males obtain no information from individual females, sperm numbers increase with q unless sperm limitation is high and one of the two competing ejaculates is strongly disfavoured. If males can distinguish between virgin and mated females, greater sperm allocation to virgins is favoured by high sperm limitation, high q, and by the second male's ejaculate being disfavoured. With high sperm limitation, sperm allocation to virgins increases and to mated females decreases with q at high q levels. With perfect information about female mating pattern, sperm allocation (i) to virgins that will mate again exceeds that to mated females and to virgins that will mate only once, (ii) to virgins that mate only once exceeds that for mated females if q is high and there is high second male disadvantage and (iii) to each type of female can decrease with q if sperm limitation is high, although the average allocation increases at least across low q levels. In general, higher sperm allocation to virgins is favoured by: strong disadvantage to the second ejaculate, high sperm limitation, high average risk and increased information (perceived risk). These conditions may apply in a few species, especially spiders.

  20. Correlation between sperm ultrastructure in infertile patients with abnormal sperm morphology and DNA damage.

    PubMed

    He, M; Tan, L

    2015-12-15

    This study explored the correlation between sperm ultrastructure in infertile patients with abnormal sperm morphology and DNA damage. Three unusual sperm morphologies were selected for the experimental group namely case 1 (95% headless sperm), case 2 (98% headless sperm), and case 3 (100% headless sperm), and the control group consisted of 2 subjects (20 and 15% headless sperm). For case 1, the patient was negative for sexually transmitted diseases and had normal semen plasma biochemistry, reproductive hormones, peripheral blood chromosomes, and azoospermia factor (AZF). The aneuploid rate of sperm chromosomes was 0.6%, and DNA damage index of sperm nuclei was 84.4%. The partner of this patient did not get pregnant after artificial reproductive technology assistance. For case 2, the aneuploid rate of sperm chromosomes was 0.8% and DNA damage index of sperm nuclei was 95%. This patient and his spouse did not choose assisted reproduction. For case 3, reproductive hormones, peripheral blood chromosomes and AZF were normal and the aneuploid rate of sperm chromosomes was 0.2%. The wife of this patient gave birth to a healthy baby after ova removal, fertilization and transplantation. For the control group, the aneuploid rate of sperm chromosomes and DNA damage index of sperm nuclei were approximately 0.3 and 30%, respectively. To sum up, sperm ultrastructure of infertile patients suffering from unusual sperm morphology is associated with DNA damage to some extent and can cause infertility. However, pregnancy is still possible through intracytoplasmic sperm injection.

  1. Influence of sperm pretreatment on the efficiency of intracytoplasmic sperm injection in pigs.

    PubMed

    García-Roselló, Empar; Matás, Carmen; Cánovas, Sebastián; Moreira, Pedro N; Gadea, Joaquín; Coy, Pilar

    2006-01-01

    The purpose of this study was to determine the influence of sperm pretreatment on the efficiency of intracytoplasmic sperm injection (ICSI) in pigs. This was done by examining the effect of 1) the conservation method (fresh vs frozen); 2) the sperm treatment preinjection (resuspension in Dulbecco phosphate-buffered saline (DPBS) vs selection by a Percoll gradient); and 3) the acrosomal and live or dead status of the spermatozoa (by incubation with or without calcium ionophore, 1 muM and 5 muM). In vitro matured porcine oocytes were injected with treated spermatozoa according to each experiment. All the experiments were done with non-artificially activated oocytes. The percentages of activation and cleavage were higher (68% vs 43% and 63% vs 43%, respectively, P < .05) in oocytes injected with fresh vs frozen spermatozoa. The DPBS treatment allowed higher cleavage proportions than the Percoll treatment (P < .05). Moreover, a boar effect was observed in the percentage of developing blastocysts. None of the studied parameters was affected by the acrosomal or the live or dead status of the spermatozoa injected. In conclusion, the use of fresh semen is recommended for porcine ICSI, as well as careful selection of the boar; Percoll treatment is only recommended for poor-quality samples or for removing toxic agents, and no exogenous form of activation or induction of the acrosome reaction is necessary for porcine oocytes to develop a male pronucleus and cleave up to the 2-cell stage after ICSI, although experimental conditions to reach the blastocyst stage need to be investigated further.

  2. Functional nonequivalence of sperm in Drosophila pseudoobscura.

    PubMed Central

    Snook, R R; Markow, T A; Karr, T L

    1994-01-01

    We report on a form of sperm polymorphism, termed polymegaly, that occurs in species of the Drosophila obscura group. Individual males of species in this group characteristically produce more than one discrete length of nucleated, motile sperm. Hypotheses suggested to explain the evolutionary significance of sperm polymorphism have been either nonadaptive or adaptive, with the latter focusing on sperm competition or nutrient provisioning. These hypotheses assume all sperm types fertilize eggs; however, no data have been gathered to test this assumption. We found that two size classes of sperm are produced and transferred to females in approximately equal numbers by the male; only long sperm persist in significant numbers in female sperm storage organs. Furthermore, we used a DNA-specific dye (bisbenzimide) and sperm-specific antibodies to ask if both sperm types fertilize eggs in Drosophila pseudoobscura. Confocal microscopy and immunofluorescent analyses of fertilized eggs using anti-sperm polyclonal antisera demonstrated that only long sperm participate in fertilization. These data falsify those hypotheses in which all sperm types are assumed to be functionally equivalent (fertilize eggs). Any remaining or new hypotheses for the evolutionary significance of polymegaly must incorporate these findings. Several new areas of research are suggested. Images PMID:7972038

  3. The heterotrimeric motor protein kinesin-II localizes to the midpiece and flagellum of sea urchin and sand dollar sperm.

    PubMed

    Henson, J H; Cole, D G; Roesener, C D; Capuano, S; Mendola, R J; Scholey, J M

    1997-01-01

    We have utilized immunoblotting and light microscopic immunofluorescent staining methods to examine the expression and localization of sea urchin kinesin-II, a heterotrimeric plus end-directed microtubule motor protein (previously referred to as KRP(85/95)), in sea urchin and sand dollar sperm. We demonstrate the presence of the 85 K and 115 K subunits of kinesin-II in sperm and localize these proteins to the sperm flagella and midpiece. The kinesin-II localization pattern is punctate and discontinuous, and in the flagella it is quite distinct from the continuous labeling present in sperm labeled with anti-flagellar dynein. The kinesin-II staining is largely insensitive to prefixation detergent extraction, suggesting that it is not associated with membranous elements in the sperm. In the midpiece the kinesin-II staining is similar to the pattern present in sperm labeled with an anti-centrosomal antibody. To our knowledge, this is the first localization of kinesin-like proteins in mature sperm and corroborates the recent identification and localization of kinesin-like proteins in the flagella and basal body of the unicellular green alga Chlamydomonas. We hypothesize that kinesin-II in the sperm may play functional roles in intraflagellar transport and/or the formation of flagella during spermatogenesis.

  4. Sperm cryopreservation of the Indian major carp, Labeo calbasu: effects of cryoprotectants, cooling rates and thawing rates on egg fertilization.

    PubMed

    Nahiduzzaman, Md; Hassan, Md Mahbubul; Roy, Pankoz Kumar; Hossain, Md Akhtar; Hossain, Mostafa Ali Reza; Tiersch, Terrence R

    2012-12-01

    A sperm cryopreservation protocol for the Indian major carp, Labeo calbasu, was developed for long-term preservation and artificial fertilization. Milt collected from mature male fish were placed in Alsever's solution (296mOsmolkg(-1)) to immobilize the sperm. Cryoprotectant toxicity was evaluated by motility assessment with dimethyl sulfoxide (DMSO) and methanol at 5, 10 and 15% concentrations. DMSO was more toxic at higher concentrations than methanol, and consequently 15% DMSO was excluded from further study. A one-step cooling protocol (from 5 to 80°C) with two cooling rates (5 and 10°C/min) was carried out in a computer-controlled freezer (FREEZE CONTROL(®) CL-3300; Australia). Based on post-thaw motility, the 10°C/min cooling rate with either 10% DMSO or 10% methanol yielded significantly higher (P=0.011) post-thaw motility than the other rate and cryoprotectant concentrations. Sperm thawed at 40°C for 15s and fresh sperm were used to fertilize freshly collected L. calbasu eggs and significant differences were observed (P=0.001) in percent fertilization between cryopreserved and fresh sperm as well as among different sperm-to-egg ratios (P=0.001). The highest fertilization and hatching rates were observed for thawed sperm at a sperm-to-egg ratio of 4.1×10(5):1. The cryopreservation protocol developed can facilitate hatchery operations and long-term conservation of genetic resources of L. calbasu.

  5. Sperm chromatin structure assay (SCSA®).

    PubMed

    Evenson, Donald P

    2013-01-01

    The SCSA(®) is the pioneering assay for the detection of damaged sperm DNA and altered proteins in sperm nuclei via flow cytometry of acridine orange (AO) stained sperm. The SCSA(®) is considered to be the most precise and repeatable test providing very unique, dual parameter data (red vs. green fluorescence) on a 1,024 × 1,024 channel scale, not only on DNA fragmentation but also on abnormal sperm characterized by lack of normal exchange of histones to protamines. Raw semen/sperm aliquots or purified sperm can be flash frozen, placed in a box with dry ice and shipped by overnight courier to an experienced SCSA(®) lab. The samples are individually thawed, prepared, and analyzed in ∼10 min. Of significance, data on 5,000 individual sperm are recorded on a 1,024 × 1,024 dot plot of green (native DNA) and red (broken DNA) fluorescence. Repeat measurements have virtually identical dot plot patterns demonstrating that the low pH treatment that opens up the DNA strands at the sites of breaks and staining by acridine orange (AO) are highly precise and repeatable (CVs of 1-3%) and the same between fresh and frozen samples. SCSAsoft(®) software transforms the X-Y data to total DNA stainability versus red/red + green fluoresence (DFI) providing a more accurate determination of % DFI as well as the more sensitive value of standard deviation of DFI (SD DFI) as demonstrated by animal fertility and dose-response toxicology studies. The current established clinical threshold is 25% DFI for placing a man into a statistical probability of the following: (a) longer time to natural pregnancy, (b) low odds of IUI pregnancy, (c) more miscarriages, or (d) no pregnancy. Changes in lifestyle as well as medical intervention can lower the %DFI to increase the probability of natural pregnancy. Couples of men with >25% DFI are counseled to try ICSI and when in the >50% range may consider TESE/ICSI. The SCSA(®) simultaneously determines the % of sperm with high DNA stainability (%HDS

  6. No evidence of sperm conjugate formation in an Australian mouse bearing sperm with three hooks

    PubMed Central

    Firman, Renée C; Bentley, Blair; Bowman, Faye; Marchant, Fernando García-Solís; Parthenay, Jahmila; Sawyer, Jessica; Stewart, Tom; O'Shea, James E

    2013-01-01

    Sperm conjugation occurs when two or more sperm physically unite for motility or transport through the female reproductive tract. In many muroid rodent species, sperm conjugates have been shown to form by a single, conspicuous apical hook located on the sperm head. These sperm “trains” have been reported to be highly variable in size and, despite all the heads pointing in roughly the same direction, exhibit a relatively disordered arrangement. In some species, sperm “trains” have been shown to enhance sperm swimming speed, and thus have been suggested to be advantageous in sperm competition. Here, we assessed the behavior of sperm in the sandy inland mouse (Pseudomys hermannsburgensis), a muroid rodent that bears sperm with three apical hooks. First, we accrued genetic evidence of multiple paternity within “wild” litters to unequivocally show that sperm competition does occur in this species. Following this we utilized both in vitro and in vivo methodologies to determine whether sandy inland mouse sperm conjugate to form motile trains. Our observations of in vitro preparations of active sperm revealed that sandy inland mouse sperm exhibit rapid, progressive motility as individual cells only. Similarly, histological sections of the reproductive tracts of mated females revealed no in vivo evidence of sperm conjugate formation. We conclude that the unique, three-hooked morphology of the sandy inland mouse sperm does not facilitate the formation of motile conjugates, and discuss our findings in relation to the different hypotheses for the evolution of the muroid rodent hook/s. PMID:23919134

  7. K+ and Cl− Channels and Transporters in Sperm Function

    PubMed Central

    Santi, C.M.; Orta, G.; Salkoff, L.

    2013-01-01

    To succeed in fertilization, spermatozoa must decode environmental cues which require a set of ion channels. Recent findings have revealed that K+ and Cl− channels participate in some of the main sperm functions. This work reviews the evidence indicating the involvement of K+ and Cl− channels in motility, maturation, and the acrosome reaction, and the advancement in identifying their molecular identity and modes of regulation. Improving our insight on how these channels operate will strengthen our ability to surmount some infertility problems, improve animal breeding, preserve biodiversity, and develop selective and secure male contraceptives. PMID:23287041

  8. Capacitation inducers act through diverse intracellular mechanisms in cryopreserved bovine sperm.

    PubMed

    Breininger, E; Cetica, P D; Beconi, M T

    2010-10-01

    The effect of various capacitation inducers, i.e. heparin, superoxide anion, bicarbonate, adenosine, and caffeine, and their role in intracellular mechanisms involved in capacitation, were studied in cryopreserved bovine sperm. Capacitation was determined by epifluorescence chlortetracycline, protein tyrosine phosphorylation, and the ability of capacitated sperm to undergo an acrosome reaction and fertilize in vitro matured oocytes. Participation of membrane adenylate cyclase and protein kinases (protein kinase A, protein kinase C, and protein tyrosine kinase) was evaluated indirectly (with specific inhibitors). Involvement of reactive oxygen species (ROS) was determined with scavengers of superoxide anion, hydrogen peroxide, or nitric oxide. Percentages of capacitated (27-29%) and acrosome-reacted sperm (23-26%) did not differ (P > 0.05) among various capacitation inducers. Significantly higher rates of IVF were obtained with heparin (43%) or bicarbonate plus caffeine (45%), when compared with control samples (17%). Adding the membrane adenylate cyclase inhibitor diminished capacitation rates with heparin (8%) or adenosine (10%). There was differential protein kinase participation in response to inducers; protein kinase inhibitors diminished cleavage rates in heparin-capacitated sperm relative to controls. There were differences between and within the studied inducers in protein tyrosine phosphorylation patterns. We inferred that capacitation in cryopreserved bovine sperm was promoted through diverse pathways. Mechanisms triggered by heparin, or caffeine plus bicarbonate-induced capacitation, involved activation of intracellular pathways to optimize fertilizing capability of cryopreserved bovine sperm.

  9. Easy sperm processing technique allowing exclusive accumulation and later usage of DNA-strandbreak-free spermatozoa.

    PubMed

    Ebner, T; Shebl, O; Moser, M; Mayer, R B; Arzt, W; Tews, G

    2011-01-01

    Sperm DNA fragmentation is increased in poor-quality semen samples and correlates with failed fertilization, impaired preimplantation development and reduced pregnancy outcome. Common sperm preparation techniques may reduce the percentage of strandbreak-positive spermatozoa, but, to date, there is no reliable approach to exclusively accumulate strandbreak-free spermatozoa. To analyse the efficiency of special sperm selection chambers (Zech-selectors made of glass or polyethylene) in terms of strandbreak reduction, 39 subfertile men were recruited and three probes (native, density gradient and Zech-selector) were used to check for strand breaks using the sperm chromatin dispersion test. The mean percentage of affected spermatozoa in the ejaculate was 15.8 ± 7.8% (range 5.0–42.1%). Density gradient did not significantly improve the quality of spermatozoa selected(14.2 ± 7.0%). However, glass chambers completely removed 90% spermatozoa showing strand breaks and polyethylene chambers removed 76%. Both types of Zech-selectors were equivalent in their efficiency, significantly reduced DNA damage (P < 0.001) and,with respect to this, performed better than density gradient centrifugation (P < 0.001). As far as is known, this is the first report ona sperm preparation technique concentrating spermatozoa unaffected in terms of DNA damage. The special chambers most probably select for sperm motility and/or maturity.

  10. Low physiological levels of prostaglandins E2 and F2α improve human sperm functions.

    PubMed

    Rios, Mariana; Carreño, Daniela V; Oses, Carolina; Barrera, Nelson; Kerr, Bredford; Villalón, Manuel

    2016-03-01

    Prostaglandins (PGs) have been reported to be present in the seminal fluid and cervical mucus, affecting different stages of sperm maturation from spermatogenesis to the acrosome reaction. This study assessed the effects of low physiological PGE2 and PGF2α concentrations on human sperm motility and on the ability of the spermatozoa to bind to the zona pellucida (ZP). Human spermatozoa were isolated from seminal samples with normal concentration and motility parameters and incubated with 1μM PGE2, 1μM PGF2α or control solution to determine sperm motility and the ability to bind to human ZP. The effects of both PGs on intracellular calcium levels were determined. Incubation for 2 or 18h with PGE2 or PGF2α resulted in a significant (P<0.05) increase in the percentage of spermatozoa with progressive motility. In contrast with PGF2α, PGE2 alone induced an increase in sperm intracellular calcium levels; however, the percentage of sperm bound to the human ZP was doubled for both PGs. These results indicate that incubation of human spermatozoa with low physiological levels of PGE2 or PGF2α increases sperm functions and could improve conditions for assisted reproduction protocols.

  11. Bioenergetics of Mammalian Sperm Capacitation

    PubMed Central

    Ferramosca, Alessandra; Zara, Vincenzo

    2014-01-01

    After ejaculation, the mammalian male gamete must undergo the capacitation process, which is a prerequisite for egg fertilization. The bioenergetics of sperm capacitation is poorly understood despite its fundamental role in sustaining the biochemical and molecular events occurring during gamete activation. Glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) are the two major metabolic pathways producing ATP which is the primary source of energy for spermatozoa. Since recent data suggest that spermatozoa have the ability to use different metabolic substrates, the main aim of this work is to present a broad overview of the current knowledge on the energy-producing metabolic pathways operating inside sperm mitochondria during capacitation in different mammalian species. Metabolism of glucose and of other energetic substrates, such as pyruvate, lactate, and citrate, is critically analyzed. Such knowledge, besides its obvious importance for basic science, could eventually translate into the development of novel strategies for treatment of male infertility, artificial reproduction, and sperm selection methods. PMID:24791005

  12. Human sperm rheotaxis: a passive physical process

    PubMed Central

    Zhang, Zhuoran; Liu, Jun; Meriano, Jim; Ru, Changhai; Xie, Shaorong; Luo, Jun; Sun, Yu

    2016-01-01

    A long-standing question in natural reproduction is how mammalian sperm navigate inside female reproductive tract and finally reach the egg cell, or oocyte. Recently, fluid flow was proposed as a long–range guidance cue for sperm navigation. Coitus induces fluid flow from oviduct to uterus, and sperm align themselves against the flow direction and swim upstream, a phenomenon termed rheotaxis. Whether sperm rheotaxis is a passive process dominated by fluid mechanics, or sperm actively sense and adapt to fluid flow remains controversial. Here we report the first quantitative study of sperm flagellar motion during human sperm rheotaxis and provide direct evidence indicating that sperm rheotaxis is a passive process. Experimental results show that there is no significant difference in flagellar beating amplitude and asymmetry between rheotaxis-turning sperm and those sperm swimming freely in the absence of fluid flow. Additionally, fluorescence image tracking shows no Ca2+ influx during sperm rheotaxis turning, further suggesting there is no active signal transduction during human sperm rheotaxis. PMID:27005727

  13. SPERM COUNT DISTRIBUTIONS IN FERTILE MEN

    EPA Science Inventory

    Sperm concentration and count are often used as indicators of environmental impacts on male reproductive health. Existing clinical databases may be biased towards subfertile men with low sperm counts and less is known about expected sperm count distributions in cohorts of fertil...

  14. Human sperm rheotaxis: a passive physical process

    NASA Astrophysics Data System (ADS)

    Zhang, Zhuoran; Liu, Jun; Meriano, Jim; Ru, Changhai; Xie, Shaorong; Luo, Jun; Sun, Yu

    2016-03-01

    A long-standing question in natural reproduction is how mammalian sperm navigate inside female reproductive tract and finally reach the egg cell, or oocyte. Recently, fluid flow was proposed as a long–range guidance cue for sperm navigation. Coitus induces fluid flow from oviduct to uterus, and sperm align themselves against the flow direction and swim upstream, a phenomenon termed rheotaxis. Whether sperm rheotaxis is a passive process dominated by fluid mechanics, or sperm actively sense and adapt to fluid flow remains controversial. Here we report the first quantitative study of sperm flagellar motion during human sperm rheotaxis and provide direct evidence indicating that sperm rheotaxis is a passive process. Experimental results show that there is no significant difference in flagellar beating amplitude and asymmetry between rheotaxis-turning sperm and those sperm swimming freely in the absence of fluid flow. Additionally, fluorescence image tracking shows no Ca2+ influx during sperm rheotaxis turning, further suggesting there is no active signal transduction during human sperm rheotaxis.

  15. Roles of the reproductive tract in modifications of the sperm membrane surface

    PubMed Central

    KUO, Yu-Wen; LI, Sheng-Hsiang; MAEDA, Kei-Ichiro; GADELLA, Bart M.; TSAI, Pei Shiue J.

    2016-01-01

    Successful fertilization requires viable and functional spermatozoa to recognize and fuse with the oocyte. In most mammalian species, mature spermatozoa are not capable of fertilizing the oocytes immediately after ejaculation. However, unlike somatic cells, spermatozoa, after leaving the testis, are transcriptionally and translationally silent; therefore, upon completion of spermiogenesis, spermatozoa carry only a minimal amount of essential proteins on their membranes as well as within their restricted volume of cytoplasm. To develop into a fully functional and competent sperm that is capable of successful fertilization, modifications of the sperm membrane surface during its transit in the reproductive tracts is critical. These post-spermatogenesis modifications advance the maturation of epididymal spermatozoa. In addition, components secreted into the lumen of the reproductive tracts that are later added onto the sperm membrane surface also regulate (inhibit or activate) the functions of the spermatozoa. This acquisition of additional proteins from the reproductive tracts may compensate for the inactivity of morphologically mature spermatozoa. In this review, we discuss the contributions of the male and female genital tracts to modifications of the sperm membrane surface at different stages of fertilization. PMID:27009019

  16. Spermiogenesis and Taxonomical Values of Sperm Ultrastructures in Male Crassostrea ariakensis (Fujita & Wakiya, 1929) (Pteroirmorphia: Ostreidae) in the Estuary of the Seomjin River, Korea

    PubMed Central

    Son, Pal Won; Chung, Jae Seung; Kim, Jin Hee; Kim, Sung Han; Chung, Ee-Yung

    2014-01-01

    Characteristics of the developmental stages of spermatids during spermiogenesis and phylogenetic classicfication of the species using sperm ultrastructures in male Crassostrea ariakensis were investigated by transmission electron microscope observations. The morphology of the spermatozoon of this species has a primitive type and is similar to those of Ostreidae. Ultrastructures of mature sperms are composed of broad, modified cap-shaped acrosomal vesicle and an axial rod in subacrosomal materials on an oval nucleus, four spherical mitochondria in the sperm midpiece, and satellite fibres which appear near the distal centriole. The axoneme of the sperm tail shows a 9+2 structure. Accordingly, the ultrastructural characteristics of mature sperm of C. ariakensis resemble to those of other investigated ostreids in Ostreidae in the subclass Pteriomorphia. In this study, particularly, two transverse bands (stripes) appear at the anterior region of the acrosomal vesicle of this species, unlike two or three transverse bands (stripes) in C. gigas. It is assumed that differences in this acrosomal substructure are associated with the inability of fertilization between the genus Crassostrea and other genus species in Ostreidae. Therefore, we can use sperm ultrastructures and morphologies in the resolution of taxonomic relationships within the Ostreidae in the subclass Pteriomorphia. These spermatozoa, which contain several ultrastructures such as acrosomal vesicle, an axial rod in the sperm head part and four mitochondria and satellite fibres in the sperm midpiece, belong to the family Ostreidae in the subclass Pteriomorphia. PMID:25949188

  17. Intracytoplasmic Sperm Injection Outcomes with Freshly Ejaculated Sperms and Testicular or Epididymal Sperm Extraction in Patients with Idiopathic Cryptozoospermia

    PubMed Central

    Ketabchi, Ali Asghar

    2016-01-01

    Background Cryptozoospermia (CO) is a situation in which spermatozoa cannot be observed in a fresh semen sample unless an extended centrifugation and microscopic search are performed. CO patients are suggested to use only intracytoplasmic sperm injection (ICSI) as infertility treatment. But still there is debate about the choice of sperm source in cryptozoospermic men candidate for ICSI. Objectives This study was conducted to evaluate fertility outcomes in men with idiopathic cryptozoospermia who were treated using ICSI with freshly ejaculated sperm and testis sperm extraction (TESE) or percutaneous epididymal sperm aspiration (PESA). Methods In this prospective cohort study carried out in an academic institution, 83 out of 92 couples with cryptozoospermia undergoing their first ICSI cycle were recruited. These patients were randomly allocated to two groups: group one (n = 42) who produced freshly ejaculated sperm and, group two (n = 41) who produced a sample by TESE or PESA. The groups were analyzed and compared in terms of fertilization rate, cleavage rate, embryo quality, implantation rate, and clinical pregnancy rate. Results There was a significant difference in fertilization rate, embryo quality, implantation rate, and pregnancy rates between the group of surgically extracted sperm and those of naturally ejaculated sperm using conventional ICSI (P < 0.05). Conclusions Sperm quality extracted by percutaneous PESA and TESE procedures increases fertility outcomes compared to naturally ejaculated sperm in men with idiopathic CO. More specifically, embryo quality, which is most relevant to fertility outcome, improved when surgically extracted sperm was used for ICSI. PMID:27896242

  18. Zn2+-stimulation of sperm capacitation and of the acrosome reaction is mediated by EGFR activation.

    PubMed

    Michailov, Yulia; Ickowicz, Debbi; Breitbart, Haim

    2014-12-15

    Extracellular zinc regulates cell proliferation via the MAP1 kinase pathway in several cell types, and has been shown to act as a signaling molecule. The testis contains a relatively high concentration of Zn(2+), required in both the early and late stages of spermatogenesis. Despite the clinical significance of this ion, its role in mature sperm cells is poorly understood. In this study, we characterized the role of Zn(2+) in sperm capacitation and in the acrosome reaction. Western blot analysis revealed the presence of ZnR of the GPR39 type in sperm cells. We previously demonstrated the presence of active epidermal growth factor receptor (EGFR) in sperm, its possible transactivation by direct activation of G-protein coupled receptor (GPCR), and its involvement in sperm capacitation and in the acrosome reaction (AR). We show here that Zn(2+) activates the EGFR during sperm capacitation, which is mediated by activation of trans-membrane adenylyl cyclase (tmAC), protein kinase A (PKA), and the tyrosine kinase, Src. Moreover, the addition of Zn(2+) to capacitated sperm caused further stimulation of EGFR and phosphatydil-inositol-3-kinase (PI3K) phosphorylation, leading to the AR. The stimulation of the AR by Zn(2+) also occurred in the absence of Ca(2+) in the incubation medium, and required the tmAC, indicating that Zn(2+) activates a GPCR. The AR stimulated by Zn(2+) is mediated by GPR39 receptor, PKA, Src and the EGFR, as well as the EGFR down-stream effectors PI3K, phospholipase C (PLC) and protein kinase C (PKC). These data support a role for extracellular zinc, acting through the ZnR, in regulating multiple signaling pathways in sperm capacitation and the acrosome reaction.

  19. Birth of domestic cat kittens of predetermined sex after transfer of embryos produced by in vitro fertilization of oocytes with flow-sorted sperm.

    PubMed

    Pope, C E; Crichton, E G; Gómez, M C; Dumas, C; Dresser, B L

    2009-03-15

    Our goals were to: (1) determine if domestic cat sperm could be sorted to high purity by flow cytometry after overnight shipment of cooled samples; (2) evaluate the efficiency with which sorted sperm could be used to generate cat embryos in vitro; and (3) determine if live kittens of predetermined sex could be produced after transfer of embryos derived by IVF using sorted sperm. Semen samples (n=5) from one male were extended in electrolyte-free solution and shipped overnight at 4 degrees C to the sorting facility. Samples were adjusted to 75x10(6)sperm/mL and stained with Hoechst 33342. After 1h at 34.5 degrees C, samples were adjusted to 50x10(6)sperm/mL with 4% egg yolk TALP+0.002% food dye and sorted by high-speed flow cytometry. Later resort analysis confirmed purities of 94% and 83% for X- and Y-chromosome bearing sperm, respectively. Sorted sperm were centrifuged, re-suspended in TEST yolk buffer and shipped overnight to the IVF laboratory. After IVF of in vivo matured oocytes with X-chromosome bearing sperm, cleavage frequency was 62% (54/87). After IVF of IVM oocytes with control, X- or Y-chromosome bearing sperm, the incidence of cleavage was 42% (48/115), 33% (40/120), and 35% (52/150), respectively, and blastocyst development was 53% (21/40), 50% (11/22), and 55% (23/42), respectively (P>0.05). On Day 2, 45 embryos produced by IVF of in vivo matured oocytes with X-chromosome bearing sperm were transferred to the oviduct of four Day 1 recipients, three of which subsequently delivered litters of one, four, and seven female kittens, respectively. In conclusion, we confirmed that sperm sorting technology can be applied to domestic cats and established that kittens of predetermined sex can be produced.

  20. No evidence for sperm priming responses under varying sperm competition risk or intensity in guppies

    NASA Astrophysics Data System (ADS)

    Evans, Jonathan P.

    2009-07-01

    Sperm competition theory predicts that males should tailor their investment in ejaculates according to the number of rival males competing to fertilize a female’s eggs. Research spanning several taxa supports this prediction by showing that males are often sensitive to the level of sperm competition and adjust their investment in sperm numbers accordingly. More recent work has revealed that males may also tailor the quality of sperm according to the number of males competing for fertilization. Here I test for both effects in guppies ( Poecilia reticulata) in an experiment that simultaneously evaluates the risk and intensity models of sperm competition. The experiment determined whether male guppies adjust the number (stripped ejaculate size) and quality (sperm velocity and viability) of sperm that are primed over a 3-day period according to experimental changes in the perceived level of sperm competition. A total of 136 focal males were initially stripped of all retrievable sperm and assayed for these sperm traits before being allocated at random to one of four treatments simulating different levels of sperm competition risk and intensity. During the 3-day treatment phase, focal males had visual and olfactory access to a sexually receptive (initially virgin) female maintained with different numbers of stimulus males to simulate variation in the risk and intensity of sperm competition. Following this, males were assayed again for the sperm traits. Contrary to predictions, there was no significant change in any of the measured variables among treatments, although qualitatively the patterns for sperm velocity and viability did conform to expectation. The lack of any trend for the number of sperm primed was unequivocal and future work examining the effects of sperm competition on sperm production should focus on whether males differentially allocate sperm numbers among matings that differ in the level of sperm competition.

  1. SLC6 family transporter SNF-10 is required for protease-mediated activation of sperm motility in C. elegans

    PubMed Central

    Fenker, Kristin E.; Hansen, Angela A.; Chong, Conrad A.; Jud, Molly C.; Duffy, Brittany A.; Norton, J. Paul; Hansen, Jody M.; Stanfield, Gillian M.

    2014-01-01

    Summary Motility of sperm is crucial for their directed migration to the egg. The acquisition and modulation of motility are regulated to ensure that sperm move when and where needed, thereby promoting reproductive success. One specific example of this phenomenon occurs during differentiation of the amoeboid sperm of C. elegans as they activate from a round spermatid to a mature, crawling spermatozoon. Sperm activation is regulated by redundant pathways to occur at a specific time and place for each sex. Here, we report the identification of the solute carrier 6 (SLC6) transporter protein SNF-10 as a key regulator of C. elegans sperm activation in response to male protease activation signals. We find that SNF-10 is present in sperm and is required for activation by the male but not by the hermaphrodite. Loss of both snf-10 and a hermaphrodite activation factor render sperm completely insensitive to activation. Using in vitro assays, we find that snf-10 mutant sperm show a specific deficit in response to protease treatment but not to other activators. Prior to activation, SNF-10 is present in the plasma membrane, where it represents a strong candidate to receive signals that lead to subcellular morphogenesis. After activation, it shows polarized localization to the cell body region that is dependent on membrane fusions mediated by the dysferlin FER-1. Our discovery of snf-10 offers insight into the mechanisms differentially employed by the two sexes to accomplish the common goal of producing functional sperm, as well as how the physiology of nematode sperm may be regulated to control motility as it is in mammals. PMID:24929237

  2. Nuclear microscopy of sperm cell elemental structure

    SciTech Connect

    Bench, G.S.

    1994-12-31

    Theories have suggested that there is a link between protamine concentrations in individual sperm and sperm fertility. At present, biochemical analyses have only been performed on bulk populations and existing methods have not been able to determine what percentage of morphologically normal sperm are biochemically defective and potentially infertile. As part of an investigation into male sperm fertility, nuclear microscopy has been utilized to measure elemental profiles at the single sperm level. By measuring the ratio of Phosphorus to Sulfur the authors have been able to determine the amount of protamine 1 and protamine 2 in individual cells from bulk fertile samples of bull and mouse sperm. Preliminary results show that, for each species, the relative amounts of protamine 1 and protamine 2 in morphologically normal sperm agree well with expected values.

  3. Sperm in "parhenogenetic" freshwater gastrotrichs.

    PubMed

    Weiss, M J; Levy, D P

    1979-07-20

    Freshwater members of the phylum Gastrotricha have been considered obligate parthenogens. In Lepidodermelia squammata, the species for which there is most evidence for parthenogenesis, sperm have been discovered. This finding will necessitate reexamination of the nature of sexuality and life cycles and of the concept of "species" in freshwater gastrotrichs.

  4. Similarities and differences between the sperm quality index and sperm mobility index of broiler breeder semen.

    PubMed

    Dumpala, P R; Parker, H M; McDaniel, C D

    2006-12-01

    The sperm quality index (SQI) and sperm mobility index (SMI) both predict sperm motility. Previous research was conducted comparing the SMI with the SQI; however, semen was diluted improperly for the SQI (40-fold). For an accurate comparison, semen must be diluted 10-fold for the SQI. Therefore, the overall objective of this study, utilizing 4 experiments, was to examine the relationship of the SQI with the SMI when: 1) active, heat-inactivated, and boiled sperm were incubated and analyzed over 60 min, 2) motile and immotile sperm were combined, 3) dead sperm (boiled or frozen) and live sperm were combined, and 4) sperm were rendered immotile by adding Zn. In experiment 1, the SQI was stable throughout incubation for active sperm and zero for heat-inactivated and boiled sperm. The SMI from active sperm steadily increased over incubation. However, the SMI from heat-inactivated sperm increased drastically resulting in values higher than active sperm after 35 min of incubation. The SMI from active sperm was higher than boiled sperm throughout incubation. For experiment 2, a cubic increase in the SQI occurred as active sperm increased from 0 to 100%. However, there was no difference in SMI readings until samples contained 80% active sperm. In experiment 3, for both boiled and frozen sperm, as the percentage of viable sperm increased, there was a logarithmic increase in the SQI. The SMI values were similar for 0 and 100% viable sperm when using boiled sperm, and all SMI readings were similar to 0% viable for frozen sperm. For experiment 4, both the SQI and SMI values from semen incubated with Zn were lower than that of saline. The SQI from sperm incubated in saline was higher than that of Zn throughout incubation, yet there were no differences in SMI values after 90 min of incubation. In conclusion, both the SQI and SMI of sperm decrease in response to Zn. However, it appears that immotile and dead sperm are capable of increasing SMI values but not SQI values.

  5. Sperm competition promotes diversity of sperm bundles in Ohomopterus ground beetles

    NASA Astrophysics Data System (ADS)

    Takami, Yasuoki; Sota, Teiji

    2007-07-01

    Diversification of sperm morphology has been investigated in the context of sperm competition, but the adaptive significance of sperm bundles is still unclear. In analyzing 10 taxa of the genus Carabus subgenus Ohomopterus and one related Carabus ground beetles, we found that dimorphic sperm bundles occurred in most species with varied degrees of bimodality, whereas sperm were generally monomorphic. Comparative analyses with phylogenetically independent contrasts revealed that the sizes of large and small sperm bundles evolved more rapidly than, and were not correlated with, the length of sperm, suggesting more intense selection on sperm bundle sizes and their independent responses to different evolutionary forces. The size of large sperm bundles was positively correlated with male genital morphology (pertinent to displacement of rival spermatophores) and postcopulatory guarding duration as well as male body length, suggesting that larger sperm bundles have been favored when the risk of spermatophore displacement is high. Larger sperm bundles may be advantageous because of their ability to migrate more rapidly into the spermatheca. In contrast, no clear association was detected between the small sperm bundle size and mating traits despite its rapid diversification. The present study provides the first record of heteromorphic sperm bundles, the diversity of which may be promoted by sperm competition.

  6. Phenotypic engineering of sperm-production rate confirms evolutionary predictions of sperm competition theory

    PubMed Central

    Sekii, Kiyono; Vizoso, Dita B.; Kuales, Georg; De Mulder, Katrien; Ladurner, Peter; Schärer, Lukas

    2013-01-01

    Sperm production is a key male reproductive trait and an important parameter in sperm competition theory. Under sperm competition, paternity success is predicted to depend strongly on male allocation to sperm production. Furthermore, because sperm production is inherently costly, individuals should economize in sperm expenditure, and conditional adjustment of the copulation frequency according to their sperm availability may be expected. However, experimental studies showing effects of sperm production on mating behaviour and paternity success have so far been scarce, mainly because sperm production is difficult to manipulate directly in animals. Here, we used phenotypic engineering to manipulate sperm-production rate, by employing dose-dependent RNA interference (RNAi) of a spermatogenesis-specific gene, macbol1, in the free-living flatworm Macrostomum lignano. We demonstrate (i) that our novel dose-dependent RNAi approach allows us to induce high variability in sperm-production rate; (ii) that a reduced sperm-production rate is associated with a decreased copulation frequency, suggesting conditional adjustment of mating behaviour; and (iii) that both sperm production and copulation frequency are important determinants of paternity success in a competitive situation, as predicted by sperm competition theory. Our study clearly documents the potential of phenotypic engineering via dose-dependent RNAi to test quantitative predictions of evolutionary theory. PMID:23446521

  7. Sperm competition promotes diversity of sperm bundles in Ohomopterus ground beetles.

    PubMed

    Takami, Yasuoki; Sota, Teiji

    2007-07-01

    Diversification of sperm morphology has been investigated in the context of sperm competition, but the adaptive significance of sperm bundles is still unclear. In analyzing 10 taxa of the genus Carabus subgenus Ohomopterus and one related Carabus ground beetles, we found that dimorphic sperm bundles occurred in most species with varied degrees of bimodality, whereas sperm were generally monomorphic. Comparative analyses with phylogenetically independent contrasts revealed that the sizes of large and small sperm bundles evolved more rapidly than, and were not correlated with, the length of sperm, suggesting more intense selection on sperm bundle sizes and their independent responses to different evolutionary forces. The size of large sperm bundles was positively correlated with male genital morphology (pertinent to displacement of rival spermatophores) and postcopulatory guarding duration as well as male body length, suggesting that larger sperm bundles have been favored when the risk of spermatophore displacement is high. Larger sperm bundles may be advantageous because of their ability to migrate more rapidly into the spermatheca. In contrast, no clear association was detected between the small sperm bundle size and mating traits despite its rapid diversification. The present study provides the first record of heteromorphic sperm bundles, the diversity of which may be promoted by sperm competition.

  8. Why small males have big sperm: dimorphic squid sperm linked to alternative mating behaviours

    PubMed Central

    2011-01-01

    Background Sperm cells are the target of strong sexual selection that may drive changes in sperm structure and function to maximize fertilisation success. Sperm evolution is regarded to be one of the major consequences of sperm competition in polyandrous species, however it can also be driven by adaptation to the environmental conditions at the site of fertilization. Strong stabilizing selection limits intra-specific variation, and therefore polymorphism, among fertile sperm (eusperm). Here we analyzed reproductive morphology differences among males employing characteristic alternative mating behaviours, and so potentially different conditions of sperm competition and fertilization environment, in the squid Loligo bleekeri. Results Large consort males transfer smaller (average total length = 73 μm) sperm to a female's internal sperm storage location, inside the oviduct; whereas small sneaker males transfer larger (99 μm) sperm to an external location around the seminal receptacle near the mouth. No significant difference in swimming speed was observed between consort and sneaker sperm. Furthermore, sperm precedence in the seminal receptacle was not biased toward longer sperm, suggesting no evidence for large sperm being favoured in competition for space in the sperm storage organ among sneaker males. Conclusions Here we report the first case, in the squid Loligo bleekeri, where distinctly dimorphic eusperm are produced by different sized males that employ alternative mating behaviours. Our results found no evidence that the distinct sperm dimorphism was driven by between- and within-tactic sperm competition. We propose that presence of alternative fertilization environments with distinct characteristics (i.e. internal or external), whether or not in combination with the effects of sperm competition, can drive the disruptive evolution of sperm size. PMID:21831296

  9. Mature Teachers Matter

    ERIC Educational Resources Information Center

    Berl, Patricia Scallan

    2005-01-01

    In this article, the author discusses the consequences of losing mature teachers due to voluntary separation or retirement and the mindset of a mature teacher that is different from younger teachers in a number of ways. Mature teachers are colleagues over 45 years of age possessing significant experience in the field. Future trends in teacher…

  10. Heterologous in vitro fertilization and sperm capacitation in an endangered African antelope, the scimitar-horned oryx (Oryx dammah).

    PubMed

    Roth, T L; Weiss, R B; Buff, J L; Bush, L M; Wildt, D E; Bush, M

    1998-02-01

    Scimitar-horned oryx sperm function was studied using protocols developed for domestic cattle. Objectives were to assess sperm 1) viability and motility in vitro over time, 2) capacitation in heparin- or calcium-supplemented medium, and 3) function in an in vitro fertilization system using heterologous (domestic cow) oocytes. Seminal aliquots were washed, and sperm were resuspended in 1) Talp with 5% fetal calf serum (TALP), 2) TALP + 10 microM heparin, 3) TALP + 20 microM heparin, and 4) TALP + 10 mM CaCl. At 0, 3, and 6 h, aliquots were evaluated for sperm motility, viability (using Hoechst 33258), and ability to acrosome-react when exposed to lysophosphatidylcholine (LC). Sperm function was assessed by evaluating fertilization and embryo development after coculture of in vitro-matured domestic cow oocytes with oryx sperm. Overall mean percentages of motile and viable sperm remained high at 6 h (> 60% and > 70%, respectively). Fewer (p < 0.05) sperm incubated in TALP + 10 microM heparin for 6 h contained intact acrosomes after exposure to LC, but there were no differences between LC and control samples after incubation in TALP without heparin. LC-treated sperm in TALP + 10 mM CaCl contained fewer (p < 0.05) intact acrosomes at 3 and 6 h (52.6% and 31.2%, respectively) than paired controls (83.6% and 70.0%, respectively). Oryx sperm from all males were capable of fertilizing cow oocytes (range 17 of 26 [65.4%] to 25 of 26 [96.2%]). Of the 55 2-cell embryos produced, 34 (61.8%) developed to > or = 8 cells. Of the 24 uncleaved oocytes, 7 (29.2%) were polyspermic. These data demonstrate that processed sperm from the endangered scimitar-horned oryx remain vigorous in vitro for at least 6 h. Capacitation can be induced using cattle sperm-processing techniques, with sperm appearing most responsive to elevated CaCl concentrations. Most interesting was the successful production and development of hybrid embryos after coincubation of oryx sperm with cow oocytes, suggesting

  11. Pregnancy rates in cattle with cryopreserved sexed sperm: effects of sperm numbers per inseminate and site of sperm deposition.

    PubMed

    Seidel, G E; Schenk, J L

    2008-04-01

    In six field trials, doses between 1.0 and 6.0 x 10(6) total sexed, frozen-thawed sperm were inseminated into the uterine body or bilaterally into the uterine horns of heifers and nursing Angus cows 12 or 24h after observed estrus. Except for one comparison in one trial in which uterine body insemination was slightly superior (P<0.05) to uterine horn insemination, there was no significant (P>0.1) difference between sites of semen deposition. Additionally, except for one small study with limited numbers, there was essentially no difference in pregnancy rates in the range between 1.5 and 6 x 10(6) sexed, frozen-thawed sperm per inseminate. Pregnancy rates with smaller doses of sexed sperm averaged about 75% of controls of 20 x 10(6) total frozen-thawed, unsexed sperm. While 1.0 x 10(6) sexed, frozen-thawed sperm per insemination dose resulted in decreased pregnancy rates compared to larger doses, the lesser fertility with sexed sperm could not be compensated by increasing sperm numbers in the range of 1.5-6 x 10(6) sperm per dose. Pregnancy rates with 2 x 10(6) sexed, frozen-thawed sperm per dose were not markedly less than control pregnancy rates with 20 x 10(6) frozen-thawed unsexed sperm/dose in well-managed herds.

  12. Lack of calcium oscillation causes failure of oocyte activation after intracytoplasmic sperm injection in pigs

    PubMed Central

    NAKAI, Michiko; ITO, Junya; SUZUKI, Shun-ichi; FUCHIMOTO, Dai-ichiro; SEMBON, Shoichiro; SUZUKI, Misae; NOGUCHI, Junko; KANEKO, Hiroyuki; ONISHI, Akira; KASHIWAZAKI, Naomi; KIKUCHI, Kazuhiro

    2016-01-01

    In pigs, the efficiency of embryo production after intracytoplasmic sperm injection (ICSI) is still low because of frequent failure of normal fertilization, which involves formation of two polar bodies and two pronuclei. To clarify the reasons for this, we hypothesized that ICSI does not properly trigger sperm-induced fertilization events, especially intracellular Ca2+ signaling, also known as Ca2+ oscillation. We also suspected that the use of in vitro-matured oocytes might negatively affect fertilization events and embryonic development of sperm-injected oocytes. Therefore, we compared the patterns of Ca2+ oscillation, the efficiency of oocyte activation and normal fertilization, and embryo development to the blastocyst stage among in vivo- or in vitro-matured oocytes after ICSI or in vitro fertilization (IVF). Unexpectedly, we found that the pattern of Ca2+ oscillation, such as the frequency and amplitude of Ca2+ rises, in oocytes after ICSI was similar to that in oocytes after IVF, irrespective of the oocyte source. However, half of the oocytes failed to become activated after ICSI and showed no Ca2+ oscillation. Moreover, the embryonic development of normal fertilized oocytes was reduced when in vitro-matured oocytes were used, irrespective of the fertilization method employed. These findings suggest that low embryo production efficiency after ICSI is attributable mainly to poor developmental ability of in vitro-matured oocytes and a lack of Ca2+ oscillation, rather than the pattern of oscillation. PMID:27725347

  13. Morphology and function of the reproductive tract of the spider crab Libinia spinosa (Crustacea, Brachyura, Majoidea): pattern of sperm storage

    NASA Astrophysics Data System (ADS)

    Sal Moyano, M. P.; Gavio, M. A.; Cuartas, E. I.

    2010-09-01

    Morphology and function of the male reproductive tract, female spermatheca and patterns of sperm storage were assessed in the crab Libinia spinosa using histological methods. Testes are characterized by the presence of peripheral spermatogonia and different sequences of sperm maturity. Spermatophores begin to be packed in the last portion. The vas deferens consists of three sections: anterior, with undeveloped spermatophores and free sperm; median, with well-developed spermatophores; and posterior with granular secretions. Female spermathecae are of the ventral type, with a velum separating dorsal and ventral chambers. Live individuals were kept in the laboratory and arranged in pairs. An experiment was conducted toward the end of the reproductive season, in which males with the right gonopod excised were placed with receptive females. After mating, females were killed and the spermathecae dissected for histological study and observation of the pattern of sperm storage. Spermatozoa were found forming discrete sperm packages. New ejaculates can fill the entire spermatheca or be restricted to the ventral chamber; sperm are rounded, with a distinguishable acrosomal core. Old ejaculates are restricted to the dorsal chamber and are of irregular shape and larger size; an acrosomal core was not distinguishable. The secretions produced by the glandular epithelium of the dorsal chamber of the spermathecae are likely to have a role in the removal of dead sperm.

  14. Phagocytosis of sperm by follicle cells of the carnivorous sponge Asbestopluma occidentalis (Porifera, Demospongiae).

    PubMed

    Riesgo, Ana

    2010-06-01

    During spermatogenesis of the carnivorous sponge Asbestopluma occidentalis, follicle cells that lined the spermatocysts phagocytosed unreleased mature sperm. Such follicle cells are part of the complex envelope that limits spermatocysts of A. occidentalis, which is also comprised of a collagen layer, a thick layer of intertwined cells, and spicules. Follicle cells showed vesicles containing single phagocytosed spermatozoa within their cytoplasm. Additionally, lipids and other inclusions were observed within the cytoplasm of follicle cells. It is likely that follicle cells recapture nutrients by phagocytosing spermatozoa and use them to form lipids and other inclusions. Such sperm phagocytosis is usually performed in higher invertebrates and vertebrates by Sertoli cells that are located in the testis wall. While Sertoli cells develop a wide range of functions such as creating a blood-testis barrier, providing crucial factors to ensure correct progression of spermatogenesis, and phagocytosis of aberrant, degenerating, and unreleased sperm cells, sponge follicle cells may only display phagocytotic activity on spermatogenic cells.

  15. Improvement of the post-thaw qualities of Okinawan native Agu pig sperm frozen in an extender supplemented with antiapoptotic PTD-FNK protein.

    PubMed

    Shimokawa, Kazuki; Oshiro, Ryuko; Yamanaka, Kenichi; Ashizawa, Koji; Ohta, Shigeo; Tatemoto, Hideki

    2012-10-15

    The technical establishment of boar sperm cryopreservation is indispensable for effective breeding of the scarce Okinawan native Agu pig. The objective was to determine whether an artificial anticell death protein (PTD-FNK protein) was capable of improving the quality of cryopreserved Agu sperm. Ejaculated Agu sperm frozen in an extender supplemented with 0, 100, 200, 300, or 400 nm PTD-FNK protein was thawed, and mitochondrial integrity and other sperm characteristics were evaluated. Treatment with 300 nm PTD-FNK protein had the most beneficial effect (P < 0.05) on mitochondrial integrity (45-59%) and sperm motility (56-67%) after freezing-thawing. In particular, the proportion of post-thaw sperm with activated caspase-9 and -3 but not caspase-8 was markedly reduced among sperm frozen in the presence of PTD-FNK protein (P < 0.05), implying protection against apoptotic-cell death in response to mitochondrial damage. There were high levels of intracellular ATP (9.4-10.5 nmol/10(8) sperm) in post-thaw sperm treated with PTD-FNK protein, and the inhibitory effect of PTD-FNK protein on activation of caspases influenced the increase in the number of sperm with intact DNA (36-53%; P < 0.05). Furthermore, the addition of PTD-FNK protein to the freezing extender strongly preserved the ability of the sperm to penetrate to mature oocytes in all individuals (60-80%; P < 0.05). In conclusion, treatment with PTD-FNK protein in the freezing extender effectively improved post-thaw qualities of fragile Agu sperm through prevention of mitochondrial dysfunction leading to apoptotic-cell death during cryopreservation.

  16. The bull sperm microRNAome and the effect of fescue toxicosis on sperm microRNA expression.

    PubMed

    Stowe, Heather M; Calcatera, Samantha M; Dimmick, Marcy A; Andrae, John G; Duckett, Susan K; Pratt, Scott L

    2014-01-01

    Tall fescue [Schedonorus phoenix (Scop.) Holub] accounts for nearly 16 million hectares of pasture in the Southeastern and Mid-Atlantic U.S. due to its heat, drought, and pest resistance, conferred to the plant by its symbiotic relationship with the endophyte Neotyphodium coenophialum. The endophyte produces ergot alkaloids that have negative effects on the growth and reproduction of animals, resulting in the syndrome known as fescue toxicosis. The objectives of our study were to identify microRNA (miRNA) present in bovine sperm and to evaluate the effects of fescue toxicosis on sperm miRNA expression. Angus bulls were assigned to treatments of either toxic or non-toxic fescue seed diets. Semen was collected and subjected to RNA isolation. Three samples from each treatment group were chosen and pooled for deep sequencing. To compare miRNA expression between treatment groups, a microarray was designed and conducted. For each of the top ten expressed miRNA, target prediction analysis was conducted using TargetScan. Gene ontology enrichment was assessed using the Database for Annotation, Visualization and Integrated Discovery. Sequencing results elucidated the presence of 1,582 unique small RNA present in sperm. Of those sequences, 382 were known Bos taurus miRNA, 22 were known but novel to Bos taurus, and 816 were predicted candidate miRNA that did not map to any currently reported miRNA. Of the sequences chosen for microarray, twenty-two showed significant differential expression between treatment groups. Gene pathways of interest included: regulation of transcription, embryonic development (including blastocyst formation), Wnt and Hedgehog signaling, oocyte meiosis, and kinase and phosphatase activity. MicroRNA present in mature sperm appears to not only be left over from spermatogenic processes, but may actually serve important regulatory roles in fertilization and early developmental processes. Further, our results indicate the possibility that environmental

  17. Laser radiation and motility patterns of human sperm

    SciTech Connect

    Lenzi, A.; Claroni, F.; Gandini, L.; Lombardo, F.; Barbieri, C.; Lino, A.; Dondero, F. )

    1989-01-01

    Human sperm were exposed in vitro to laser radiation. An increase in progressive sperm motility was associated with a faster rate of sperm ATP consumption. Computer-assisted analysis of sperm motility confirmed the positive effect of laser irradiation on velocity and linearity of sperm.

  18. The reproductive system of Osedax (Annelida, Siboglinidae): ovary structure, sperm ultrastructure, and fertilization mode

    PubMed Central

    Katz, Sigrid; Rouse, Greg W

    2013-01-01

    Osedax is a genus of siboglinid annelids in which the females live on dead vertebrate bones on the seafloor. These females have a posterior end that lies within the bone and contains the ovarian tissue, as well as the “roots” involved with bone degradation and nutrition. The males are microscopic and live as “harems” in the lumen of the gelatinous tube that surrounds the female trunk, well away from the ovary. Females are known to spawn fertilized primary oocytes, suggesting internal fertilization. However, little is known about sperm transfer, sperm storage, or the location of fertilization, and the morphology of the female reproductive system has not been described and compared with the reproductive systems of other siboglinids. A 3D-reconstruction of the ovisac of Osedax showed ovarian tissue with multiple lobes and mature oocytes stored in a “uterus” before being released through the single oviduct. The oviduct emerges as a gonopore on the trunk and travels along the trunk to finally open to the seawater as a thin cylindrical tube among the crown of palps. Light and transmission electron microscopy of mature Osedax sperm revealed elongate heads consisting of a nucleus with helical grooves occupied by mitochondria. In contrast to other Siboglinidae, Osedax sperm are not packaged into spermatophores or spermatozeugmata, and Osedax females lack a discrete region for sperm storage. Transmission electron microscopy and fluorescence microscopy allowed detection of sperm associated with ovarian tissue of the female ovisac of four different Osedax species. This provides the first evidence for the site of internal fertilization in Osedax. A heart body was found in the circulatory system, as seen in other siboglinids and some other annelids. The possible presence of nephridia in the anterior ovisac region was also documented. These morphological features provide new insights for comparing the regionalization of Osedax females in relation to other siboglinids

  19. Ficoll-400 density gradient method as an effective sperm preparation technique for assisted reproductive techniques

    PubMed Central

    Highland, Hyacinth N.; Rishika, A. Sharma; Almira, S. Shaikh; Kanthi, P. Bansal

    2016-01-01

    BACKGROUND: Infertility being a burning issue, the male itself contributes about 40% as a cause, as evident by statistical data. However, Assisted Reproductive Technology (ART) has emerged as a powerful tool in the management of infertility. Sperm preparation techniques govern the selection procedure to separate functional spermatozoa which can then be used in IUI, IVF, and ART and for cryopreservation. AIM: The present study was aimed at evaluation of sperm preparation techniques for reliability, performance and to determine the most effective, feasible and economical technique. MATERIALS AND METHODS: The subjects under study includes males with normal proven fertility (n=40) and the males with unexplained infertility (n=40). Four sperm separation techniques, viz., Swim-up, Swim-down, Sucrose and Ficoll-400 density gradient techniques were evaluated for their efficacy in separation of good quality fraction of spermatozoa. Sperm viability, morphology and maturation status of spermatozoa were taken as evaluation parameters following the standard methods (WHO 2010). STATISTICAL ANALYSIS: Data was analyzed using student's t-test and the four selected techniques were compared with the normal semen samples for scoring the efficiency of the techniques. RESULTS AND DISCUSSION: Out of the several techniques used, Ficoll-400 was found to be more efficient method for separation of spermatozoa. The percentage of change in each parameter was calculated and taken as the index for recovery of potent sperm from the original sperm. Ficoll-400 density gradient yielded higher percentage of live, mature, morphologically normal spermatozoa in an isolated fraction as compared to other three techniques. CONCLUSION: It was observed that a combination of Ficoll-400 gradient separation with Swim-up technique could give quality spermatozoa which in-turn would directly have an impact on the success of IVF and other ART techniques. PMID:27803588

  20. Variability in sperm form and function in the context of sperm competition risk in two Tupinambis lizards.

    PubMed

    Blengini, Cecilia S; Sergio, Naretto; Gabriela, Cardozo; Giojalas, Laura C; Margarita, Chiaraviglio

    2014-11-01

    In polyandrous species, sperm morphometry and sperm velocity are under strong sexual selection. Although several hypotheses have been proposed to explain the role of sperm competition in sperm trait variation, this aspect is still poorly understood. It has been suggested that an increase in sperm competition pressure could reduce sperm size variation or produce a diversity of sperm to maximize male fertilization success. We aim at elucidating the variability of sperm morphometric traits and velocity in two Tupinambis lizards in the context of sperm competition risk. Sperm traits showed substantial variation at all levels examined: between species, among males within species, and within the ejaculate of individual males. Sperm velocity was found to be positively correlated with flagellum: midpiece ratio, with relatively longer flagella associated with faster sperm. Our results document high variability in sperm form and function in lizards.

  1. Variability in sperm form and function in the context of sperm competition risk in two Tupinambis lizards

    PubMed Central

    Blengini, Cecilia S; Sergio, Naretto; Gabriela, Cardozo; Giojalas, Laura C; Margarita, Chiaraviglio

    2014-01-01

    In polyandrous species, sperm morphometry and sperm velocity are under strong sexual selection. Although several hypotheses have been proposed to explain the role of sperm competition in sperm trait variation, this aspect is still poorly understood. It has been suggested that an increase in sperm competition pressure could reduce sperm size variation or produce a diversity of sperm to maximize male fertilization success. We aim at elucidating the variability of sperm morphometric traits and velocity in two Tupinambis lizards in the context of sperm competition risk. Sperm traits showed substantial variation at all levels examined: between species, among males within species, and within the ejaculate of individual males. Sperm velocity was found to be positively correlated with flagellum: midpiece ratio, with relatively longer flagella associated with faster sperm. Our results document high variability in sperm form and function in lizards. PMID:25505535

  2. Delayed sexual maturity in males of Vespa velutina.

    PubMed

    Poidatz, Juliette; Bressac, Christophe; Bonnard, Olivier; Thiéry, Denis

    2017-03-01

    Vespa velutina var nigrithorax (lepelletier, 1835) is an invasive predator of bees accidentally introduced in France in 2004, and it is having a serious impact on apiculture and ecosystems. Studying the reproduction of an invasive species is key to assess its population dynamic. This study explores the sexual maturation of V. velutina males and the evolution of their fertility. The main studied parameters were physiologic (spermiogenesis, spermatogenesis) and anatomic (testes size and structure, head width). Two populations of males were described based on their emergence period: early males in early summer or classic males in autumn. Each testis has an average of 108 testicular follicles. Spermatogenesis is synchronous, with only one sperm production wave, and completed, on average, at 10.3 days after emergence with the degeneration of the testes. The sperm counts in seminal vesicles of mature males are 3×10(6) in October/November and 0.8× 10(6) in June. In comparison, females store 0.1 10(6) sperm in their spermathecae. The early males emerged from colonies made by fertilized queens. The reproductive potential of these early males seemed limited, and their function in the colony is discussed. The sperm stock evolution in autumn males suggests the occurrence of a reproductive pattern of male competition for the access to females and a single copulation per male. The synchronicity of male and foundress emergences and sexual maturation is of primary importance for the mating success and the future colony development. This article is protected by copyright. All rights reserved.

  3. Sperm protamine-status correlates to the fertility of breeding bulls.

    PubMed

    Dogan, Sule; Vargovic, Peter; Oliveira, Rodrigo; Belser, Lauren E; Kaya, Abdullah; Moura, Arlindo; Sutovsky, Peter; Parrish, John; Topper, Einko; Memili, Erdoğan

    2015-04-01

    During fertilization, spermatozoa make essential contributions to embryo development by providing oocyte activating factors, centrosomal components, and paternal chromosomes. Protamines are essential for proper packaging of sperm DNA; however, in contrast to the studies of oocyte-related female infertility, the influence of sperm chromatin structure on male infertility has not been evaluated extensively. The objective of this study was to determine the sperm chromatin content of bull spermatozoa by evaluating DNA fragmentation, chromatin maturity/protamination, PRM1 protein status, and nuclear shape in spermatozoa from bulls with different fertility. Relationships between protamine 1 (PRM1) and the chromatin integrity were ascertained in spermatozoa from Holstein bulls with varied (high vs. low) but acceptable fertility. Sperm DNA fragmentation and chromatin maturity (protamination) were tested using Halomax assay and toluidine blue staining, respectively. The PRM1 content was assayed using Western blotting and in-gel densitometry, flow cytometry, and immunocytochemistry. Fragmentation of DNA was increased and chromatin maturity significantly reduced in spermatozoa from low-fertility bulls compared to those from high-fertility bulls. Field fertility scores of the bulls were negatively correlated with the percentage of spermatozoa displaying reduced protamination and fragmented DNA using toluidine blue and Halomax, respectively. Bull fertility was also positively correlated with PRM1 content by Western blotting and flow cytometry. However, detection of PRM1 content by Western blotting alone was not predictive of bull fertility. In immunocytochemistry, abnormal spermatozoa showed either a lack of PRM1 or scattered localization in the apical/acrosomal region of the nuclei. The nuclear shape was distorted in spermatozoa from low-fertility bulls. In conclusion, we showed that inadequate amount and localization of PRM1 were associated with defects in sperm chromatin

  4. Protective effects of ethyl pyruvate on sperm quality in cyclophosphamide treated mice

    PubMed Central

    Bakhtiary, Zahra; Shahrooz, Rasoul; Ahmadi, Abbas; Zarei, Leila

    2015-01-01

    Background: One of the affecting factors in disturbance process of spermatogenesis is chemotherapeutic-induced oxidative stress resulted from cyclophosphamide (CP) treatment which leads to diminished sperm quality via interference in spermatogenesis process. Objective: This study was conducted to investigate the effects of ethyl pyruvate (EP) in reducing the CP-induced side effects on reproductive system. Materials and Methods: 24 mature male mice were randomly divided into 3 equal groups and were undergone therapy for 35 days. Control group received normal saline (0.1 ml/day, IP). CP group were injected CP (15 mg/kg/week, IP) and CP+EP group received EP (40 mg/kg/day, IP) as well as CP. In the end of the treatment period, the mice were euthanized by cervical dislocation. Then, the epididymis was incubated with CO2 in a human tubal fluid medium (1 ml) for half an hour in order to float sperm. Then, the number, motility, viability (eosin-nigrosin staining), DNA breakage (acridine orange staining), nucleus maturity, and sperm morphology (aniline blue staining) were analyzed. Results: The average (15.87±1.28), motility (35.77±2.75), viability (40±3.03), nucleus maturity (36±2.79) and sperm morphology (61.75±0.85) were decreased significantly in CP group in comparison with control and EP groups, whereas EP caused significant increase of these parameters. Also, the percentage of DNA damage was increased significantly in CP group (41.75±3.75) in comparison with control (2±0.71) and EP groups (22.5±4.13). Conclusion: The results of this study revealed ameliorating effects of EP on sperm quality of CP treated animals. PMID:26221128

  5. Sperm competition and maternal effects differentially influence testis and sperm size in Callosobruchus maculatus.

    PubMed

    Gay, L; Hosken, D J; Vasudev, R; Tregenza, T; Eady, P E

    2009-05-01

    The evolutionary factors affecting testis size are well documented, with sperm competition being of major importance. However, the factors affecting sperm length are not well understood; there are no clear theoretical predictions and the empirical evidence is inconsistent. Recently, maternal effects have been implicated in sperm length variation, a finding that may offer insights into its evolution. We investigated potential proximate and microevolutionary factors influencing testis and sperm size in the bruchid beetle Callosobruchus maculatus using a combined approach of an artificial evolution experiment over 90 generations and an environmental effects study. We found that while polyandry seems to select for larger testes, it had no detectable effect on sperm length. Furthermore, population density, a proximate indicator of sperm competition risk, was not significantly associated with sperm length or testis size variation. However, there were strong maternal effects influencing sperm length.

  6. Efficient utilization of very dilute aquatic sperm: sperm competition may be more likely than sperm limitation when eggs are retained.

    PubMed Central

    Pemberton, Andrew J; Hughes, Roger N; Manríquez, Patricio H; Bishop, John D D

    2003-01-01

    Fertilization success may be severely limited in marine invertebrates that spawn both male and female gametes. In a diverse group of aquatic organisms only sperm are released, with sperm-egg fusion occurring at the mother. Here, we report fertilization kinetics data for two such 'brooding' or 'spermcast' species--representing each major clade of the animal kingdom. High levels of fertilization were achieved at sperm concentrations of two or three orders of magnitude lower than is common with broadcast spawning species. At a concentration of 100 sperm ml(-1), fertilization rates of a bryozoan and colonial ascidian were near maximum, whereas most broadcast spawners would have displayed near complete reproductive failure. A further experiment looked at the rate of uptake of sperm under natural conditions. Results suggested that sperm released at ca. 0.9 m from an acting female could be collected at a rate of 3-12 times greater than the minimum required simply to avoid sperm limitation. Thus, evolutionary pressures on gametic and other reproductive characteristics of many species that release sperm but retain eggs may be quite different from those of broadcast spawners and may confer on the former an enhanced scope for sperm competition and female choice. PMID:14667389

  7. Sperm Bundles in the Seminal Vesicle of the Crematogaster victima (Smith) Adult Males (Hymenoptera: Formicidae).

    PubMed

    Oliveira, C M; Moreira, J; Gomes, L F; Camargo-Mathias, M I; Lino-Neto, J

    2014-06-01

    This study establishes the presence of spermatodesm in the seminal vesicles of sexually mature males of Crematogaster victima (Smith). In this species, the spermatozoa are maintained together by an extracellular matrix in which the acrosomal regions are embedded. This characteristic has not yet been observed in any other Aculeata. However, the sperm morphology in this species is similar to that described for other ants. The spermatozoa measure on average 100 μm in length, and the number of sperm per bundle is up to 256. They are composed of a head formed by the acrosome and nucleus; this is followed by the flagellum, which is formed by the centriolar adjunct, an axoneme with a 9 + 9 + 2 microtubule pattern, two mitochondrial derivatives, and two accessory bodies. The acrosome is formed by the acrosomal vesicle and perforatorium. The nucleus is filled with compact chromatin with many areas of thick and non-compacted filaments. Both mitochondrial derivatives have the same shape and diameters. The presence of sperm bundles in sexually mature males differentiates C. victima from other ants; however, the similarities in the sperm ultrastructure support the monophyly of this insect group.

  8. Aquaporin biology of spermatogenesis and sperm physiology in mammals and teleosts.

    PubMed

    Boj, Mónica; Chauvigné, François; Cerdà, Joan

    2015-08-01

    Fluid homeostasis is recognized as a critical factor during the development, maturation, and function of vertebrate male germ cells. These processes have been associated with the presence of multiple members of the aquaporin superfamily of water and solute channels in different cell types along the reproductive tract as well as in spermatozoa. We present a comparative analysis of the existing knowledge of aquaporin biology in the male reproductive tissues of mammals and teleosts. Current data suggest that in both vertebrate groups, aquaporins may have similar functions during differentiation of spermatozoa in the germinal epithelium, in the concentration and maturation of sperm in the testicular ducts, and in the regulation of osmotically induced volume changes in ejaculated spermatozoa. Recent studies have also provided insight into the possible function of aquaporins beyond water transport, such as in signaling pathways during spermatogenesis or the sensing of cell swelling and mitochondrial peroxide transport in activated sperm. However, an understanding of the specific physiological functions of the various aquaporins during germ cell development and sperm motility, as well as the molecular mechanisms involved, remains elusive. Novel experimental approaches need to be developed to elucidate these processes and to dissect the regulatory intracellular pathways implicated, which will greatly help to uncover the molecular basis of sperm physiology and male fertility in vertebrates.

  9. Dynamics of vegetative cytoplasm during generative cell formation and pollen maturation in Arabidopsis thaliana

    NASA Technical Reports Server (NTRS)

    Kuang, A.; Musgrave, M. E.

    1996-01-01

    Ultrastructural changes of pollen cytoplasm during generative cell formation and pollen maturation in Arabidopsis thaliana were studied. The pollen cytoplasm develops a complicated ultrastructure and changes dramatically during these stages. Lipid droplets increase after generative cell formation and their organization and distribution change with the developmental stage. Starch grains in amyloplasts increase in number and size during generative and sperm cell formation and decrease at pollen maturity. The shape and membrane system of mitochondria change only slightly. Dictyosomes become very prominent, and numerous associated vesicles are observed during and after sperm cell formation. Endoplasmic reticulum appears extensively as stacks during sperm cell formation. Free and polyribosomes are abundant in the cytoplasm at all developmental stages although they appear denser at certain stages and in some areas. In mature pollen, all organelles are randomly distributed throughout the vegetative cytoplasm and numerous small particles appear. Organization and distribution of storage substances and appearance of these small particles during generative and sperm cell formation and pollen maturation are discussed.

  10. Sperm navigation in complex environments

    NASA Astrophysics Data System (ADS)

    Olson, Sarah

    2016-11-01

    Sperm can swim in a variety of environments, interacting with chemicals and other proteins in the fluid. Some of these extra proteins or cells may act as friction, possibly preventing or enhancing forward progression of swimmers. The homogenized fluid flow is assumed to be governed by the incompressible Brinkman equation, where a friction term with a resistance parameter represents a sparse array of obstacles. Representing the swimmers with a centerline approximation, we employ regularized fundamental solutions to investigate swimming speeds, trajectories, and interactions of swimmers. Asymmetric waveforms due to an increase in flagellar calcium is known to be important for sperm to reach and fertilize the egg. The trajectories of hyperactivated swimmers are found to have a decreased path curvature. Although attraction of two swimmers is more efficient in the Stokes regime, we find that attraction does not occur for larger resistance. Additionally, we study interactions of swimmers in a channel. NSF DMS 1413110.

  11. Diazinon alters sperm chromatin structure in mice by phosphorylating nuclear protamines

    SciTech Connect

    Pina-Guzman, B.; Solis-Heredia, M.J.; Quintanilla-Vega, B. . E-mail: mquintan@mail.cinvestav.mx

    2005-01-15

    Organophosphorus (OP) pesticides, widely used in agriculture and pest control, are associated with male reproductive effects, including sperm chromatin alterations, but the mechanisms underlying these effects are unknown. The main toxic action of OP is related to phosphorylation of proteins. Chemical alterations in sperm nuclear proteins (protamines), which pack DNA during the last steps of spermatogenesis, contribute to male reproductive toxicity. Therefore, in the present study, we tested the ability of diazinon (DZN), an OP compound, to alter sperm chromatin by phosphorylating nuclear protamines. Mice were injected with a single dose of DZN (8.12 mg/kg, i.p.), and killed 8 and 15 days after treatment. Quality of sperm from epididymis and vas deferens was evaluated through standard methods and chromatin condensation by flow cytometry (DNA Fragmented Index parameters: DFI and DFI%) and fluorescence microscopy using chromomycin-A{sub 3} (CMA{sub 3}). Increases in DFI (15%), DFI% (4.5-fold), and CMA{sub 3} (2-fold) were observed only at 8 days post-treatment, indicating an alteration in sperm chromatin condensation and DNA damage during late spermatid differentiation. In addition, an increase of phosphorous content (approximately 50%) in protamines, especially in the phosphoserine content (approximately 73%), was found at 8 days post-treatment. Sperm viability, motility, and morphology showed significant alterations at this time. These data strongly suggest that spermatozoa exposed during the late steps of maturation were the targets of DZN exposure. The correlation observed between the phosphorous content in nuclear protamines with DFI%, DFI, and CMA{sub 3} provides evidence that phosphorylation of nuclear protamines is involved in the OP effects on sperm chromatin.

  12. Normal reproductive development of offspring derived by intracytoplasmic injection of porcine sperm grown in host mice.

    PubMed

    Kaneko, Hiroyuki; Kikuchi, Kazuhiro; Nakai, Michiko; Tanihara, Fuminori; Noguchi, Junko; Noguchi, Michiko; Ito, Junya; Kashiwazaki, Naomi

    2012-09-01

    For establishment of gonadal xenografting, it is essential to clarify whether offspring derived from gametes grown in host mice harboring xenografts have normal reproductive development. This study examined the secretory profiles of gonadal hormones in relation to sexual maturation or ovarian cyclicity in pigs generated by intracytoplasmic sperm injection using xenogeneic sperm (Xeno-ICSI pigs, four males and one female). We also assessed the developmental activity of gametes obtained from these pigs using in vitro culture systems, or by mating with conventionally produced (conventional) pigs. During the growth of male Xeno-ICSI pigs, serum inhibin and testosterone concentrations were generally within ranges for those hormones in conventional pigs. Histologically, there were no differences in the growth and differentiation of seminiferous tubules between Xeno-ICSI and conventional pigs. Parameters of semen quality, including volume, pH, sperm concentration, and the percentage of motile sperm were not different from those in conventional pigs. Among the Xeno-ICSI pigs, individual differences were noted in the ability of sperm to penetrate oocytes and to produce blastocysts. However, oocytes after in vitro fertilization using these sperm developed into blastocysts containing more than 31 cells. One conventional sow delivered 12 piglets after being mated with a male Xeno-ICSI pig. During growth of the female Xeno-ICSI pig, serum progesterone concentrations had a sudden increase at 41 wk of age, suggesting CL formation. After puberty, this animal showed cyclic changes in the serum concentrations of progesterone and inhibin, and delivered 10 piglets after AI using fresh sperm obtained from a conventional boar. In conclusion, these findings demonstrated that both male and female Xeno-ICSI pigs had normal reproductive abilities.

  13. Evaluation of sperm recovered after slaughter from cauda epididymides of red Sokoto bucks

    PubMed Central

    Abu, A. H.; Kisani, A. I.; Ahemen, T.

    2016-01-01

    Aim: Viable spermatozoa could be recovered from the cauda epididymides for the purpose of preservation of genetic material of male animals with desirable traits and for use in reproductive biotechnology. The aim of this study was to determine the effect of storage time on testicular and epididymal biometry, sperm reserves and epididymal sperm characteristics of red Sokoto bucks post mortem. Materials and Methods: Testes-epididymides were collected immediately after slaughter of mature red Sokoto bucks and transported in ice chest to the laboratory. The samples were either processed immediately or stored at 5°C in refrigerator for 24, 48 h and then processed. The testes and epididymides were measured and weighed. Sperm motility, concentration, livability, morphology, intact acrosome, and sperm reserves from different treatment groups including control were evaluated and means (±standard error of mean) were recorded. Results: There was no significant difference (p>0.05) in the testicular and epididymal dimensions determined between the means of the groups. Percent sperm motility and viability decreased significantly (p<0.05) after 24 h from 69.00±0.46 and 71.27±0.50% to 50.60±0.48 and 60.47±0.70% at 48 h, respectively. Significant decreases (p<0.05) in epididymal sperm concentration and intact acrosome from 2.86±0.08 and 92.87±0.39 at 0 to 24 h of storage, respectively, were observed. Conclusion: The results of this study suggest that spermatozoa recovered from the epididymides of red Sokoto bucks were viable after storage for up to 48 h. Furthermore, this finding offers some hope that epididymal sperm recovered post-mortem can be used in assisted reproductive technologies. PMID:28096618

  14. Nuclear microscopy of sperm cell elemental structure

    NASA Astrophysics Data System (ADS)

    Bench, Graham S.; Balhorn, Rod; Friz, Alexander M.

    1995-05-01

    Theories suggest there is a link between protamine concentrations in individual sperm and male fertility. Previously, biochemical analyses have used pooled samples containing millions of sperm to determine protamine concentrations. These methods have not been able to determine what percentage of morphologically normal sperm are biochemically defective and potentially infertile. Nuclear microscopy has been utilized to measure elemental profiles at the single sperm level. By measuring the amount of phosphorus and sulfur, the total DNA and protamine content in individual sperm from fertile bull and mouse semen have been determined. These values agree with results obtained from other biochemical analyses. Nuclear microscopy shows promise for measuring elemental profiles in the chromatin of individual sperm. The technique may be able to resolve theories regarding the importance of protamines to male fertility and identify biochemical defects responsible for certain types of male infertility.

  15. Nuclear microscopy of sperm cell elemental structure

    SciTech Connect

    Bench, G.S.; Balhorn, R.; Friz, A.M.; Freeman, S.P.H.T.

    1994-09-28

    Theories suggest there is a link between protamine concentrations in individual sperm and male fertility. Previously, biochemical analyses have used pooled samples containing millions of sperm to determine protamine concentrations. These methods have not been able to determine what percentage of morphologically normal sperm are biochemically defective and potentially infertile. Nuclear microscopy has been utilized to measure elemental profiles at the single sperm level. By measuring the amount of phosphorus and sulfur, the total DNA and protamine content in individual sperm from fertile bull and mouse semen have been determined. These values agree with results obtained from other biochemical analyses. Nuclear microscopy shows promise for measuring elemental profiles in the chromatin of individual sperm. The technique may be able to resolve theories regarding the importance of protamines to male fertility and identify biochemical defects responsible for certain types of male infertility.

  16. Bovine binder-of-sperm protein BSP1 promotes protrusion and nanotube formation from liposomes

    SciTech Connect

    Lafleur, Michel; Courtemanche, Lesley; Karlsson, Goeran; Edwards, Katarina; Schwartz, Jean-Louis; Manjunath, Puttaswamy

    2010-08-27

    Research highlights: {yields} Binder-of-sperm protein 1 (BSP1) modifies the morphology of lipidic vesicles inducing bead necklace-like and thread-like structures. {yields} In the presence of multilamellar liposomes, BSP1 leads to the formation of long nanotubes. {yields} The insertion of BSP1 in the external lipid leaflet of membranes induces local changes in bilayer curvature. -- Abstract: Binder-of-sperm (BSP) proteins interact with sperm membranes and are proposed to extract selectively phosphatidylcholine and cholesterol from these. This change in lipid composition is a key step in sperm capacitation. The present work demonstrates that the interactions between the protein BSP1 and model membranes composed with phosphatidylcholine lead to drastic changes in the morphology of the lipidic self-assemblies. Using cryo-electron microscopy and fluorescence microscopy, we show that, in the presence of the protein, the lipid vesicles elongate, and form bead necklace-like structures that evolve toward small vesicles or thread-like structures. In the presence of multilamellar vesicles, where a large reservoir of lipid is available, the presence of BSP proteins lead to the formation of long nanotubes. Long spiral-like threads, associated with lipid/protein complexes, are also observed. The local curvature of lipid membranes induced by the BSP proteins may be involved in lipid domain formation and the extraction of some lipids during the sperm maturation process.

  17. Identification and characterization of an oocyte factor required for sperm decondensation in pig.

    PubMed

    Li, Jingyu; Huan, Yanjun; Xie, Bingteng; Wang, Jiaqiang; Zhao, Yanhua; Jiao, Mingxia; Huang, Tianqing; Kong, Qingran; Liu, Zhonghua

    2014-10-01

    Mammalian oocytes possess factors to support fertilization and embryonic development, but knowledge on these oocyte-specific factors is limited. In the current study, we demonstrated that porcine oocytes with the first polar body collected at 33 h of in vitro maturation sustain IVF with higher sperm decondensation and pronuclear formation rates and support in vitro development with higher cleavage and blastocyst rates, compared with those collected at 42 h (P<0.05). Proteomic analysis performed to clarify the mechanisms underlying the differences in developmental competence between oocytes collected at 33 and 42 h led to the identification of 18 differentially expressed proteins, among which protein disulfide isomerase associated 3 (PDIA3) was selected for further study. Inhibition of maternal PDIA3 via antibody injection disrupted sperm decondensation; conversely, overexpression of PDIA3 in oocytes improved sperm decondensation. In addition, sperm decondensation failure in PDIA3 antibody-injected oocytes was rescued by dithiothreitol, a commonly used disulfide bond reducer. Our results collectively report that maternal PDIA3 plays a crucial role in sperm decondensation by reducing protamine disulfide bonds in porcine oocytes, supporting its utility as a potential tool for oocyte selection in assisted reproduction techniques.

  18. Ejaculated Mouse Sperm Enter Cumulus-Oocyte Complexes More Efficiently In Vitro than Epididymal Sperm

    PubMed Central

    Suarez, Susan S.

    2015-01-01

    The mouse is an established and popular animal model for studying reproductive biology. Epididymal mouse sperm, which lack exposure to secretions of male accessory glands and do not precisely represent ejaculated sperm for the study of sperm functions, have been almost exclusively used in studies. We compared ejaculated and epididymal sperm in an in vitro fertilization setting to examine whether ejaculated sperm enter cumulus-oocyte complexes more efficiently. In order to prepare sperm for fertilization, they were incubated under capacitating conditions. At the outset of incubation, ejaculated sperm stuck to the glass surfaces of slides and the incidences of sticking decreased with time; whereas, very few epididymal sperm stuck to glass at any time point, indicating differences in surface charge. At the end of the capacitating incubation, when sperm were added to cumulus-oocyte complexes, the form of flagellar movement differed dramatically; specifically, ejaculated sperm predominantly exhibited increased bending on one side of the flagellum (a process termed pro-hook hyperactivation), while epididymal sperm equally exhibited increased bending on one or the other side of the flagellum (pro-hook or anti-hook hyperactivation). This indicates that accessory sex gland secretions might have modified Ca2+ signaling activities in sperm, because the two forms of hyperactivation are reported to be triggered by different Ca2+ signaling patterns. Lastly, over time, more ejaculated than epididymal sperm entered the cumulus oocyte complexes. We concluded that modification of sperm by male accessory gland secretions affects the behavior of ejaculated sperm, possibly providing them with an advantage over epididymal sperm for reaching the eggs in vivo. PMID:25996155

  19. Relationship between the nuclear morphology of the sperm of 10 bulls and their fertility.

    PubMed

    Vieytes, A L; Cisale, H O; Ferrari, M R

    2008-11-22

    The relationships between the fertility and nuclear morphology, chromatin maturity and chromatin condensation of the sperm of three bulls with a calving rate over a year of more than 65 per cent, four bulls with a calving rate between 65 per cent and 35 per cent, and three bulls with a calving rate of less than 35 per cent were studied. The sperm nuclei were stained with the Feulgen reaction, and chromatin condensation and maturation were evaluated in situ by staining with toluidine blue and acid aniline blue. Nuclear chromatin decondensation was induced with dithiothreitol; this showed that in the bulls with low fertility, more than 35 per cent of nuclei were decondensed, and that one of them had the lowest percentage of normal nuclei (64.9 per cent) and stronger positive reactions to the acid aniline blue and toluidine blue stains than the other bulls.

  20. The egg nucleus regulates the behavior of sperm nuclei as well as cycling of MPF in physiologically polyspermic newt eggs.

    PubMed

    Iwao, Y; Sakamoto, N; Takahara, K; Yamashita, M; Nagahama, Y

    1993-11-01

    The possible role of the egg nucleus in regulating the behavior of sperm nuclei and the cycling of maturation-promoting factor (MPF) was investigated in the physiologically polyspermic eggs of the newt Cynops pyrrhogaster. Many sperm entered all areas of the egg, but only one sperm pronucleus, the principal sperm pronucleus, moved to the center of the animal hemisphere to form a zygote nucleus with the egg pronucleus. All sperm and egg pronuclei synthesized DNA, but the zygote nucleus completed the synthesis of DNA 0.5-1 hr earlier than the accessory sperm nuclei. Entrance into M phase by the accessory sperm nuclei was delayed to a greater and greater extent with increasing distance of these nuclei from the zygote nucleus. When DNA in the egg nucleus was damaged by uv irradiation, not only were both prophase and M phase in the zygote nucleus prolonged, but also the MPF cycle was delayed. Some accessory sperm nuclei in the animal hemisphere escaped degeneration to form additional bipolar spindles, so that delayed multipolar cleavage occurred. The MPF activity in the vegetal hemisphere was less than 25% of that found in the animal hemisphere at M phase in normally fertilized eggs. The levels of immunologically detectable proteins that contained the sequence PSTAIR in vegetal hemispheres were less than 25% of those in animal hemispheres. These results indicate that the egg nucleus of the Cynops egg is involved in the control of the activation of MPF and that the accessory sperm nuclei in the vegetal hemisphere degenerate as a result of the lack of components that are indispensable for entry into M phase.

  1. Leptin Improves Sperm Cryopreservation via Antioxidant Defense

    PubMed Central

    Fontoura, Paula; Mello, Mariana Duque; Gallo-Sá, Paulo; Erthal-Martins, Maria Cecília; Cardoso, Maria Cecília Almeida; Ramos, Cristiane

    2017-01-01

    Background: Leptin and its receptor are present in spermatozoa; however, the role of leptin in sperm function is still controversial. Our present study aimed at demonstrating the effect of cryopreservation on sperm DNA fragmentation (DNAf) and investigating the possible effects of sperm capacitation techniques and leptin in vitro incubation on frozen-thawed sperm DNAf and oxidative stress. Methods: Samples of 45 normospermic men attending for infertility investigation at Vida Centro de Fertilidade, Rio de Janeiro, Brazil, were frozen and thawed with or without capacitation and leptin incubation prior to freezing. Sperm DNA fragmentation was evaluated by Sperm Chromatin Dispersion Assay before and after cryopreservation and oxidative stress parameters were measured by spectrophotometry with and without leptin incubation. Statistical analysis was performed using paired t test to compare DNAf between groups before and after freeze-thaw cycle, to compare groups before and after capacitation and leptin incubation and oxidative measurements before and after leptin incubation. Statistical significance was considered when p≤0.05. Results: Our results revealed a significant post-thaw rise in sperm DNAf compared with fresh samples (p=0.0003). Sperm DNAf was significantly reduced when sperm capacitation was performed before freezing, when compared to those frozen with no previous capacitation (p=0.01). The addition of leptin to capacitated sperm before freezing reduced DNAf (p<0.0001) and enhanced superoxide dismutase (p=0.001) and glutathione peroxidase (p=0.02) antioxidant enzymes activity. Conclusion: The addition of leptin to capacitated sperm can improve sperm DNA quality following cryopreservation, possibly by inducing the activity of certain antioxidant enzymes. PMID:28377896

  2. Signaling in Sperm: More Different than Similar.

    PubMed

    Kaupp, U B; Strünker, T

    2017-02-01

    For a given sensory cell type, signaling motifs are rather uniform across phyla. By contrast, sperm from different species use diverse repertoires of sperm-specific signaling molecules and even closely related protein isoforms feature different properties and serve different functions. This surprising diversity has consequences for strategies in fertilization research and it will take some time to get the big picture. We discuss the function of receptors, ion channels, and exchangers embedded in cellular pathways from different sperm species.

  3. Effect of organic selenium in the diet on sperm quality of boars.

    PubMed

    López, A; Rijsselaere, T; Van Soom, A; Leroy, J L M R; De Clercq, J B P; Bols, P E J; Maes, D

    2010-12-01

    The effect of a diet supplemented with organic selenium (Se) on sperm production and quality of boars was investigated. Sixty mature boars from a commercial artificial insemination centre were randomly allocated at Day (D) 0 into Group A and B. Group A received the regular ration supplemented with inorganic Se (0.4 mg/kg feed as Na(2) SeO(3)) whereas Group B was switched to the same diet but with organic Se (0.4 mg/kg fed as Se-yeast). The sperm was investigated during 4 months (D0, D30, D60, D75, D90, D105 and D120). Sperm concentration and motility were objectively measured using a photometer and Computer Assisted Semen Analysis (CASA) respectively. Morphology of the sperm was assessed using eosin-nigrosin staining and the resistance to induction of oxidative stress (production of malonaldehyde, MDA) through thiobarbituric acid reagent species analysis. Additionally, the Se concentration in sperm and blood plasma were measured. Repeated measures analysis of variance (anova) from D60 to 120 (spermatogenesis of approximately 2 months) or anova at D120 (Se concentrations) were used for statistical analysis. The total number of ejaculated sperm was not significantly different between both groups, but boars of Group B had a significantly higher sperm concentration (434.6 vs 514.1 × 10(6) sperm/ml; p < 0.05). Small differences (p < 0.05) were observed between both groups for some CASA parameters, namely straight line velocity (μm/s) (Group A: 48.3, Group B: 45.1), straightness (%) (Group A: 65.6, Group B: 62.2) and linearity (%) (Group A: 32.2, Group B: 29.3). The sperm of Group B showed more oxidative stress (4.1 vs 4.9 μmol MDA/l; p < 0.05) compared with those of Group A. No significant differences (p > 0.05) were observed for the other parameters. Under the present study conditions, changing from inorganic Se to organic Se in the diet of boars increased sperm concentration but reduced some motility parameters and resistance to

  4. Presence and release of bovine sperm histone H1 during chromatin decondensation by heparin-glutathione.

    PubMed

    Sánchez-Vázquez, María Luisa; Flores-Alonso, Juan Carlos; Merchant-Larios, Horacio; Reyes, Rosalina

    2008-01-01

    During spermatogenesis, changes in sperm nuclear morphology are associated with the replacement of core somatic histones by protamines. Although protamines are the major nucleoproteins of mature sperm, not all species totally replace the histones. Histone H1, along with protamines, mediates chromatin condensation into an insoluble complex that is transcriptionally inactive. In vitro, heparin-reduced glutathione causes sperm decondensation, and the structures formed are morphologically similar to the in vivo male pronucleus. To study the participation of histone H1 in bovine sperm chromatin remodelling, we measured the presence and release of histone H1 by immunofluorescence, acetic acid-urea-triton-polyacrylamide gel electrophoresis, and immunoblotting. Nuclear decondensation was induced by 80 microM heparin and 15.0 mM reduced glutathione (GSH) for 7, 14, and 21 h at 37 degrees C. Additionally, nucleons, composed of nuclei isolated from the sperm, were decondensed with 20.0 microM heparin and 5.0 mM GSH for 4.0 h at 37 degrees C. Controls were incubated in buffer for similar periods of time. Immunofluorescent localization of histone H1 was carried out with mouse monoclonal antibody, and DNA localization was visualized by 0.001% quinacrine staining. Chromatin decondensation was accompanied by increased sperm nuclei and nucleon surface area. We observed that histone H1 was localized exclusively in the nuclei of intact sperm and nucleons. Histone H1 immunofluorescent intensity did not change in control samples but decreased over time in samples treated with heparin-GSH. There was a negative correlation between the surface area of sperm nuclei and immunohistochemical intensity of histone H1 (P < 0.05). Nucleon decondensation showed a similar relationship. By electrophoresis and immunoblotting, we verified the loss of histone H1 from the sperm and nucleons and its release into the incubation media. Based on these results, we propose that histone H1 is present in the

  5. Morphologic changes in boar sperm nuclei with reduced disulfide bonds in electrostimulated porcine oocytes.

    PubMed

    Nakai, Michiko; Kashiwazaki, Naomi; Takizawa, Akiko; Maedomari, Naoki; Ozawa, Manabu; Noguchi, Junko; Kaneko, Hiroyuki; Shino, Masao; Kikuchi, Kazuhiro

    2006-03-01

    In pigs, failure of sperm nuclear decondensation has been reported after injection into oocytes. We examined the effects of pretreating sperm heads with Triton X-100 (TX-100) and dithiothreitol (DTT) and of electrical stimulation of oocytes after sperm head injection on time-dependent morphologic changes in sperm nuclei and in vitro development to the blastocyst stage. In experiment 1, spermatozoa were pretreated with 1% TX-100 and 5 mM DTT (T + D) or not treated, and then injected into in vitro matured oocytes. Electrical stimulation (1.5 kV/cm, 20 mus DC pulse) was applied to the oocytes 1 h after injection (stimulated group) or was not applied (unstimulated group). Some of the oocytes in each group were evaluated at hourly intervals until 10 h after injection for morphologic changes in the sperm nuclei. Unstimulated oocytes injected with untreated spermatozoa showed a delayed peak in the rate of nuclear decondensation (39.4-44.1%, 3-6 h after injection) compared with oocytes injected with T + D-treated spermatozoa (57.0% and 52.6%, 1 and 2 h, respectively). The rate of male pronucleus formation peaked 6 h after stimulation (by 40-60%) after injected oocytes had been stimulated with an electrical pulse, irrespective of whether or not the spermatozoa had been pretreated. In unstimulated oocytes, the rate of male pronucleus formation did not increase and stayed at the basal level (less than 20%) throughout the culture period, regardless of the sperm treatment. Thus, T + D treatment of spermatozoa did not affect completion of fertilization. In experiment 2, we evaluated the effects of electrical stimulation and sperm treatment with T + D on the rate of blastocyst formation and the mean number of cells per blastocyst. Oocytes stimulated after injection with either T + D-treated or untreated spermatozoa showed significantly higher percentages of blastocyst formation (24.8% and 27.1% respectively) than did unstimulated oocytes (1.1% and 4.1% for T + D-treated and

  6. 21 CFR 173.275 - Hydrogenated sperm oil.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... from rendering the fatty tissue of the sperm whale or is prepared by synthesis of fatty acids and fatty alcohols derived from the sperm whale. The sperm oil obtained by rendering is refined. The oil...

  7. Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm

    SciTech Connect

    Bleil, J.D.; Wassarman, P.M.

    1986-04-01

    The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O-linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetrate the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound 125I-ZP3 were found localized to the acrosomal cap region of heads of acrosome-reacted sperm. Under the same conditions, 125I-fetuin bound at only background levels to heads of both acrosome-intact and -reacted sperm, and 125I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor.

  8. Sperm cryopreservation update: Cryodamage, markers, and factors affecting the sperm freezability in pigs.

    PubMed

    Yeste, Marc

    2016-01-01

    Cryopreservation is the most efficient method for long-term preservation of mammalian sperm. However, freeze-thawing procedures may strongly impair the sperm function and survival and thus decrease the reproductive performance. In addition, the sperm resilience to withstand cryopreservation, also known as freezability, presents a high individual variability. The present work summarizes the principles of cryoinjury and the relevance of permeating and nonpermeating cryoprotective agents. Descriptions about sperm cryodamage are mainly focused on boar sperm, but reference to other mammalian species is also made when relevant. Main cryoinjuries not only regard to sperm motility and membrane integrity, but also to the degradation effect exerted by freeze-thawing on other important components for sperm fertilizing ability, such as mRNAs. After delving into the main differences between good and poor freezability boar ejaculates, those protein markers predicting the sperm ability to sustain cryopreservation are also mentioned. Moreover, factors that may influence sperm freezability, such as season, diet, breed, or ejaculate fractions are discussed, together with the effects of different additives, like seminal plasma and antioxidants. After briefly referring to the effects of long-term sperm preservation in frozen state and the reproductive performance of frozen-thawed boar sperm, this work speculates with new research horizons on the preservation of boar sperm, such as vitrification and freeze-drying.

  9. Relationship between Porcine Sperm Motility and Sperm Enzymatic Activity using Paper-based Devices.

    PubMed

    Matsuura, Koji; Huang, Han-Wei; Chen, Ming-Cheng; Chen, Yu; Cheng, Chao-Min

    2017-04-07

    Mammalian sperm motility has traditionally been analyzed to determine fertility using computer-assisted semen analysis (CASA) systems. To develop low-cost and robust male fertility diagnostics, we created a paper-based MTT assay and used it to estimate motile sperm concentration. When porcine sperm motility was inhibited using sperm enzyme inhibitors for sperm enzymes related to mitochondrial activity and glycolysis, we simultaneously recorded sperm motility and enzymatic reactivity using a portable motility analysis system (iSperm) and a paper-based MTT assay, respectively. When using our paper-based MTT-assay, we calculated the area mean value signal intensity (AMV) to evaluate enzymatic reactivity. Both sperm motility and AMV decreased following treatment with iodoacetamide (IODO) and 3-bromopyruvic acid (3BP), both of which are inhibitors of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a correlation between recorded motility using iSperm and AMV from our paper-based assay (P < 0.05), suggesting that a sperm-related enzymatic reaction is involved in sperm motility. Under this protocol, MTT reduction was coupled with catalysis of GAPDH and was promoted by electron transfer from NADH. Based on this inhibitor study, sperm motility can be estimated using our paper-based MTT-assay.

  10. Artificial insemination with donor sperm (AID): heterogeneity in sperm banking facilities in a single country (Belgium)

    PubMed Central

    Thijssen, A.; Dhont, N.; Vandormael, E.; Cox, A.; Klerkx, E.; Creemers, E.; Ombelet, W.

    2014-01-01

    Due to the high inflow of foreign patients seeking cross-border reproductive care in Belgium and the increased number of lesbian couples and single women who call for artificial insemination with donor sperm (AID), Belgian sperm banks nowadays face a shortage in donor sperm. However, since there is no central registration system for sperm donors in Belgium, no figures are currently available supporting this statement. Therefore a study was performed to obtain a detailed overview of the sperm banking facilities in Belgium. Questionnaires were sent to all Belgian centres for assisted reproduction with laboratory facilities (n = 18) to report on their sperm banking methods. The results showed that 82% of the centres rely partially or completely on foreign donor sperm. Moreover, four of the thirteen centres that have their own sperm bank use imported donor sperm in > 95% AID cycles. Our results show that in 63% of the Belgian AID cycles imported Danish donor sperm is being used. Donor recruitment is mainly performed through the centre’s website (61%) or by distributing flyers in the centre (46%) and 9 to 180 potential donors have been recruited per centre in 2013. Eventually, 15 to 50% of these candidate donors were accepted. Different criteria for donor acceptance are handled by the centres: donor age limits range from 18-25 to 36-46 years old, and thresholds for sperm normality differ considerably. We can conclude that a wide variation in methods associated with sperm banking is observed in Belgian centres. PMID:25009728

  11. Relationship between Porcine Sperm Motility and Sperm Enzymatic Activity using Paper-based Devices

    PubMed Central

    Matsuura, Koji; Huang, Han-Wei; Chen, Ming-Cheng; Chen, Yu; Cheng, Chao-Min

    2017-01-01

    Mammalian sperm motility has traditionally been analyzed to determine fertility using computer-assisted semen analysis (CASA) systems. To develop low-cost and robust male fertility diagnostics, we created a paper-based MTT assay and used it to estimate motile sperm concentration. When porcine sperm motility was inhibited using sperm enzyme inhibitors for sperm enzymes related to mitochondrial activity and glycolysis, we simultaneously recorded sperm motility and enzymatic reactivity using a portable motility analysis system (iSperm) and a paper-based MTT assay, respectively. When using our paper-based MTT-assay, we calculated the area mean value signal intensity (AMV) to evaluate enzymatic reactivity. Both sperm motility and AMV decreased following treatment with iodoacetamide (IODO) and 3-bromopyruvic acid (3BP), both of which are inhibitors of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a correlation between recorded motility using iSperm and AMV from our paper-based assay (P < 0.05), suggesting that a sperm-related enzymatic reaction is involved in sperm motility. Under this protocol, MTT reduction was coupled with catalysis of GAPDH and was promoted by electron transfer from NADH. Based on this inhibitor study, sperm motility can be estimated using our paper-based MTT-assay. PMID:28387379

  12. Hypercholesterolemia Impaired Sperm Functionality in Rabbits

    PubMed Central

    Monclus, Maria A.; Cabrillana, Maria E.; Clementi, Marisa A.; Espínola, Leandro S.; Cid Barría, Jose L.; Vincenti, Amanda E.; Santi, Analia G.; Fornés, Miguel W.

    2010-01-01

    Hypercholesterolemia represents a high risk factor for frequent diseases and it has also been associated with poor semen quality that may lead to male infertility. The aim of this study was to analyze semen and sperm function in diet-induced hypercholesterolemic rabbits. Twelve adult White New Zealand male rabbits were fed ad libitum a control diet or a diet supplemented with 0.05% cholesterol. Rabbits under cholesterol-enriched diet significantly increased total cholesterol level in the serum. Semen examination revealed a significant reduction in semen volume and sperm motility in hypercholesterolemic rabbits (HCR). Sperm cell morphology was seriously affected, displaying primarily a “folded head”-head fold along the major axe-, and the presence of cytoplasmic droplet on sperm flagellum. Cholesterol was particularly increased in acrosomal region when detected by filipin probe. The rise in cholesterol concentration in sperm cells was determined quantitatively by Gas chromatographic-mass spectrometric analyses. We also found a reduction of protein tyrosine phosphorylation in sperm incubated under capacitating conditions from HCR. Interestingly, the addition of Protein Kinase A pathway activators -dibutyryl-cyclic AMP and iso-butylmethylxanthine- to the medium restored sperm capacitation. Finally, it was also reported a significant decrease in the percentage of reacted sperm in the presence of progesterone. In conclusion, our data showed that diet-induced hypercholesterolemia adversely affects semen quality and sperm motility, capacitation and acrosomal reaction in rabbits; probably due to an increase in cellular cholesterol content that alters membrane related events. PMID:20976152

  13. Alterations to the Bull Sperm Surface Proteins That Bind Sperm to Oviductal Epithelium1

    PubMed Central

    Hung, Pei-hsuan; Suarez, Susan S.

    2012-01-01

    ABSTRACT Three Binder of SPerm proteins (BSP1, BSP3, BSP5) are secreted by bovine seminal vesicles into seminal plasma and adsorbed onto sperm. When sperm inseminated into the female reach the oviduct, the BSP proteins bind them to its epithelial lining, forming a sperm storage reservoir. Previously, we reported that binding of capacitated sperm to oviductal epithelium in vitro is lower than that of uncapacitated sperm and we proposed that reduced binding was due to loss of BSP proteins during capacitation. Because of differences in amino acid sequences, we predicted that each BSP would respond differently to capacitating conditions. To test whether all three BSP proteins were lost from sperm during capacitation and whether the kinetics of loss differed among the three BSP proteins, ejaculated bull sperm were incubated under various capacitating conditions, and then the amounts of BSP proteins remaining on the sperm were assayed by Western blotting. Capacitation was assayed by analysis of protein tyrosine phosphorylation. While loss of BSP1 was not detected, most of the BSP5 was lost from sperm during incubation in TALP medium, even without addition of the capacitation enhancers heparin and dbcAMP-IBMX. Surprisingly, a smaller molecular mass was detected by anti-BSP3 antibodies in extracts of incubated sperm. Its identity was confirmed as BSP3 by mass spectrometry, indicating that BSP3 undergoes modification on the sperm surface. These changes in the composition of BSP proteins on sperm could play a role in releasing sperm from the storage reservoir by modifying sperm interactions with the oviductal epithelium. PMID:22837481

  14. Sexual selection and the evolution of sperm quality.

    PubMed

    Fitzpatrick, John L; Lüpold, Stefan

    2014-12-01

    Sperm experience intense and varied selection that dramatically impacts the evolution of sperm quality. Selection acts to ensure that sperm are fertilization-competent and able to overcome the many challenges experienced on their way towards eggs. However, simply being able to fertilize an egg is not enough to ensure male fertility in most species. Owing to the prevalence of female multiple mating throughout the animal kingdom, successful fertilization requires sperm to outcompete rival sperm. In addition, females can actively influence sperm quality, storage or utilization to influence male fertility. This review provides an overview of how these selective forces influence the evolution of sperm quality. After exploring the link between sperm traits and male fertility, we examine how post-mating competition between rival ejaculates influences the evolution of sperm quality. We then describe how complex genetic, social and sexual interactions influence sperm quality, focusing on the importance of seminal fluid and interactions between sperm and the female's reproductive tract. In light of the complexities of selection on sperm traits, greater use of multivariate approaches that incorporate male-male, sperm-sperm and sperm-female interactions to study sperm quality will enhance our understanding of how selection acts on sperm traits and factors influencing male fertility. Because the metric of male reproductive success--fertilization--is the same across the animal kingdom, we argue that information about sperm evolution gained from non-human animals has enormous potential to further our understanding of the factors that impact human fertility.

  15. Current status and potential of morphometric sperm analysis

    PubMed Central

    Maroto-Morales, Alejandro; García-Álvarez, Olga; Ramón, Manuel; Martínez-Pastor, Felipe; Fernández-Santos, M Rocío; Soler, A Josefa; Garde, José Julián

    2016-01-01

    The spermatozoon is the most diverse cell type known and this diversity is considered to reflect differences in sperm function. How the diversity in sperm morphology arose during speciation and what role the different specializations play in sperm function, however, remain incompletely characterized. This work reviews the hypotheses proposed to explain sperm morphological evolution, with a focus on some aspects of sperm morphometric evaluation; the ability of morphometrics to predict sperm cryoresistance and male fertility is also discussed. For this, the evaluation of patterns of change of sperm head morphometry throughout a process, instead of the study of the morphometric characteristics of the sperm head at different stages, allows a better identification of the males with different sperm cryoconservation ability. These new approaches, together with more studies employing a greater number of individuals, are needed to obtain novel results concerning the role of sperm morphometry on sperm function. Future studies should aim at understanding the causes of sperm design diversity and the mechanisms that generate them, giving increased attention to other sperm structures besides the sperm head. The implementation of scientific and technological advances could benefit the simultaneous examination of sperm phenotype and sperm function, demonstrating that sperm morphometry could be a useful tool for sperm assessment. PMID:27678465

  16. Identification and characterization of genes differentially expressed in X and Y sperm using suppression subtractive hybridization and cDNA microarray.

    PubMed

    Chen, Xiaoli; Yue, Yang; He, Yanan; Zhu, Huabin; Hao, Haisheng; Zhao, Xueming; Qin, Tong; Wang, Dong

    2014-10-01

    Differential expression of genes leads to variations in the phenotypes of X and Y sperm, although some differentially expressed gene products are shared through intercellular bridges. Genes differentially expressed in bovine X and Y sperm were identified by a combination of suppression subtractive hybridization (SSH), cDNA microarray, and sequence-homology analysis. Microarray data and Significance Analysis of Microarrays software were used to identify 31 differentially expressed genes, only four of which were previously identified. These genes are involved in fundamental life processes of mature sperm, and may be associated with the differences between X and Y sperm since 27 versus 4 were upregulated in X versus Y sperm, respectively. The levels of expression of seven genes-including the known genes UTY, DPH3, CYTB, and ISCU, and the unknown genes X + Y contig 41, X + Y contig 18, and Y + X contig 16-were validated by quantitative real-time PCR, and some genes were clearly differentially expressed by X and Y sperm, despite the presence of intercellular bridges among spermatids. These results provide a theoretical basis for research on gene expression during sperm development, as well as on sex control at the level of sperm.

  17. Methamidophos alters sperm function and DNA at different stages of spermatogenesis in mice

    SciTech Connect

    Urióstegui-Acosta, Mayrut; Hernández-Ochoa, Isabel; Sánchez-Gutiérrez, Manuel; Piña-Guzmán, Belem; Rafael-Vázquez, Leticia; Solís-Heredia, M.J.; Martínez-Aguilar, Gerardo; Quintanilla-Vega, Betzabet

    2014-09-15

    Methamidophos (MET) is a highly toxic organophosphate (OP) pesticide that is widely used in developing countries. MET has male reproductive effects, including decreased fertility. We evaluated MET effects on sperm quality, fertilization and DNA integrity, exploring the sensitivity of different stages of spermatogenesis. Adult male mice received MET (3.75 or 5 mg/kg-bw/ip/day/4 days) and were euthanized 1, 28 or 45 days post-treatment (dpt) to evaluate MET's effects on epididymal maturation, meiosis or mitosis, respectively. Spermatozoa were obtained from the cauda epididymis–vas deferens and were evaluated for sperm quality, acrosome reaction (AR; Coomassie staining), mitochondrial membrane potential (by JC-1), DNA damage (comet assay), oxidative damage (malondialdehyde (MDA) production), in vitro fertilization and protein phosphorylation (immunodetection), and erythrocyte acetylcholinesterase (AChE) activity. At 1-dpt, MET inhibited AChE (43–57%) and increased abnormal cells (6%). While at 28- and 45-dpt, sperm motility and viability were significantly reduced with an increasing MET dose, and abnormal morphology increased at 5 mg/kg/day/4 days. MDA and mitochondrial activity were not affected at any dose or time. DNA damage (OTM and %DNA) was observed at 5 mg/kg/day/4 days in a time-dependent manner, whereas both parameters were altered in cells from mice exposed to 3.75 mg/kg/day/4 days only at 28-dpt. Depending on the time of collection, initial-, spontaneous- and induced-AR were altered at 5 mg/kg/day/4 days, and the fertilization capacity also decreased. Sperm phosphorylation (at serine and tyrosine residues) was observed at all time points. Data suggest that meiosis and mitosis are the more sensitive stages of spermatogenesis for MET reproductive toxicity compared to epididymal maturation. - Highlights: • Methamidophos alters sperm cell function at different stages of spermatogenesis. • Testicular stages of spermatogenesis are more sensitive to

  18. Sperm dynamics in spiders (Araneae): ultrastructural analysis of the sperm activation process in the garden spider Argiope bruennichi (Scopoli, 1772).

    PubMed

    Vöcking, Oliver; Uhl, Gabriele; Michalik, Peter

    2013-01-01

    Storage of sperm inside the female genital tract is an integral phase of reproduction in many animal species. The sperm storage site constitutes the arena for sperm activation, sperm competition and female sperm choice. Consequently, to understand animal mating systems information on the processes that occur from sperm transfer to fertilization is required. Here, we focus on sperm activation in spiders. Male spiders produce sperm whose cell components are coiled within the sperm cell and that are surrounded by a proteinaceous sheath. These inactive and encapsulated sperm are transferred to the female spermathecae where they are stored for later fertilization. We analyzed the ultrastructural changes of sperm cells during residency time in the female genital system of the orb-web spider Argiope bruennichi. We found three clearly distinguishable sperm conditions: encapsulated sperm (secretion sheath present), decapsulated (secretion sheath absent) and uncoiled sperm (cell components uncoiled, presumably activated). After insemination, sperm remain in the encapsulated condition for several days and become decapsulated after variable periods of time. A variable portion of the decapsulated sperm transforms rapidly to the uncoiled condition resulting in a simultaneous occurrence of decapsulated and uncoiled sperm. After oviposition, only decapsulated and uncoiled sperm are left in the spermathecae, strongly suggesting that the activation process is not reversible. Furthermore, we found four different types of secretion in the spermathecae which might play a role in the decapsulation and activation process.

  19. Panning for sperm gold: Isolation and purification of apyrene and eupyrene sperm from lepidopterans.

    PubMed

    Karr, Timothy L; Walters, James R

    2015-08-01

    We describe a simple and straightforward procedure for the purification and separation of apyrene and eupyrene forms of lepidopteran sperm. The procedure is generally applicable to both butterfly and moth species with results varying according to the relative amounts of sperm produced and size of sperm storage organs. The technique relies upon inherent differences between eupyene sperm bundles and free apyrene sperm morphology. These differences allow for separation of the sperm morphs by repeated "panning" of sperm bundles into the center of a plastic dish. The purified eupyrene sperm bundles can then be removed and apyrene sperm collected from the supernatant by centrifugation. Efficacy of the purification process was confirmed by light microscopy and gel electrophoresis of the resulting fractions. Both one- and two-dimensional gel electrophoresis identified significant protein differences between the fractions further suggesting that the panning procedure effectively separated eurpyrene from apyrene sperm. The panning procedure should provide a convenient and accessible technique for further studies of sperm biology in lepidopterans.

  20. [Adolescent brain maturation].

    PubMed

    Holzer, L; Halfon, O; Thoua, V

    2011-05-01

    Recent progress in neuroscience has yielded major findings regarding brain maturation during adolescence. Unlike the body, which reaches adult size and morphology during this period, the adolescent brain is still maturing. The prefrontal cortex appears to be an important locus of maturational change subserving executive functions that may regulate emotional and motivational issues. The recent expansion of the adolescent period has increased the lag between the onset of emotional and motivational changes activated by puberty and the completion of cognitive development-the maturation of self-regulatory capacities and skills that are continuing to develop long after puberty has occurred. This "disconnect" predicts risk for a broad set of behavioral and emotional problems. Adolescence is a critical period for high-level cognitive functions such as socialization that rely on maturation of the prefrontal cortex. Intervention during the period of adolescent brain development provides opportunities and requires an interdisciplinary approach.

  1. In vitro maturation and in vitro fertilization of mouse oocytes and preimplantation embryo culture.

    PubMed

    Kidder, Benjamin L

    2014-01-01

    Epigenetic regulation of gene expression in the germline is important for reproductive success of mammals. Misregulation of genes whose expression is correlated with reproductive success may result in subfertility or infertility. To study epigenetic events that occur during oocyte maturation and preimplantation embryo development, it is important to generate large numbers of ovarian follicles and embryos. Oocyte maturation can be modeled using in vitro maturation (IVM), which is a system of maturing ovarian follicles in a culture dish. In addition, fertilization and early embryogenesis can be modeled using in vitro fertilization (IVF), which involves the fertilization of mature oocytes with capacitated sperm in a culture dish. Here, we describe protocols for in vitro maturation (IVM) and in vitro fertilization (IVF) of mouse oocytes and preimplantation embryo culture. These protocols are suitable for the study of oocyte and embryo biology and the production of embryonic mice.

  2. In vitro differentiation of fertile sperm from cryopreserved spermatogonia of the endangered endemic cyprinid honmoroko (Gnathopogon caerulescens)

    PubMed Central

    Higaki, Shogo; Shimada, Manami; Kawamoto, Kazuaki; Todo, Takaaki; Kawasaki, Toshihiro; Tooyama, Ikuo; Fujioka, Yasuhiro; Sakai, Noriyoshi; Takada, Tatsuyuki

    2017-01-01

    Many endemic fish species are threatened with extinction. Conservation strategies and the restoration of endemic fish after extinction must therefore be investigated. Although sperm cryopreservation is indispensable for the conservation of endangered fishes, the limited number of mature fish and limited availability (volume and period) of sperm from small endemic fish hinders the optimization and practical use of this material. In this report, we demonstrate the in vitro differentiation of fertile sperm from cryopreserved spermatogonia of juveniles of the endangered small cyprinid honmoroko (Gnathopogon caerulescens), which is endemic to Lake Biwa in Japan. The entire process of spermatogenesis was recapitulated in vitro using cryopreserved spermatogonia of non-spawning adult and juvenile fish. The differentiation of sperm from spermatogonia was captured as a time-lapse video and confirmed by 5-ethynyl-2′-deoxyuridine (EdU) incorporation into sperm. Fertility was demonstrated by artificial insemination. These results suggest that the combination of cryopreservation of spermatogonia and in vitro sperm differentiation will provide a new and promising strategy for the preservation of paternal genetic materials. PMID:28211534

  3. In vitro differentiation of fertile sperm from cryopreserved spermatogonia of the endangered endemic cyprinid honmoroko (Gnathopogon caerulescens)

    NASA Astrophysics Data System (ADS)

    Higaki, Shogo; Shimada, Manami; Kawamoto, Kazuaki; Todo, Takaaki; Kawasaki, Toshihiro; Tooyama, Ikuo; Fujioka, Yasuhiro; Sakai, Noriyoshi; Takada, Tatsuyuki

    2017-02-01

    Many endemic fish species are threatened with extinction. Conservation strategies and the restoration of endemic fish after extinction must therefore be investigated. Although sperm cryopreservation is indispensable for the conservation of endangered fishes, the limited number of mature fish and limited availability (volume and period) of sperm from small endemic fish hinders the optimization and practical use of this material. In this report, we demonstrate the in vitro differentiation of fertile sperm from cryopreserved spermatogonia of juveniles of the endangered small cyprinid honmoroko (Gnathopogon caerulescens), which is endemic to Lake Biwa in Japan. The entire process of spermatogenesis was recapitulated in vitro using cryopreserved spermatogonia of non-spawning adult and juvenile fish. The differentiation of sperm from spermatogonia was captured as a time-lapse video and confirmed by 5-ethynyl-2‧-deoxyuridine (EdU) incorporation into sperm. Fertility was demonstrated by artificial insemination. These results suggest that the combination of cryopreservation of spermatogonia and in vitro sperm differentiation will provide a new and promising strategy for the preservation of paternal genetic materials.

  4. Semen characteristics and sperm morphology in the Arabian leopard (Panthera pardus nimr) and how these vary with age and season.

    PubMed

    de Haas van Dorsser, Florine J; Strick, Jacqueline A

    2005-01-01

    The Arabian leopard is a critically endangered species. Since there are only an estimated 200 animals remaining in the wild, careful management of the captive population is necessary to minimise inbreeding. The objective of this study was to characterise sperm morphology and ejaculate quality in captive males. Semen was collected by electroejaculation from 8 adult captive male leopards (aged 2-16 years) during the summer and winter months, and semen parameters, including sperm morphology, were assessed. Two-year-old leopards showed lower total sperm counts per ejaculate than older animals and these counts declined at > 8 years. Ejaculates collected during the hot summer showed significantly lower sperm concentrations, total sperm counts, sperm motility and viability and percentage of spermatozoa showing normal morphology than ejaculates collected in the cooler winter. The results showed that the male leopard attains sexual maturity between 2 and 3 years of age and exhibits good semen quality until 8 years. Collection of semen for artificial breeding or banking would best be carried out in the cooler winter months.

  5. Predominance of sperm motion in corners

    PubMed Central

    Nosrati, Reza; Graham, Percival J.; Liu, Qiaozhi; Sinton, David

    2016-01-01

    Sperm migration through the female tract is crucial to fertilization, but the role of the complex and confined structure of the fallopian tube in sperm guidance remains unknown. Here, by confocal imaging microchannels head-on, we distinguish corner- vs. wall- vs. bulk-swimming bull sperm in confined geometries. Corner-swimming dominates with local areal concentrations as high as 200-fold that of the bulk. The relative degree of corner-swimming is strongest in small channels, decreases with increasing channel size, and plateaus for channels above 200 μm. Corner-swimming remains predominant across the physiologically-relevant range of viscosity and pH. Together, boundary-following sperm account for over 95% of the sperm distribution in small rectangular channels, which is similar to the percentage of wall swimmers in circular channels of similar size. We also demonstrate that wall-swimming sperm travel closer to walls in smaller channels (~100 μm), where the opposite wall is within the hydrodynamic interaction length-scale. The corner accumulation effect is more than the superposition of the influence of two walls, and over 5-fold stronger than that of a single wall. These findings suggest that folds and corners are dominant in sperm migration in the narrow (sub-mm) lumen of the fallopian tube and microchannel-based sperm selection devices. PMID:27211846

  6. Geometric Morphometrics of Rodent Sperm Head Shape

    PubMed Central

    Varea Sánchez, María; Bastir, Markus; Roldan, Eduardo R. S.

    2013-01-01

    Mammalian spermatozoa, particularly those of rodent species, are extremely complex cells and differ greatly in form and dimensions. Thus, characterization of sperm size and, particularly, sperm shape represents a major challenge. No consensus exists on a method to objectively assess size and shape of spermatozoa. In this study we apply the principles of geometric morphometrics to analyze rodent sperm head morphology and compare them with two traditional morphometry methods, that is, measurements of linear dimensions and dimensions-derived parameters calculated using formulae employed in sperm morphometry assessments. Our results show that geometric morphometrics clearly identifies shape differences among rodent spermatozoa. It is also capable of discriminating between size and shape and to analyze these two variables separately. Thus, it provides an accurate method to assess sperm head shape. Furthermore, it can identify which sperm morphology traits differ between species, such as the protrusion or retraction of the base of the head, the orientation and relative position of the site of flagellum insertion, the degree of curvature of the hook, and other distinct anatomical features and appendices. We envisage that the use of geometric morphometrics may have a major impact on future studies focused on the characterization of sperm head formation, diversity of sperm head shape among species (and underlying evolutionary forces), the effects of reprotoxicants on changes in cell shape, and phenotyping of genetically-modified individuals. PMID:24312234

  7. Predominance of sperm motion in corners

    NASA Astrophysics Data System (ADS)

    Nosrati, Reza; Graham, Percival J.; Liu, Qiaozhi; Sinton, David

    2016-05-01

    Sperm migration through the female tract is crucial to fertilization, but the role of the complex and confined structure of the fallopian tube in sperm guidance remains unknown. Here, by confocal imaging microchannels head-on, we distinguish corner- vs. wall- vs. bulk-swimming bull sperm in confined geometries. Corner-swimming dominates with local areal concentrations as high as 200-fold that of the bulk. The relative degree of corner-swimming is strongest in small channels, decreases with increasing channel size, and plateaus for channels above 200 μm. Corner-swimming remains predominant across the physiologically-relevant range of viscosity and pH. Together, boundary-following sperm account for over 95% of the sperm distribution in small rectangular channels, which is similar to the percentage of wall swimmers in circular channels of similar size. We also demonstrate that wall-swimming sperm travel closer to walls in smaller channels (~100 μm), where the opposite wall is within the hydrodynamic interaction length-scale. The corner accumulation effect is more than the superposition of the influence of two walls, and over 5-fold stronger than that of a single wall. These findings suggest that folds and corners are dominant in sperm migration in the narrow (sub-mm) lumen of the fallopian tube and microchannel-based sperm selection devices.

  8. Hydrodynamics of Sperm Cells near Surfaces

    PubMed Central

    Elgeti, Jens; Kaupp, U. Benjamin; Gompper, Gerhard

    2010-01-01

    Sperm are propelled by an actively beating tail, and display a wide variety of swimming patterns. When confined between two parallel walls, sperm swim either in circles or on curvilinear trajectories close to the walls. We employ mesoscale hydrodynamics simulations in combination with a mechanical sperm model to study the swimming behavior near walls. The simulations show that sperm become captured at the wall due to the hydrodynamic flow fields which are generated by the flagellar beat. The circular trajectories are determined by the chiral asymmetry of the sperm shape. For strong (weak) chirality, sperm swim in tight (wide) circles, with the beating plane of the flagellum oriented perpendicular (parallel) to the wall. For comparison, we also perform simulations based on a local anisotropic friction of the flagellum. In this resistive force approximation, surface adhesion and circular swimming patterns are obtained as well. However, the adhesion mechanism is now due to steric repulsion, and the orientation of the beating plane is different. Our model provides a theoretical framework that explains several distinct swimming behaviors of sperm near and far from a wall. Moreover, the model suggests a mechanism by which sperm navigate in a chemical gradient via a change of their shape. PMID:20712984

  9. Mass-Specific Metabolic Rate and Sperm Competition Determine Sperm Size in Marsupial Mammals

    PubMed Central

    Tourmente, Maximiliano; Gomendio, Montserrat; Roldan, Eduardo R. S.

    2011-01-01

    Two complementary hypotheses have been proposed to explain variation in sperm size. The first proposes that post-copulatory sexual selection favors an increase in sperm size because it enhances sperm swimming speed, which is an important determinant of fertilization success in competitive contexts. The second hypothesis proposes that mass-specific metabolic rate acts as a constraint, because large animals with low mass-specific metabolic rates will not be able to process resources at the rates needed to produce large sperm. This constraint is expected to be particularly pronounced among mammals, given that this group contains some of the largest species on Earth. We tested these hypotheses among marsupials, a group in which mass-specific metabolic rates are roughly 30% lower than those of eutherian mammals of similar size, leading to the expectation that metabolic rate should be a major constraint. Our findings support both hypotheses because levels of sperm competition are associated with increases in sperm size, but low mass-specific metabolic rate constrains sperm size among large species. We also found that the relationship between sperm size and mass-specific metabolic rate is steeper among marsupials and shallower among eutherian mammals. This finding has two implications: marsupials respond to changes in mass-specific metabolic rate by modifying sperm length to a greater extent, suggesting that they are more constrained by metabolic rate. In addition, for any given mass-specific metabolic rate, marsupials produce longer sperm. We suggest that this is the consequence of marsupials diverting resources away from sperm numbers and into sperm size, due to their efficient sperm transport along the female tract and the existence of mechanisms to protect sperm. PMID:21731682

  10. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation.

    PubMed

    Fernández, Jose Luis; Muriel, Lourdes; Rivero, Maria Teresa; Goyanes, Vicente; Vazquez, Rosana; Alvarez, Juan G

    2003-01-01

    Sperm DNA fragmentation is being increasingly recognized as an important cause of infertility. We herein describe the Sperm Chromatin Dispersion (SCD) test, a novel assay for sperm DNA fragmentation in semen. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm with non-fragmented DNA, following acid denaturation and removal of nuclear proteins. This was confirmed by the analysis of DNA fragmentation using the specific DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) assay, which allows the detection of DNA breaks in lysed sperm nuclei. Sperm suspensions either prepared from semen or isolated from semen by gradient centrifugation were embedded in an agarose microgel on slides and treated with 0.08 N HCl and lysing solutions containing 0.8 M dithiothreitol (DTT), 1% sodium dodecyl sulfate (SDS), and 2 M NaCl. Then, the slides were sequentially stained with DAPI (4',6-diamidino-2-phenylindole) and/or the Diff-Quik reagent, and the percentages of sperm with nondispersed and dispersed chromatin loops were monitored by fluorescence and brightfield microscopy, respectively. The results indicate that all sperm with nondispersed chromatin displayed DNA fragmentation, as measured by DBD-FISH. Conversely, all sperm with dispersed chromatin had very low to undetectable DBD-FISH labeling. SCD test values were significantly higher in patients being screened for infertility than in normozoospermic sperm donors who had participated in a donor insemination program. The coefficient of variation obtained using 2 different observers, either by digital image analysis (DIA) or by brightfield microscopy scoring, was less than 3%. In conclusion, the SCD test is a simple, accurate, highly reproducible, and inexpensive method for the analysis of sperm DNA fragmentation in semen and processed sperm. Therefore, the SCD test could potentially be used as a routine test

  11. Cofilin is correlated with sperm quality and influences sperm fertilizing capacity in humans.

    PubMed

    Chen, S M; Chen, X M; Lu, Y L; Liu, B; Jiang, M; Ma, Y X

    2016-11-01

    Spermatozoa should undergo a series of biochemical modifications in female reproduction tract, which is collectively called sperm capacitation. The capacitated spermatozoa can bind to the egg zona pellucida, resulting in the occurrence of acrosome reaction which enabled spermatozoa penetrate into the egg. The formation of actin plays an important role in these processes. Actin polymerized during sperm capacitation, but the polymers dispersed before acrosome reaction. In this study, we take our focus on actin-binding protein, cofilin. Our results showed that the % and intensity of sperm expressing cofilin in normal sperm were significantly higher than in abnormal sperm, and the sperm expressing cofilin was correlated with sperm quality. Furthermore, treatment with anti-cofilin antibody increased the percentage of sperm capacitation and inhibited progesterone- or A23187- induced acrosome reaction in a dose-dependent manner. The presence of 100 ng/mL anti-cofilin antibodies markedly blocked the sperm penetration of zona-free hamster eggs. Besides, immunofluorescence results revealed that cofilin was colocalized with F-actin in the midpiece of spermatozoa; however, phospho-cofilin was expressed in the tail rather than in the midpiece of spermatozoa, which was not colocalized with F-actin in spermatozoa. Moreover, western blot revealed that phospho-cofilin increased in sperm capacitation, and the total cofilin and cofilin in insoluble fraction increased in acrosome reaction; immunofluorescence results showed that the amount of cofilin in acrosome increased in sperm capacitation. In conclusion, our study revealed that cofilin expression in human sperm is correlated with sperm quality and the alterations of cofilin and phospho-cofilin in fertilization affects sperm capacitation, acrosome reaction, and spermatozoa-oocyte fusion.

  12. Sperm wars and the evolution of male fertility.

    PubMed

    Simmons, Leigh W; Fitzpatrick, John L

    2012-11-01

    Females frequently mate with several males, whose sperm then compete to fertilize available ova. Sperm competition represents a potent selective force that is expected to shape male expenditure on the ejaculate. Here, we review empirical data that illustrate the evolutionary consequences of sperm competition. Sperm competition favors the evolution of increased testes size and sperm production. In some species, males appear capable of adjusting the number of sperm ejaculated, depending on the perceived levels of sperm competition. Selection is also expected to act on sperm form and function, although the evidence for this remains equivocal. Comparative studies suggest that sperm length and swimming speed may increase in response to selection from sperm competition. However, the mechanisms driving this pattern remain unclear. Evidence that sperm length influences sperm swimming speed is mixed and fertilization trials performed across a broad range of species demonstrate inconsistent relationships between sperm form and function. This ambiguity may in part reflect the important role that seminal fluid proteins (sfps) play in affecting sperm function. There is good evidence that sfps are subject to selection from sperm competition, and recent work is pointing to an ability of males to adjust their seminal fluid chemistry in response to sperm competition from rival males. We argue that future research must consider sperm and seminal fluid components of the ejaculate as a functional unity. Research at the genomic level will identify the genes that ultimately control male fertility.

  13. The use of complimentary assays to evaluate the enrichment of human sperm quality in asthenoteratozoospermic and teratozoospermic samples processed with Annexin-V magnetic activated cell sorting.

    PubMed

    Delbes, G; Herrero, M B; Troeung, E-T; Chan, P T K

    2013-09-01

    Sperm chromatin integrity may affect the outcomes of assisted reproductive technology (ART). Developing a clinically reliable strategy to enrich sperm samples with high chromatin quality spermatozoa prior to sperm banking or use in ART would thus be advantageous. The objectives of this study were to: (i) assess the sperm chromatin quality in men with different categories of semen parameters; and (ii) evaluate the extents of Annexin-V magnetic-activated cell sorting (MACS) technology coupled with differential density gradient centrifugation (DGC) in improving sperm chromatin quality. Three categories of men from couples attending a university-based fertility clinic were recruited based on their semen parameters: normozoospermic (n = 13), asthenoteratozoospermic (n = 17) and teratozoospermic (n = 12). For each patient, spermatozoa in semen samples were processed first by DGC to enrich the motility and further by MACS to remove spermatozoa showing apoptotic features. The yield and enrichment of sperm quality was evaluated at each step with conventional semen parameters in conjunction with a combination of five complementary assays, to assess sperm maturity, chromatin structure, compaction and DNA integrity (Hyaluronic Binding Assay, SCSA, chromomycine A3 staining and TUNEL and COMET assays). Our results demonstrated that, compared with normozoospermic samples, raw asthenoteratozoospermic and teratozoospermic samples had a higher proportion of spermatozoa containing DNA breaks, but only asthenoteratozoospermic exhibited altered chromatin structure and decreased binding to hyaluronic acid. Interestingly, the DGC appeared to select for more mature spermatozoa with high DNA compaction. More importantly, in all categories of semen samples, Annexin-V MACS allows enrichment of spermatozoa with good chromatin quality as measured by the TUNEL and SCSA. Because effective treatment modalities to improve sperm DNA damage are limited, our results suggest a potential clinical

  14. Sperm count and sperm motility decrease in old rats.

    PubMed

    Lucio, Rosa Angélica; Tlachi-López, José L; Eguibar, Jose R; Ågmo, Anders

    2013-02-17

    Sexual behavior declines with age in male rats. The rate and magnitude of this decline may depend on the amount of prior sexual experience and a number of other, unidentified factors. Age-dependent changes in the characteristics of ejaculate quality in rats have not been described earlier, and the relationship between such changes and modifications of sexual behavior is likewise unknown. We have recently developed a technique for the detailed analysis of parameters of ejaculate in rats, and this technique was used for the determination of semen and seminal plug characteristics in rats of different ages. Sexually experienced Wistar rats were tested for sex behavior at the ages of 3, 12 and 24 months. Semen was obtained from the female partner immediately after ejaculation at these tests. Between tests, the males were offered the opportunity to copulate once every 3-4 weeks. The behavioral data showed that the latency to ejaculation was increased only at 24 months. Concerning the characteristics of semen, there was a substantial increase in the proportion of immobile spermatozoa and motility of those moving was much reduced, both at 12 and 24 months of age. There was no relationship between parameters of sexual behavior and those of the ejaculate. Likewise, the size of the seminal plug did not affect the amount of intrauterine spermatozoa. The reduced sperm number together with the increased sperm immobility diminishes the ejaculate quality of old males, which could influence fertility.

  15. Mammalian sperm nuclear organization: resiliencies and vulnerabilities.

    PubMed

    Champroux, A; Torres-Carreira, J; Gharagozloo, P; Drevet, J R; Kocer, A

    2016-01-01

    Sperm cells are remarkably complex and highly specialized compared to somatic cells. Their function is to deliver to the oocyte the paternal genomic blueprint along with a pool of proteins and RNAs so a new generation can begin. Reproductive success, including optimal embryonic development and healthy offspring, greatly depends on the integrity of the sperm chromatin structure. It is now well documented that DNA damage in sperm is linked to reproductive failures both in natural and assisted conception (Assisted Reproductive Technologies [ART]). This manuscript reviews recent important findings concerning - the unusual organization of mammalian sperm chromatin and its impact on reproductive success when modified. This review is focused on sperm chromatin damage and their impact on embryonic development and transgenerational inheritance.

  16. Induction of spermatogenesis by rhFSH for azoospermia due to spermatogenic dysfunction with maturation arrest: five case series.

    PubMed

    Kobori, Yoshitomo; Suzuki, Keisuke; Iwahata, Toshiyuki; Shin, Takeshi; Sato, Ryo; Nishio, Kojiro; Yagi, Hiroshi; Arai, Gaku; Soh, Shigehiro; Okada, Hiroshi

    2015-06-01

    When sperm cannot be retrieved from the testes of patients with azoospermia due to spermatogenic dysfunction (ASD), there is no rational way for the patient to become a biological father. We investigated the possibility of inducing spermatogenesis in such patients by hormonal therapy with recombinant human follicle-stimulating hormone (rhFSH) alone. Twenty-six ASD patients who could not obtain spermatozoa by microdissection testicular sperm extraction (micro-TESE) were confirmed to have arrested spermatogenesis at the late stage of maturation arrest. They were subsequently treated with 75-150 IU two times/week rhFSH alone for 12 months. The primary endpoint was the appearance of sperm in ejaculate, and we followed the patients to determine the outcome of inseminating their partners. After rhFSH treatment, mature spermatozoa were found in the ejaculate in five of 26 (19.2%) patients, all of whom showed histology of non-uniform type maturation arrest. Intracytoplasmic sperm injection of the mature spermatozoa resulted in two ongoing clinical pregnancies (insemination success rate, 40.0%). Recombinant human follicle-stimulating hormone treatment can be used as an advanced assisted reproductive technology to improve spermatogenesis in some azoospermic patients with maturation arrest of spermatogenesis and is a potential treatment option after unsuccessful micro-TESE.

  17. The effects of cooling rates and type of freezing extenders on cryosurvival of rat sperm

    PubMed Central

    Varisli, Omer; Scott, Hollie; Agca, Cansu; Agca, Yuksel

    2013-01-01

    Cryopreservation of rat sperm is very challenging due to its sensitivity to various stress factors. The objective of this study was to determine the optimal cooling rate and extender for epididymal sperm of outbred Sprague Dawley (SD) and inbred Fischer 344 (F344) rat strains. The epididymal sperm from 10–12 weeks old sexually mature SD and F344 strains were suspended in five different freezing extenders, namely HEPES buffered Tyrode’s lactate (TL-HEPES), modified Kreb’s Ringer bicarbonate (mKRB), 3% dehydrated skim milk (SM), Salamon’s Tris-citrate (TRIS), and tes/tris (TES). All extenders contained 20% egg yolk, 0.75% Equex Paste and 0.1 M raffinose or 0.1 M sucrose. The sperm samples in each extender were cooled to 4°C and held for 45 min for equilibration before freezing. The equilibrated sperm samples in each extender were placed onto a shallow quartz dish inserted into Linkam Cryostage (BCS 196). The samples were then cooled to a final temperature of −150 °C by using various cooling rates (10, 40, 70, and 100 °C/min). For thawing, the quartz dish containing the sperm samples were rapidly removed from the Linkam cryo-stage and placed on a 37 °C slide warmer and held for 1 min before motility analysis. Sperm membrane and acrosomal integrity and mitochondrial membrane potential (MMP) were assessed by SYBR-14/Propidium iodide, Alexa Fluor-488-PNA conjugate and JC-1, respectively. The total motility, acrosomal integrity, membrane integrity and MMP values were compared among cooling rates and extenders. Both cooling rate and type of extender had significant effect on cryosurvival (P<0.05). Sperm motility increased as cooling rate was increased for both strains (P<0.05). Highest cryosurvival was achieved when 100 °C/min cooling rate was used in combination with TES extender containing 20% egg yolk, 0.75% Equex paste and either 0.1 M sucrose or raffinose (P < 0.05). This study showed that TES extender containing 0.1 M raffinose or sucrose with 70

  18. Sperm Chromatin Immaturity Observed in Short Abstinence Ejaculates Affects DNA Integrity and Longevity In Vitro

    PubMed Central

    Salian, Sujith Raj; Kumar, Dayanidhi; Singh, Vikram Jeet; D’Souza, Fiona; Kalthur, Guruprasad; Kamath, Asha; Adiga, Satish Kumar

    2016-01-01

    Background The influence of ejaculatory abstinence (EA) on semen parameters and subsequent reproductive outcome is still debatable; hence understanding the impact of EA on sperm structural and functional integrity may provide a valuable information on predicting successful clinical outcome. Objective To understand the influence of EA on sperm chromatin maturity, integrity, longevity and global methylation status. Methods This experimental prospective study included 76 ejaculates from 19 healthy volunteers who provided ejaculates after observing 1, 3, 5 and 7 days of abstinence. Sperm chromatin maturity, DNA integrity and global methylation status were assessed in the neat ejaculate. Sperm motility, DNA integrity and longevity were assessed in the processed fraction of the fresh and frozen-thawed ejaculates to determine their association with the length of EA. Results Spermatozoa from 1 day ejaculatory abstinence (EA-1) displayed significantly higher level of sperm chromatin immaturity in comparison to EA-3 (P < 0.05) and EA-5 (P < 0.01) whereas; the number of 5-methyl cytosine immunostained spermatozoa did not vary significantly across groups. On the other hand, in vitro incubation of processed ejaculate from EA-1 resulted in approximately 20 and 40 fold increase in the DNA fragmented spermatozoa at the end of 6 and 24h respectively (P < 0.01–0.001). Conclusion Use of short-term EA for therapeutic fertilization would be a clinically valuable strategy to improve the DNA quality. However, use of such spermatozoa after prolonged incubation in vitro should be avoided as it can carry a substantial risk of transmitting DNA fragmentation to the oocytes. PMID:27043437

  19. Nuclear status of immature and mature stallion spermatozoa.

    PubMed

    Dias, G M; Retamal, C A; Tobella, L; Arnholdt, A C V; López, M L

    2006-07-15

    'The highly packed chromatin of mature spermatozoa results from replacement of somatic-like histones by highly basic arginine- and cysteine-rich protamines during spermatogenesis, with additional conformational changes in chromatin structure during epididymal transit. The objective of the present study was to compare the nuclear characteristics of immature and mature epididymal stallion spermatozoa, using a variety of experimental approaches. Resistance to in vitro decondensation of chromatin, following exposure to SDS-DTT and alkaline thioglycolate, increased significantly in mature spermatozoa. Evaluation of the thiol-disulfide status (monobromobimane labeling) demonstrated that immature cells obtained from ductulli efferentes contained mostly thiol groups, whereas these groups were oxidized in mature cells collected from the cauda epididymidis. Based on atomic absorption spectrophotometry, maturation of stallion spermatozoa was accompanied by a 60% reduction in the Zn(2+) content of sperm cells, concomitant with increased concentrations of this ion in epididymal fluid. Furthermore, the degree of disulfide bonding was inversely correlated with susceptibility of chromatin to acid denaturation (SCSA). Collectively, these data were consistent with the hypothesis that maturation of stallion spermatozoa involves oxidation of sulphydryl groups to form intra- and intermolecular disulfide links between adjacent protamines, with loss of zinc as an integral feature. These changes endow mechanical and chemical resistance to the nucleus, ensuring efficient transmission of the paternal genome at fertilization.

  20. Cysteamine supplementation revealed detrimental effect on cryosurvival of buffalo sperm based on computer-assisted semen analysis and oxidative parameters.

    PubMed

    Swami, Dheer Singh; Kumar, Pradeep; Malik, R K; Saini, Monika; Kumar, Dharmendra; Jan, M H

    2017-02-01

    The aim of this study was to investigate the effect of addition of cysteamine to the semen extender on post-thaw semen quality. A total of 30 ejaculates were collected from six bulls. Each ejaculate was divided into five equal parts and diluted to final concentration of 80 million sperms/mL using Optixcell(®)(IMV, France) semen extender supplemented with different concentrations of cysteamine (0, 0.75, 1.25, 2.5 and 5mM) and cryopreserved. In the frozen-thawed samples, the VAP, VSL, VCL ALH and sperm motility of control samples was greater (P<0.05) than cysteamine treated samples. The sperm abnormality and malondialdehyde (MDA) concentration were found highest in 5mM cysteamine treated samples. The cysteamine treated samples travelled significantly less distance in cervical mucus as compared to control. Further, cysteamine decreased acrosomal integrity of sperm. In incubation test, control samples showed better sperm motility as compared to treatment groups. Further, cysteamine supplementation decreased the total antioxidants and increased the MDA concentration of sperm. From the study, we hypothesized that cysteamine cannot stimulate synthesis of glutathione (GSH) intracellularly in sperm to combat free radicals because during the maturation, sperm lost its cytoplasm which is necessary for biochemical reaction in which cysteamine reacts with cystine to form a mixed disulfide which taken up by cells and split into cysteine in the cytoplasm. Synthesis of GSH depends on the availability of cysteine. In conclusion, the results of our study strongly emphasize that cysteamine would not be a suitable additive in extender for freezing buffalo bull semen.

  1. Effects of cytoplasmic genes on sperm viability and sperm morphology in a seed beetle: implications for sperm competition theory?

    PubMed

    Dowling, D K; Nowostawski, A Larkeson; Arnqvist, G

    2007-01-01

    Sperm competition theory predicts that sperm traits influencing male fertilizing ability will evolve adaptively. However, it has been suggested that some sperm traits may be at least partly encoded by mitochondrial genes. If true, this may constrain the adaptive evolution of such traits because mitochondrial DNA (mtDNA) is maternally inherited and there is thus no selection on mtDNA in males. Phenotypic variation in such traits may nevertheless be high because mutations in mtDNA that have deleterious effects on male traits, but neutral or beneficial effects in females, may be maintained by random processes or selection in females. We used backcrossing to create introgression lines of seed beetles (Callosobruchus maculatus), carrying orthogonal combinations of distinct lineages of cytoplasmic and nuclear genes, and then assayed sperm viability and sperm length in all lines. We found sizeable cytoplasmic effects on both sperm traits and our analyses also suggested that the cytoplasmic effects varied across nuclear genetic backgrounds. We discuss some potential implications of these findings for sperm competition theory.

  2. Selenoprotein P in seminal fluid is a novel biomarker of sperm quality.

    PubMed

    Michaelis, Marten; Gralla, Oliver; Behrends, Thomas; Scharpf, Marcus; Endermann, Tobias; Rijntjes, Eddy; Pietschmann, Nicole; Hollenbach, Birgit; Schomburg, Lutz

    2014-01-17

    Hepatically-derived selenoprotein P (SePP) transports selenium (Se) via blood to other tissues including the testes. Male Sepp-knockout mice are infertile. SePP-mediated Se transport to Sertoli cells is needed for supporting biosynthesis of the selenoenzyme glutathione peroxidase-4 (GPX4) in spermatozoa. GPX4 becomes a structural component of sperm midpiece during sperm maturation, and its expression correlates to semen quality. We tested whether SePP is also present in seminal plasma, potentially correlating to fertility parameters. Semen quality was assessed by sperm density, morphology and motility. SePP was measured by an immunoluminometric assay, and trace elements were determined by X-ray fluorescence spectroscopy. SePP levels were considerably lower in seminal plasma as compared to serum (0.4±0.1 mg/l vs. 3.5±1.0 mg/l); Se concentrations showed a similar but less pronounced difference (48.9±20.7 μg/l vs. 106.7±17.3 μg/l). Se and Zn correlated positively in seminal fluid but not in serum. Seminal plasma SePP concentrations were independent of serum SePP concentrations, but correlated positively to sperm density and fraction of vital sperm. SePP concentrations in seminal plasma of vasectomized men were similar to controls indicating that accessory sex glands are a testes-independent source of SePP. This notion was corroborated by histochemical analyses localizing SePP in epithelial cells of seminal vesicles. We conclude that SePP is not only involved in Se transport to testes supporting GPX4 biosynthesis but it also becomes secreted into seminal plasma, likely important to protect sperm during storage, genital tract passage and final journey.

  3. Formation and Dissociation of Sperm Bundles in Monotremes.

    PubMed

    Nixon, Brett; Ecroyd, Heath; Dacheux, Jean-Louis; Dacheux, Francoise; Labas, Valerie; Johnston, Steve D; Jones, Russell C

    2016-10-01

    Because monotremes are the earliest offshoot of the mammalian lineage, the platypus and short-beaked echidna were studied as model animals to assess the origin and biological significance of adaptations considered unique to therian mammals: epididymal sperm maturation and subsequent capacitation. We show that spermatozoa from both species assemble into bundles of approximately 100 cells during passage through the epididymis and that an epididymal protein-secreted protein, acidic, cysteine-rich (osteonectin; SPARC)-is involved in bundle formation. The bundles persisted during incubation in vitro for at least 1 h under conditions that capacitate therian spermatozoa, and then underwent a time-dependent dissociation to release spermatozoa capable of fertilization. Only after this dissociation could the spermatozoa bind to the perivitelline membrane of a hen's egg, display an altered form of motility reminiscent of hyperactivation, and be induced to undergo an acrosome reaction. It is concluded that the development of sperm bundles in the monotreme epididymis mandates that they require a time-dependent process to be capable of fertilizing an ovum. However, because this functional end point was achieved without overt changes in protein tyrosine phosphorylation (a hallmark of capacitation in therians), it is concluded that the process in monotremes is distinctly different from capacitation in therian mammals.

  4. The sperm nucleus: chromatin, RNA and the nuclear matrix

    PubMed Central

    Johnson, Graham D.; Lalancette, Claudia; Linnemann, Amelia K.; Leduc, Frédéric; Boissonneault, Guylain; Krawetz, Stephen A.

    2017-01-01

    Within the sperm nucleus the paternal genome remains functionally inert and protected following protamination. This is marked by a structural morphogenesis that is heralded by a striking reduction in nuclear volume. Despite these changes, both human and mouse spermatozoa maintain low levels of nucleosomes that appear non-randomly distributed throughout the genome. These regions may be necessary for organizing higher order genomic structure through interactions with the nuclear matrix. The promoters of this transcriptionally quiescent genome are differentially marked by modified histones that may poise downstream epigenetic effects. This notion is supported by increasing evidence that the embryo inherits these differing levels of chromatin organization. In concert with the suite of RNAs retained in the mature sperm they may synergistically interact to direct early embryonic gene expression. Irrespective, these features reflect the transcriptional history of spermatogenic differentiation. As such they may soon be utilized as clinical markers of male fertility. In this review we explore and discuss how this may be orchestrated. PMID:20876223

  5. Protein and carbohydrate intake influence sperm number and fertility in male cockroaches, but not sperm viability

    PubMed Central

    Bunning, Harriet; Rapkin, James; Belcher, Laurence; Archer, C. Ruth; Jensen, Kim; Hunt, John

    2015-01-01

    It is commonly assumed that because males produce many, tiny sperm, they are cheap to produce. Recent work, however, suggests that sperm production is not cost-free. If sperm are costly to produce, sperm number and/or viability should be influenced by diet, and this has been documented in numerous species. Yet few studies have examined the exact nutrients responsible for mediating these effects. Here, we quantify the effects of protein (P) and carbohydrate (C) intake on sperm number and viability in the cockroach Nauphoeta cinerea, as well as the consequences for male fertility. We found the intake of P and C influenced sperm number, being maximized at a high intake of diets with a P : C ratio of 1 : 2, but not sperm viability. The nutritional landscapes for male fertility and sperm number were closely aligned, suggesting that sperm number is the major determinant of male fertility in N. cinerea. Under dietary choice, males regulate nutrient intake at a P : C ratio of 1 : 4.95, which is midway between the ratios needed to maximize sperm production and pre-copulatory attractiveness in this species. This raises the possibility that males regulate nutrient intake to balance the trade-off between pre- and post-copulatory traits in this species. PMID:25608881

  6. Cryopreservation of European catfish Silurus glanis sperm: sperm motility, viability, and hatching success of embryos.

    PubMed

    Linhart, Otomar; Rodina, Marek; Flajshans, Martin; Gela, David; Kocour, Martin

    2005-12-01

    The aim of this study was to elaborate cryopreservation methods for ex situ conservation of European catfish. The success of sperm cryopreservation was evaluated by post-thaw sperm motility and velocity, percentage of live spermatozoa and fertility (hatching rates) using frozen/thawed sperm. The best hatching rates of 82-86% were obtained with sperm stored for 5 h before freezing in immobilizing solution and frozen with Me2SO in concentrations of 8, 10, and 12%, or with a mixture of 5% Me2SO and 5% propandiole. These results did not significantly differ from the fresh sperm control sample. The percentage of live spermatozoa in frozen/thawed sperm did not correlate with hatching rate or motility of spermatozoa, but was negatively correlated with velocity of spermatozoa (r=-0.47, P=0.05). The percentage motility in frozen/thawed sperm ranged from 8 to 62%, when sperm was stored in immobilizing solution 5h before freezing. The average value in the fresh sperm (control) was 96%. The frozen/thawed sperm motility rate significantly correlated with the hatching rate (r=0.76, P=0.0002), but not with the percentage of live spermatozoa (r=0.16, P=0.52) or the sperm velocity (r=0.07, P=0.79). The velocity of frozen/thawed spermatozoa ranged from 37 to 85 microm/s, whereby methanol concentrations of 7.5 and 10% resulted in highest velocities. Freezing sperm volumes of 1-4 ml did not affect the quality of frozen/thawed sperm.

  7. Sperm characterization and identification of sperm sub-populations in ejaculates from pampas deer (Ozotoceros bezoarticus).

    PubMed

    Beracochea, F; Gil, J; Sestelo, A; Garde, J J; Santiago-Moreno, J; Fumagalli, F; Ungerfeld, R

    2014-10-01

    Pampas deer (Ozotoceros bezoarticus) is a native endangered species. Knowledge of the basic spermiogram characteristics and the morphometric descriptors is necessary to effectively develop sperm cryopreservation. In other species, sperm sub-population is related to sperm cryo-resistance. The objective was to provide a general description of the sperm, including sperm head morphometric descriptors, its repeatability, and the existence of sperm sub-populations. Sperm were obtained from adult males by electroejaculation during the breeding season. The motility score was 3.4 ± 0.2 (mean ± SEM) and progressive motility was 59.4 ± 3.7%. Ejaculated volume was 413.9 ± 51.0 μl, the total number of sperm ejaculated was 321.2 ± 55.4 × 10(6). Also, 63.3 ± 3.1% of the sperm were morphologically abnormal and 23.7 ± 2.3% had acrosome damage. The sperm head length was 7.6 ± 0.01 μm, width 4.4 ± 0.01 μm, area 28.1 ± 0.07 μm(2) and the perimeter was 21.9 ± 0.04 μm. There was a positive relationship among morphometric descriptors and the motility score, overall motility and progressive motility. Also length (P=0.011), width (P=0.003), area (P=0.006) and perimeter (P=0.009) of sperm head obtained in two different collections were positively related. Overall, the low concentration, volume, overall quality and abnormal morphology, and wide variation of these variables may be a limitation for the development of sperm cryopreserved banks. There were three sperm sub-populations with different morphometric characteristics. The morphometric descriptors are maintained similarly among different collections.

  8. Cryopreservation of sperm in common carp Cyprinus carpio: sperm motility and hatching success of embryos.

    PubMed

    Linhart, O; Rodina, M; Cosson, J

    2000-11-01

    In this study, fish sperm cryopreservation methods were elaborated upon for ex situ conservation of nine strains of Bohemian common carp. Common carp sperm were diluted in Kurokura medium and chilled to 4 degrees C and dimethyl sulfoxide was added. Cryotubes of sperm with media were then cooled from +4 to -9 degrees C at a rate of 4 degrees C min(-1) and then from -9 to -80 degrees C at a rate of 11 degrees C min(-1), held for 6 min at -80 degrees C, and finally transferred into liquid N(2). The spermatozoa were thawed in a water bath at 35 degrees C for 110 s and checked for fertilization yield, hatching yield of embryos, and larval malformations. Fresh and frozen/thawed sperm were evaluated for the percentage and for the velocity of motile sperm from video frames using image analysis. The percentage and velocity of sperm motility at 15 s after activation of frozen/thawed sperm was significantly lower than that of fresh sperm (nine males). ANOVA showed a significant influence of fresh vs frozen/thawed sperm on fertilization rate (P < 0.0001), but differences in hatching rate and in larval malformation (0-6.8%) were not significant, and different males had a significant influence on fertilization and hatching rate (P < 0.003 and P < 0.007, respectively). Multiple range analysis (LSD) showed significant differences between fresh and frozen/thawed sperm regarding fertilization rate (68 +/- 11 and 56 +/- 10%, respectively) and insignificant differences between fresh and frozen/thawed sperm on the hatching rate (50 +/- 18 and 52 +/- 9%, respectively). The percentage and velocity of fresh sperm motility were correlated, respectively, with the fertilization yield of frozen/thawed sperm at the levels r = 0.51 and r = 0.54.

  9. Toward Teacher Maturity.

    ERIC Educational Resources Information Center

    Pickle, Judy

    1985-01-01

    The essence of teacher maturity can be synthesized into personal, professional, and process domains. Although overlapping, these categories add a multidimensional approach to the search for what is good in teaching and provide a model for professional development. (MT)

  10. Effect of melatonin on maturation capacity and fertilization of Nili-Ravi buffalo (Bubalus bubalis) oocytes

    PubMed Central

    Nagina, G.; Asima, A.; Nemat, U.; Shamim, A.

    2016-01-01

    This study evaluated the effect of melatonin supplementation of in vitro maturation media on in vitro maturation (IVM) and in vitro fertilization (IVF) rate of buffalo oocytes. Cumulus oocytes complexes (COCs) were aspirated from follicles of 2-8 mm diameter. In experiment I, COCs were matured in IVM medium supplemented with 0 (control), 250, 500, and 1000 μM melatonin for 22-24 hours in CO2 incubator at 38.5°C with 5% CO2 and at 95% relative humidity. The maturation rate did not differ in media supplemented with melatonin at 250 μM, 500 μM, 1000 μM and control (0 μM). In experiment II, the matured oocytes were fertilized in 50 μl droplets of Tyrode’s Albumin Lactate Pyruvate (TALP) medium having 10 ug/ml heparin for sperm (2 million/ml) capacitation. The fertilization droplets were then kept for incubation at 5% CO2, 39°C and at 95% relative humidity for 18 hours. The fertilization rate was assessed by sperm penetration and pronuclear formation. Fertilization rate was improved when maturation medium was supplemented with 250 μM melatonin compared to control. In conclusion, melatonin supplementation to serum free maturation media at 250 μM improved the fertilization rate of buffalo oocytes. PMID:27540514

  11. SpermBlue: a new universal stain for human and animal sperm which is also amenable to automated sperm morphology analysis.

    PubMed

    van der Horst, G; Maree, L

    2009-12-01

    Our study was aimed at exploring a simple procedure to stain differentially the acrosome, head, midpiece, and flagellum of human and animal sperm. A further prerequisite was that sperm morphology of the stained samples could be analyzed using automated sperm morphology analysis (ASMA). We developed a new staining process using SpermBlue fixative and SpermBlue stain, which are iso-osmotic in relation to semen. The entire fixation and staining processes requires only 25 min. Three main steps are required. First, a routine sperm smear is made by either using semen or sperm in a diluting medium. The smear is allowed to air dry at room temperature. Second, the smear is fixed for 10 min by either placing the slide with the dried smear in a staining tray containing SpermBlue fixative or by adding 1 ml SpermBlue fixative to the slide. Third, the fixed smear is stained for 15 min by either immersing the slide in a staining tray containing SpermBlue stain or adding four drops of SpermBlue stain to the fixed smear. The stained slide is dipped gently in distilled water followed by air drying and mounting in DPX or an equivalent medium. The method is simple and suitable for field conditions. Sperm of human, three monkey species, horse, boar, bull, ram, mouse, rat, domestic chicken, fish, and invertebrate species were stained successfully using the SpermBlue staining process. SpermBlue stains human and animal sperm different hues or intensities of blue. It is possible to distinguish clearly the acrosome, sperm head, midpiece, principal piece of the tail, and even the short end piece. The Sperm Class Analyzer ASMA system was used successfully to quantify sperm head and midpiece measurements automatically at either 600 x or 1000 x magnification for most of the species studied.

  12. Suspended sediments limit coral sperm availability

    PubMed Central

    Ricardo, Gerard F.; Jones, Ross J.; Clode, Peta L.; Humanes, Adriana; Negri, Andrew P.

    2015-01-01

    Suspended sediment from dredging activities and natural resuspension events represent a risk to the reproductive processes of coral, and therefore the ongoing maintenance of reefal populations. To investigate the underlying mechanisms that could reduce the fertilisation success in turbid water, we conducted several experiments exposing gametes of the corals Acropora tenuis and A. millepora to two sediment types. Sperm limitation was identified in the presence of siliciclastic sediment (230 and ~700 mg L−1), with 2–37 fold more sperm required to achieve maximum fertilisation rates, when compared with sediment-free treatments. This effect was more pronounced at sub-optimum sperm concentrations. Considerable (>45%) decreases in sperm concentration at the water’s surface was recorded in the presence of siliciclastic sediment and a >20% decrease for carbonate sediment. Electron microscopy then confirmed sediment entangled sperm and we propose entrapment and sinking is the primary mechanism reducing sperm available to the egg. Longer exposure to suspended sediments and gamete aging further decreased fertilisation success when compared with a shorter exposure. Collectively, these findings demonstrate that high concentrations of suspended sediments effectively remove sperm from the water’s surface during coral spawning events, reducing the window for fertilisation with potential subsequent flow-on effects for recruitment. PMID:26659008

  13. Redox regulation of mammalian sperm capacitation

    PubMed Central

    O’Flaherty, Cristian

    2015-01-01

    Capacitation is a series of morphological and metabolic changes necessary for the spermatozoon to achieve fertilizing ability. One of the earlier happenings during mammalian sperm capacitation is the production of reactive oxygen species (ROS) that will trigger and regulate a series of events including protein phosphorylation, in a time-dependent fashion. The identity of the sperm oxidase responsible for the production of ROS involved in capacitation is still elusive, and several candidates are discussed in this review. Interestingly, ROS-induced ROS formation has been described during human sperm capacitation. Redox signaling during capacitation is associated with changes in thiol groups of proteins located on the plasma membrane and subcellular compartments of the spermatozoon. Both, oxidation of thiols forming disulfide bridges and the increase on thiol content are necessary to regulate different sperm proteins associated with capacitation. Reducing equivalents such as NADH and NADPH are necessary to support capacitation in many species including humans. Lactate dehydrogenase, glucose-6-phospohate dehydrogenase, and isocitrate dehydrogenase are responsible in supplying NAD (P) H for sperm capacitation. Peroxiredoxins (PRDXs) are newly described enzymes with antioxidant properties that can protect mammalian spermatozoa; however, they are also candidates for assuring the regulation of redox signaling required for sperm capacitation. The dysregulation of PRDXs and of enzymes needed for their reactivation such as thioredoxin/thioredoxin reductase system and glutathione-S-transferases impairs sperm motility, capacitation, and promotes DNA damage in spermatozoa leading to male infertility. PMID:25926608

  14. Comparative profiling of the sperm proteome.

    PubMed

    Holland, Ashling; Ohlendieck, Kay

    2015-02-01

    The highly complex and species-selective mechanism of fertilization is a central theme of developmental biology. Gametogenesis, sperm activation, and egg-sperm recognition are fundamental biological processes, warranting detailed studies into the molecular composition of gametes. Biological MS has been instrumental for the comprehensive itemizing of gamete proteomes. The protein constellation of sperm cells and its subcellular structures has been established for a variety of animal species. Spermatogenesis and the crucial activation of sperm cells as a prerequisite of successful fertilization and physiological adaptations to external stressors was investigated using proteomics, as well as the underlying mechanisms of male infertility with respect to proteome-wide alterations. This review outlines recent achievements of sperm proteomics and exemplifies the usefulness of gel-based surveys by outlining the comparative analysis of abnormal spermatozoa in globozoospermia. Besides label-free MS techniques and cell-based labeling methodology, high-resolution fluorescence 2DE has been shown to be highly suitable as a proteomic biomarker discovery tool in sperm protein research. The appropriateness of novel protein markers for improving our understanding of normal spermatogenesis and sperm activation versus the molecular pathogenesis of male infertility will be discussed. New biomarker candidates might be useful to improve diagnostic, prognostic, and therapeutic aspects of infertility.

  15. Suspended sediments limit coral sperm availability.

    PubMed

    Ricardo, Gerard F; Jones, Ross J; Clode, Peta L; Humanes, Adriana; Negri, Andrew P

    2015-12-14

    Suspended sediment from dredging activities and natural resuspension events represent a risk to the reproductive processes of coral, and therefore the ongoing maintenance of reefal populations. To investigate the underlying mechanisms that could reduce the fertilisation success in turbid water, we conducted several experiments exposing gametes of the corals Acropora tenuis and A. millepora to two sediment types. Sperm limitation was identified in the presence of siliciclastic sediment (230 and ~700 mg L(-1)), with 2-37 fold more sperm required to achieve maximum fertilisation rates, when compared with sediment-free treatments. This effect was more pronounced at sub-optimum sperm concentrations. Considerable (>45%) decreases in sperm concentration at the water's surface was recorded in the presence of siliciclastic sediment and a >20% decrease for carbonate sediment. Electron microscopy then confirmed sediment entangled sperm and we propose entrapment and sinking is the primary mechanism reducing sperm available to the egg. Longer exposure to suspended sediments and gamete aging further decreased fertilisation success when compared with a shorter exposure. Collectively, these findings demonstrate that high concentrations of suspended sediments effectively remove sperm from the water's surface during coral spawning events, reducing the window for fertilisation with potential subsequent flow-on effects for recruitment.

  16. Tolerance to environmental desiccation in moss sperm.

    PubMed

    Shortlidge, Erin E; Rosenstiel, Todd N; Eppley, Sarah M

    2012-05-01

    • Sexual reproduction in mosses requires that sperm be released freely into the environment before finding and fertilizing a receptive female. After release from the male plant, moss sperm may experience a range of abiotic stresses; however, few data are available examining stress tolerance of moss sperm and whether there is genetic variation for stress tolerance in this important life stage. • Here, we investigated the effects of environmental desiccation and recovery on the sperm cells of three moss species (Bryum argenteum, Campylopus introflexus, and Ceratodon purpureus). • We found that a fraction of sperm cells were tolerant to environmental desiccation for extended periods (d) and that tolerance did not vary among species. We found that this tolerance occurs irrespective of ambient dehydration conditions, and that the addition of sucrose during dry-down improved cell recovery. Although we observed no interspecific variation, significant variation among individuals within species in sperm cell tolerance to environmental desiccation was observed, suggesting selection could potentially act on this basic reproductive trait. • The observation of desiccation-tolerant sperm in multiple moss species has important implications for understanding bryophyte reproduction, suggesting the presence of a significant, uncharacterized complexity in the ecology of moss mating systems.

  17. Patterns of sperm damage in Chernobyl passerine birds suggest a trade-off between sperm length and integrity.

    PubMed

    Hermosell, Ignacio G; Laskemoen, Terje; Rowe, Melissah; Møller, Anders P; Mousseau, Timothy A; Albrecht, Tomás; Lifjeld, Jan T

    2013-10-23

    Interspecific variation in sperm size is enigmatic, but generally assumed to reflect species-specific trade-offs in selection pressures. Among passerine birds, sperm length varies sevenfold, and sperm competition risk seems to drive the evolution of longer sperm. However, little is known about factors favouring short sperm or constraining the evolution of longer sperm. Here, we report a comparative analysis of sperm head abnormalities among 11 species of passerine bird in Chernobyl, presumably resulting from chronic irradiation following the 1986 accident. Frequencies of sperm abnormalities varied between 15.7 and 77.3% among species, more than fourfold higher than in uncontaminated areas. Nonetheless, species ranked similarly in sperm abnormalities in unpolluted areas as in Chernobyl, pointing to intrinsic factors underlying variation in sperm damage among species. Scanning electron microscopy of abnormal spermatozoa revealed patterns of acrosome damage consistent with premature acrosome reaction. Sperm length, but not sperm competition risk explained variation in sperm damage among species. This suggests that longer spermatozoa are more susceptible to premature acrosome reaction. Therefore, we hypothesize a trade-off between sperm length and sperm integrity affecting sperm evolution in passerine birds.

  18. Oxidative stress in zebrafish (Danio rerio) sperm.

    PubMed

    Hagedorn, Mary; McCarthy, Megan; Carter, Virginia L; Meyers, Stuart A

    2012-01-01

    Laboratories around the world have produced tens of thousands of mutant and transgenic zebrafish lines. As with mice, maintaining all of these valuable zebrafish genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. Because reducing oxidative stress has become an important aspect of reducing the variability in mouse sperm cryopreservation, we examined whether antioxidants might improve cryopreservation of zebrafish sperm. Four experiments were conducted in this study. First, we used the xanthine-xanthine oxidase (X-XO) system to generate reactive oxygen species (ROS). The X-XO system was capable of producing a stress reaction in zebrafish sperm reducing its sperm motility in a concentration dependent manner (P<0.05). Second, we examined X-XO and the impact of antioxidants on sperm viability, ROS and motility. Catalase (CAT) mitigated stress and maintained viability and sperm motility (P>0.05), whereas superoxide dismutase (SOD) and vitamin E did not (P<0.05). Third, we evaluated ROS in zebrafish spermatozoa during cryopreservation and its effect on viability and motility. Methanol (8%) reduced viability and sperm motility (P<0.05), but the addition of CAT mitigated these effects (P>0.05), producing a mean 2.0 to 2.9-fold increase in post-thaw motility. Fourth, we examined the effect of additional cryoprotectants and CAT on fresh sperm motility. Cryoprotectants, 8% methanol and 10% dimethylacetamide (DMA), reduced the motility over the control value (P<0.5), whereas 10% dimethylformamide (DMF) with or without CAT did not (P>0.05). Zebrafish sperm protocols should be modified to improve the reliability of the cryopreservation process, perhaps using a different cryoprotectant. Regardless, the simple addition of CAT to present-day procedures will significantly improve this process, assuring increased and less variable fertilization success and allowing resource managers to dependably plan how many straws are needed to safely

  19. Turbidity as a method of preparing sperm dilutions in the echinoid sperm/egg bioassay

    SciTech Connect

    Hall, T.J.; Haley, R.K.; Battan, K.J. )

    1993-11-01

    The use of turbidimeter for preparing sperm dilutions used in the echinoid sperm/egg bioassay was evaluated. Regression analyses of the relationship between sperm density and turbidity for the sea urchins Strongylocentrotus purpuratus and Strongylocentrotus droebachiensis and the sand dollar Dendraster excentricus indicated that although there were slope differences for each species, each coefficient of determination was highly significant. For Dendraster excentricus, triplicate hemacytometer counts over a range of turbidities as well as repeated preparations of a single sperm turbidity indicated similar variability for each. The use of the turbidimeter has time-saving advantages over conventional hemacytometer methods without sacrificing precision. Sperm dilutions can be prepared rapidly, minimizing seawater sperm preactivation before test initiation, and may therefore contribute to increased test precision.

  20. Using sperm posthumously: national guidelines versus practice.

    PubMed

    Raziel, Arieh; Friedler, Shevach; Strassburger, Deborah; Kaufman, Sarit; Umansky, Ana; Ron-El, Raphael

    2010-08-01

    Under pressure from involved families and time limitations, Israeli judges tend to authorize postmortem sperm retrieval and freezing, even in unmarried men and in contradiction to current, albeit non-legally binding, guidelines of the Attorney General. Postmortem sperm retrieval in our institution (2003 to June 2009) was successful in 17 cases, motile sperm was found in 16/17, and cryopreservation was performed in 15/16: no application for use of any of the postmortem frozen samples has been performed thus far.

  1. Heat shock suppresses mating and sperm transfer in the rice leaf folder Cnaphalocrocis medinalis.

    PubMed

    Liao, H J; Qian, Q; Liu, X D

    2014-06-01

    Temperature is a key environmental factor in determining the population size of Cnaphalocrocis medinalis in summer. High temperatures inhibit survival, development and fecundity of this insect. However, biological responses of female and male adults to heat shock, and physiological mechanism of high temperature suppressing population development are still ambiguous. We experimentally tested the impact of heat shock (5 h day-1) on biological traits, spermatogenesis and sperm transfer of adults of C. medinalis. The result showed that heat exposure to 39 and 40 °C for 5 h reduced longevity and copulation frequency of adults, and hatchability of eggs. Immediate survival rate of males was lower than that of females after 3 days of exposure to 41 °C. The oviposition period, copulation frequency, fecundity of adults and hatchability of eggs were significantly lower when male adults were exposed to 40 or 41 °C for 3 days. Heat shock decreased frequency and success rate of mating when males were exposed, and it also resulted in postponement of mating behaviour and prolongation of mating duration as both the female and male adults were exposed. Heat shock did not affect spermatogenesis, but significantly inhibited sperms maturation. Moreover, males could not ejaculate sperm into females during copulation when these male moths received heat shock. Heat shock remarkably suppressed mating behaviour and sperm transfer, which led to a dramatic decline of rice leaf folder populations.

  2. Participation of the sperm proteasome during in vitro fertilisation and the acrosome reaction in cattle.

    PubMed

    Sánchez, R; Deppe, M; Schulz, M; Bravo, P; Villegas, J; Morales, P; Risopatrón, J

    2011-04-01

    In this work, we have investigated the role of the bovine sperm proteasome during in vitro fertilisation (IVF) and the acrosome reaction (AR). Motile spermatozoa, obtained by a swim-up method in Sperm-Talp medium, were capacitated for 3.5 h and incubated in the presence or absence of the specific proteasome inhibitor epoxomicin for 30 and 60 min. Then, the spermatozoa were co-incubated with mature bovine cumulus oocytes and after 48 h the cleavage rate of inseminated oocytes was evaluated. In addition, we evaluated the participation of the sperm proteasome during the progesterone-induced AR. Capacitated spermatozoa were incubated for 30 min with or without epoxomicin, then progesterone was added and the ARs were evaluated using the dual fluorescent staining technique 'Hoechst and chlortetracycline'. The results indicate that the proteasome inhibitor decreased the cleavage rate of oocytes inseminated with treated spermatozoa. In addition, acrosomal exocytosis levels were statistically significantly higher in the samples treated with the AR inducer progesterone than in control samples in the absence of the inducer. However, the progesterone-induced AR was significantly reduced by previous treatment of the spermatozoa with epoxomicin (P < 0.001). These observations indicate that the bovine sperm proteasome participates in the IVF and AR processes.

  3. Ovarian Fluid Mediates the Temporal Decline in Sperm Viability in a Fish with Sperm Storage

    PubMed Central

    Gasparini, Clelia; Evans, Jonathan P.

    2013-01-01

    A loss of sperm viability and functionality during sperm transfer and storage within the female reproductive tract can have important fitness implications by disrupting fertilization and impairing offspring development and survival. Consequently, mechanisms that mitigate the temporal decline in sperm function are likely to be important targets of selection. In many species, ovarian fluid is known to regulate and maintain sperm quality. In this paper, we use the guppy Poecilia reticulata, a highly polyandrous freshwater fish exhibiting internal fertilization and sperm storage, to determine whether ovarian fluid (OF) influences the decline in sperm viability (the proportion of live sperm in the ejaculate) over time and whether any observed effects depend on male sexual ornamentation. To address these questions we used a paired experimental design in which ejaculates from individual males were tested in vitro both in presence and absence of OF. Our results revealed that the temporal decline in sperm viability was significantly reduced in the presence of OF compared to a saline control. This finding raises the intriguing possibility that OF may play a role in mediating the decline in sperm quality due to the deleterious effects of sperm ageing, although other possible explanations for this observation are discussed. Interestingly, we also show that the age-related decline in sperm viability was contingent on male sexual ornamentation; males with relatively high levels of iridescence (indicating higher sexual attractiveness) exhibited a more pronounced decline in sperm viability over time than their less ornamented counterparts. This latter finding offers possible insights into the functional basis for the previously observed trade-off between these key components of pre- and postcopulatory sexual selection. PMID:23691216

  4. Tactic-specific differences in seminal fluid influence sperm performance.

    PubMed

    Locatello, Lisa; Poli, Federica; Rasotto, Maria B

    2013-03-22

    Seminal fluid often makes up a large part of an ejaculate, yet most empirical and theoretical studies on sperm competition have focused on how sperm characteristics (number and quality) affect fertilization success. However, seminal fluid influences own sperm performance and may potentially influence the outcome of sperm competition, by also affecting that of rivals. As a consequence males may be expected to allocate their investment in both sperm and seminal fluid in relation to the potential level of competition. Grass goby (Zosterisessor ophiocephalus) is an external fertilizer with guard-sneaker mating tactics, where sperm competition risk varies according to the tactic adopted. Here, we experimentally manipulated grass goby ejaculates by separately combining sperm and seminal fluid from territorial and sneaker males. While sperm of sneaker and territorial males did not differ in their performance when they interacted with their own seminal fluid only, sperm of sneakers increased their velocity and fertilization rate in the presence of territorial males' seminal fluid. By contrast, sneaker males' seminal fluid had a detrimental effect on the performance of territorial males' sperm. Sperm velocity was unaffected by the seminal fluid of males employing the same tactic, suggesting that seminal fluid's effect on rival-tactic sperm is not based on a self/non-self recognition mechanism. Our findings show that cross interactions of sperm and seminal fluid may influence the fertilization success of competing ejaculates with males investing in both sperm and seminal fluid in response to sperm competition risk.

  5. Mating behavior and the evolution of sperm design

    PubMed Central

    Schärer, Lukas; Littlewood, D. Timothy J.; Waeschenbach, Andrea; Yoshida, Wataru; Vizoso, Dita B.

    2011-01-01

    Sperm are the most diverse of all animal cell types, and much of the diversity in sperm design is thought to reflect adaptations to the highly variable conditions under which sperm function and compete to achieve fertilization. Recent work has shown that these conditions often evolve rapidly as a consequence of multiple mating, suggesting a role for sexual selection and sexual conflict in the evolution of sperm design. However, very little of the striking diversity in sperm design is understood functionally, particularly in internally fertilizing organisms. We use phylogenetic comparative analyses covering 16 species of the hermaphroditic flatworm genus Macrostomum to show that a complex sperm design is associated with reciprocal mating and that this complexity is lost secondarily when hypodermic insemination—sperm injection through the epidermis—evolves. Specifically, the complex sperm design, which includes stiff lateral bristles, is likely a male persistence trait associated with sexual conflicts over the fate of received ejaculates and linked to female resistance traits, namely an intriguing postcopulatory sucking behavior and a thickened epithelium of the sperm-receiving organ. Our results suggest that the interactions between sperm donor, sperm, and sperm recipient can change drastically when hypodermic insemination evolves, involving convergent evolution of a needle-like copulatory organ, a simpler sperm design, and a simpler female genital morphology. Our study documents that a shift in the mating behavior may alter fundamentally the conditions under which sperm compete and thereby lead to a drastic change in sperm design. PMID:21220334

  6. Long sperm fertilize more eggs in a bird

    PubMed Central

    Bennison, Clair; Hemmings, Nicola; Slate, Jon; Birkhead, Tim

    2015-01-01

    Sperm competition, in which the ejaculates of multiple males compete to fertilize a female's ova, results in strong selection on sperm traits. Although sperm size and swimming velocity are known to independently affect fertilization success in certain species, exploring the relationship between sperm length, swimming velocity and fertilization success still remains a challenge. Here, we use the zebra finch (Taeniopygia guttata), where sperm size influences sperm swimming velocity, to determine the effect of sperm total length on fertilization success. Sperm competition experiments, in which pairs of males whose sperm differed only in length and swimming speed, revealed that males producing long sperm were more successful in terms of (i) the number of sperm reaching the ova and (ii) fertilizing those ova. Our results reveal that although sperm length is the main factor determining the outcome of sperm competition, complex interactions between male and female reproductive traits may also be important. The mechanisms underlying these interactions are poorly understood, but we suggest that differences in sperm storage and utilization by females may contribute to the outcome of sperm competition. PMID:25621327

  7. Mouse Y-Encoded Transcription Factor Zfy2 Is Essential for Sperm Formation and Function in Assisted Fertilization

    PubMed Central

    Yamauchi, Yasuhiro; Riel, Jonathan M.; Ruthig, Victor; Ward, Monika A.

    2015-01-01

    Abstract Spermatogenesis is a key developmental process allowing for a formation of a mature male gamete. During its final phase, spermiogenesis, haploid round spermatids undergo cellular differentiation into spermatozoa, which involves extensive restructuring of cell morphology, DNA, and epigenome. Using mouse models with abrogated Y chromosome gene complements and Y-derived transgene we identified Y chromosome encoded Zfy2 as the gene responsible for sperm formation and function. In the presence of a Zfy2 transgene, mice lacking the Y chromosome and transgenic for two other Y-derived genes, Sry driving sex determination and Eif2s3y initiating spermatogenesis, are capable of producing sperm which when injected into the oocytes yield live offspring. Therefore, only three Y chromosome genes, Sry, Eif2s3y and Zfy2, constitute the minimum Y chromosome complement compatible with successful intracytoplasmic sperm injection in the mouse. PMID:26719889

  8. Lactic acid is a sperm motility inactivation factor in the sperm storage tubules

    PubMed Central

    Matsuzaki, Mei; Mizushima, Shusei; Hiyama, Gen; Hirohashi, Noritaka; Shiba, Kogiku; Inaba, Kazuo; Suzuki, Tomohiro; Dohra, Hideo; Ohnishi, Toshiyuki; Sato, Yoshikatsu; Kohsaka, Tetsuya; Ichikawa, Yoshinobu; Atsumi, Yusuke; Yoshimura, Takashi; Sasanami, Tomohiro

    2015-01-01

    Although successful fertilization depends on timely encounters between sperm and egg, the decoupling of mating and fertilization often confers reproductive advantages to internally fertilizing animals. In several vertebrate groups, postcopulatory sperm viability is prolonged by storage in specialized organs within the female reproductive tract. In birds, ejaculated sperm can be stored in a quiescent state within oviductal sperm storage tubules (SSTs), thereby retaining fertilizability for up to 15 weeks at body temperature (41 °C); however, the mechanism by which motile sperm become quiescent within SSTs is unknown. Here, we show that low oxygen and high lactic acid concentrations are established in quail SSTs. Flagellar quiescence was induced by lactic acid in the concentration range found in SSTs through flagellar dynein ATPase inactivation following cytoplasmic acidification (sperm morphology under hypoxic and high temperature conditions indicates that a combination of these factors enables sperm cells to survive during the ovulation cycles. Our findings suggested a novel physiological role for lactic acid in promoting sperm quiescence in SSTs and opened up a new opportunity for technological improvement in prolonging sperm longevity at ambient or body temperature. PMID:26619826

  9. Sperm competition affects male behaviour and sperm output in the rainbow darter

    PubMed Central

    Fuller, R. C.

    1998-01-01

    Rainbow darters, Etheostoma caeruleum, are promiscuous fish with moderate rates of group spawning (between one and five males may simultaneously mate with one female). In this study, I examined male sperm output and male willingness to spawn under different levels of sperm competition intensity. One male and one female were allowed to spawn in an aquarium where they had visual and olfactory access to one of four treatments: four males, one male, zero males, or one female. Theory predicts that males should reduce sperm output when there are more than the average number of males at a group spawning (four-male treatment) and should increase sperm output when there are fewer than average males at a group spawning (one-male treatment). Mean sperm output did not differ among treatments. However, males released more sperm when spawning in the presence of competing males (four-male and one-male treatments pooled) than when spawning in the absence of competing males (zero-male and one-female treatments pooled). Males were also most likely to forego spawning opportunities when sperm competition intensity was high. Furthermore, male willingness to spawn was size dependent. Large males were more likely to forego spawning opportunities under high sperm competition intensity. Large males may be better off waiting for future spawning opportunities when there is a lower potential for sperm competition intensity.

  10. Boar seminal plasma exosomes maintain sperm function by infiltrating into the sperm membrane

    PubMed Central

    Du, Jian; Shen, Jian; Wang, Yuanxian; Pan, Chuanying; Pang, Weijun; Diao, Hua; Dong, Wuzi

    2016-01-01

    Seminal plasma ingredients are important for maintenance of sperm viability. This study focuses on the effect of boar seminal plasma exosomes on sperm function during long-term liquid storage. Boar seminal plasma exosomes had typical nano-structure morphology as measured by scanning electron microscopy (SEM) and molecular markers such as AWN, CD9 and CD63 by western blot analysis. The effect on sperm parameters of adding different ratio of boar seminal plasma exosomes to boar sperm preparations was analyzed. Compared to the diluent without exosomes, the diluent with four times or sixteen times exosomes compared to original semen had higher sperm motility, prolonged effective survival time, improved sperm plasma membrane integrity (p < 0.05), increased total antioxidant capacity (T-AOC) activity and decreased malondialdehyde (MDA) content. The diluent containing four times concentration of exosomes compared to original semen was determined to inhibit premature capacitation, but not to influence capacitation induced in vitro. Inhibition of premature capacitation is likely related to the concentration of exosomes which had been demonstrated to transfer proteins including AWN and PSP-1 into sperm. In addition, using fluorescence microscopy and scanning electron microscopy analysis, it was demonstrated that exosomes in diluent were directly binding to the membrane of sperm head which could improve sperm plasma membrane integrity. PMID:27542209

  11. Sperm competition in a fish with external fertilization: the contribution of sperm number, speed and length.

    PubMed

    Stoltz, J A; Neff, B D

    2006-11-01

    The role of sperm number and quality in male competitiveness was investigated using in vitro fertilization experiments with bluegill (Lepomis macrochirus). Bluegill males use one of three mating tactics: 'sneakers', which streak spawn; 'satellites', which mimic females; and 'parentals', which are territorial. The in vitro experiments mimicked natural spawning by incorporating these males' mean proximity to eggs and timing of sperm release. Using a maximum-likelihood algorithm, raffle equations were fit to paternity data, which revealed a strong effect of sperm number on male competitiveness. There was no difference in sperm flagellum length, curvilinear swim speed or path linearity among the three male mating types, and these traits did not explain any additional variation in male competitiveness. It was estimated that, given closer proximity to eggs, satellites need release only 0.34 times as many sperm as parentals to obtain equal paternity. Despite being farther from the eggs and releasing sperm about half a second after parentals, sneakers need only release 0.58 times as many sperm as parentals to obtain equal paternity. Thus, the increased competitiveness of sneakers' sperm must come from a component of sperm quality other than speed or length.

  12. Female sperm use and storage between fertilization events drive sperm competition and male ejaculate allocation.

    PubMed

    Requena, Gustavo S; Alonzo, Suzanne H

    2014-12-01

    Sperm competition theory has traditionally focused on how male allocation responds to female promiscuity, when males compete to fertilize a single clutch of eggs. Here, we develop a model to ask how female sperm use and storage across consecutive reproductive events affect male ejaculate allocation and patterns of mating and paternity. In our model, sperm use (a single parameter under female control) is the main determinant of sperm competition, which alters the effect of female promiscuity on male success and, ultimately, male reproductive allocation. Our theory reproduces the general pattern predicted by existing theory that increased sperm competition favors increased allocation to ejaculates. However, our model predicts a negative correlation between male ejaculate allocation and female promiscuity, challenging the generality of a prevailing expectation of sperm competition theory. Early models assumed that the energetic costs of precopulatory competition and the level of sperm competition are both determined by female promiscuity, which leads to an assumed covariation between these two processes. By modeling precopulatory costs and sperm competition independently, our theoretical framework allows us to examine how male allocation should respond independently to variation in sperm competition and energetic trade-offs in mating systems that have been overlooked in the past.

  13. Importance of sperm morphology during sperm transport and fertilization in mammals

    PubMed Central

    García-Vázquez, Francisco A; Gadea, Joaquín; Matás, Carmen; Holt, William V

    2016-01-01

    After natural or artificial insemination, the spermatozoon starts a journey from the site of deposition to the place of fertilization. However, only a small subset of the spermatozoa deposited achieves their goal: to reach and fertilize the egg. Factors involved in controlling sperm transport and fertilization include the female reproductive tract environment, cell-cell interactions, gene expression, and phenotypic sperm traits. Some of the significant determinants of fertilization are known (i.e., motility or DNA status), but many sperm traits are still indecipherable. One example is the influence of sperm dimensions and shape upon transport within the female genital tract towards the oocyte. Biophysical associations between sperm size and motility may influence the progression of spermatozoa through the female reproductive tract, but uncertainties remain concerning how sperm morphology influences the fertilization process, and whether only the sperm dimensions per se are involved. Moreover, such explanations do not allow the possibility that the female tract is capable of distinguishing fertile spermatozoa on the basis of their morphology, as seems to be the case with biochemical, molecular, and genetic properties. This review focuses on the influence of sperm size and shape in evolution and their putative role in sperm transport and selection within the uterus and the ability to fertilize the oocyte. PMID:27624988

  14. Sperm morphology: consistency of assessment of the same sperm by different observers.

    PubMed

    Baker, H W; Clarke, G N

    1987-01-01

    Photographic slides of 36 sperm were shown to a group of 28 observers with different lengths of experience in assessing sperm morphology. Experienced observers were generally consistent (10 or more of the 17 agreeing) in classifying sperm as normal, amorphous, small heads, lacking acrosomes, and having tail defects or cytoplasmic droplets but categorization was more variable for large, tapering and pyriform heads. This study highlights the need for more widespread agreement about definition of sperm shapes and the development of practical objective methods of assessment.

  15. Epididymis seleno-independent glutathione peroxidase 5 maintains sperm DNA integrity in mice

    PubMed Central

    Chabory, Eléonore; Damon, Christelle; Lenoir, Alain; Kauselmann, Gary; Kern, Hedrun; Zevnik, Branko; Garrel, Catherine; Saez, Fabrice; Cadet, Rémi; Henry-Berger, Joelle; Schoor, Michael; Gottwald, Ulrich; Habenicht, Ursula; Drevet, Joël R.; Vernet, Patrick

    2009-01-01

    The mammalian epididymis provides sperm with an environment that promotes their maturation and protects them from external stresses. For example, it harbors an array of antioxidants, including non-conventional glutathione peroxidase 5 (GPX5), to protect them from oxidative stress. To explore the role of GPX5 in the epididymis, we generated mice that lack epididymal expression of the enzyme. Histological analyses of Gpx5–/– epididymides and sperm cells revealed no obvious defects. Furthermore, there were no apparent differences in the fertilization rate of sexually mature Gpx5–/– male mice compared with WT male mice. However, a higher incidence of miscarriages and developmental defects were observed when WT female mice were mated with Gpx5-deficient males over 1 year old compared with WT males of the same age. Flow cytometric analysis of spermatozoa recovered from Gpx5-null and WT male mice revealed that sperm DNA compaction was substantially lower in the cauda epididymides of Gpx5-null animals and that they suffered from DNA oxidative attacks. Real-time PCR analysis of enzymatic scavengers expressed in the mouse epididymis indicated that the cauda epididymidis epithelium of Gpx5-null male mice mounted an antioxidant response to cope with an excess of ROS. These observations suggest that GPX5 is a potent antioxidant scavenger in the luminal compartment of the mouse cauda epididymidis that protects spermatozoa from oxidative injuries that could compromise their integrity and, consequently, embryo viability. PMID:19546506

  16. The effect of polymer dots on bioactivity of mouse sperm in vitro

    NASA Astrophysics Data System (ADS)

    Feng, Gang; Chen, Qiang; Zhai, Peng; Wang, Xiaomei; Lin, Guimiao; Xu, Gaixia; Chen, Danni

    2014-09-01

    Objective: In recent years, semiconducting polymer dots (Pdots)have caught considerable attention for their outstanding optical characteristics in biomedical imaging applications. Not as semiconductor quantum dots, Pdots are composed of nonmetallic material and their biological effects remain unclear. In this work, we investigated the effects of a band new polymer dots on bioactivity of mouse sperm using a computer-aided sperm analysis system(CASA) and an in vitro fertilization (IVF) model. Methods: The semiconducting polymer dots used in this study is CN-PPV Pdots, which emits in the orange wavelength range with high brightness. Epididymal mouse sperm were collected from 7-8weeks old Balb/c mouse. Firstly, CN-PPV Pdots was added into the Human Tubal Fluid (HTF) media at various concentrations (0, 1, 10, 100 nmol/L respectively ), then sperm bioactivity and vitality were evaluated every 10 minutes. Secondly, the treated sperm were co-cultured with matured oocytes in HTF media, fertilization rate and oocytes development were recorded after 24 hours co-incubation. Results: Sperm viability in the control group (0 nmol/L) and experimental group (1, 10,100 nmol/L) were 57.20+/-4.51%, 58.17+/-4.81%, 55.50+/-4.52%, 46.26%+/-3.83%, respectively. Fertilization rate in different groups showed no obvious differences, control group (0 nmol/L) and experimental group (1, 10, 100 nmol/L) were 38.75+/-1.71%, 37.01+/-4.69%, 32.75+/-1.71%, 35.24+/-2.37%, respectively. Conclusion: Our data indicated that the CN-PPV Pdots had a very high biocompatibility on sperm in both the activation and the IVF process, even in extreme high Pdots concentration,the sperm bioactivity only got slight restrained. The effect of CN-PPV Pdots seems has no or little toxicity,and the long-term embryonic development has yet to be verified.

  17. Characterisation of Caenorhabditis elegans sperm transcriptome and proteome

    PubMed Central

    2014-01-01

    Background Although sperm is transcriptionally and translationally quiescent, complex populations of RNAs, including mRNAs and non-coding RNAs, exist in sperm. Previous microarray analysis of germ cell mutants identified hundreds of sperm genes in Caenorhabditis elegans. To take a more comprehensive view on C. elegans sperm genes, here, we isolate highly pure sperm cells and employ high-throughput technologies to obtain sperm transcriptome and proteome. Results First, sperm transcriptome consists of considerable amounts of non-coding RNAs, many of which have not been annotated and may play functional roles during spermatogenesis. Second, apart from kinases/phosphatases as previously reported, ion binding proteins are also enriched in sperm, underlying the crucial roles of intracellular ions in post-translational regulation in sperm. Third, while the majority of sperm genes/proteins have low abundance, a small number of sperm genes/proteins are hugely enriched in sperm, implying that sperm only rely on a small set of proteins for post-translational regulation. Lastly, by extensive RNAi screening of sperm enriched genes, we identified a few genes that control fertility. Our further analysis reveals a tight correlation between sperm transcriptome and sperm small RNAome, suggesting that the endogenous siRNAs strongly repress sperm genes. This leads to an idea that the inefficient RNAi screening of sperm genes, a phenomenon currently with unknown causes, might result from the competition between the endogenous RNAi pathway and the exogenous RNAi pathway. Conclusions Together, the obtained sperm transcriptome and proteome serve as valuable resources to systematically study spermatogenesis in C. elegans. PMID:24581041

  18. Testicular fine needle aspiration: the alternative method for sperm retrieval in non-obstructive azoospermia.

    PubMed

    Lewin, A; Reubinoff, B; Porat-Katz, A; Weiss, D; Eisenberg, V; Arbel, R; Bar-el, H; Safran, A

    1999-07-01

    The objective of this prospective open study was to determine the feasibility of obtaining mature spermatozoa for intracytoplasmic sperm injection (ICSI) by testicular fine needle aspiration (TEFNA) in men diagnosed with non-obstructive azoospermia. TEFNA consisted of a mean of 15 punctures and aspirations in each testis, using 23 gauge butterfly needles, connected to a 20 ml syringe with an aspiration handle. Patients (n = 85) underwent 111 TEFNA cycles. Mature testicular spermatozoa were recovered in 65 (58.5%) cycles from 50 (58.8%) patients. The sperm recovery rate by testicular histology was 14 out of 29 (48.3%) in patients with Sertoli cell-only, 13 out of 28 (46.4%) in patients with maturation arrest, 19 out of 20 (95%) in patients with hypospermatogenesis, four out of six (66.6%) in patients with tubular hyalinization due to non-mosaic Klinefelter's syndrome. No spermatozoa were found in two cases with post-irradiation fibrosis. ICSI was performed in all 65 cycles. In 58 cycles in which only the husbands' spermatozoa were used, 406 mature oocytes were injected, and 154 (37.9%) were normally fertilized. Of the 143 embryos that developed (92.8%), 119 were transferred in 42 cycles resulting in 18 clinical pregnancies (42. 8%), with 31 gestational sacs, providing an implantation rate of 26%. One abortion of a singleton pregnancy occurred (5.6%). No major side-effects, such as haematoma or infection were recorded. In conclusion, we have found TEFNA to be efficient, easy to learn, safe and well tolerated by all patients. In our opinion, TEFNA should be considered the first choice whenever sperm recovery is attempted in patients with non-obstructive azoospermia.

  19. Transmembrane Signal Transduction in Oocyte Maturation and Fertilization: Focusing on Xenopus laevis as a Model Animal

    PubMed Central

    Sato, Ken-ichi

    2014-01-01

    Fertilization is a cell biological phenomenon of crucial importance for the birth of new life in a variety of multicellular and sexual reproduction species such as algae, animal and plants. Fertilization involves a sequence of events, in which the female gamete “egg” and the male gamete “spermatozoon (sperm)” develop, acquire their functions, meet and fuse with each other, to initiate embryonic and zygotic development. Here, it will be briefly reviewed how oocyte cytoplasmic components are orchestrated to undergo hormone-induced oocyte maturation and sperm-induced activation of development. I then review how sperm-egg membrane interaction/fusion and activation of development in the fertilized egg are accomplished and regulated through egg coat- or egg plasma membrane-associated components, highlighting recent findings and future directions in the studies using Xenopus laevis as a model experimental animal. PMID:25546390

  20. Sperm Shape (Morphology): Does It Affect Fertility?

    MedlinePlus

    ... decide whether a couple should use in vitro fertilization (IVF) to attempt a pregnancy. It is best ... genetic material. Once the sperm enters the egg, fertilization has a good chance of taking place. However, ...

  1. Sperm motility under conditions of weightlessness.

    PubMed

    Engelmann, U; Krassnigg, F; Schill, W B

    1992-01-01

    The aim of this study was to determine the differences in motility of frozen and thawed bull spermatozoa under conditions of weightlessness compared with ground conditions. The tests were performed within a series of scientific and technologic experiments under microgravity using sounding rockets in the Technologische Experimente unter Schwerelosigkeit (TEXUS) program launched in Kiruna, North Sweden. Using a computerized sperm motility analyzer, significant differences were found in sperm motility under microgravity compared with sperm under gravitational conditions on earth. Computer analysis showed alterations in straight line and curvilinear velocity, as well as in linearity values. The amount of progressively motile spermatozoa, including all spermatozoa with a velocity > 20 microns/second, increased significantly from 24% +/- 9.5% in the reference test to 49% +/- 7.6% in the microgravity test. In conclusion, there is strong evidence that gravity influences sperm motility.

  2. Sperm harvesting and post-mortem fatherhood.

    PubMed

    Murphy, Timothy F

    1995-10-01

    The motives and consequences of harvesting sperm from brain dead males for the purpose of effecting post mortem fatherhood are examined. I argue that sperm harvesting and post mortem fatherhood raise no harms of a magnitude that would justify forbidding the practice outright. Dead men are not obviously harmed by the practice; children need not be harmed by this kind of birth; and the practice enlarges rather than diminishes the reproductive choices of surviving partners. Certain ethical and legal issues nevertheless require attention. As a matter of consistency with other harvesting protocols, there ought to be a mechanism for respecting the wishes of men who when alive do not wish to become fathers post mortem. Mechanisms governing entitlement to harvest and use sperm will also be required. I note that the law is unlikely to recognize the paternity of children born from harvested sperm, though there may be reasons to recognize that paternity in some instances.

  3. Organophosphorous pesticide exposures and sperm quality

    PubMed Central

    Perry, Melissa J.; Venners, Scott A.; Chen, Xing; Liu, Xue; Tang, Genfu; Xing, Houxun; Barr, Dana Boyd; Xu, Xiping

    2010-01-01

    Many Americans are exposed to low levels of organophosphorous (OP) pesticides. In is unclear whether these exposures impact sperm production. We investigated whether there was an association between urinary OP insecticide metabolites and sperm concentration and motility in newly married men from a rural area of eastern People’s Republic of China. Ninety-four cases and 95 controls were included based on their median residual value of sperm concentration and motility after adjusting for relevant covariates. Their urine was analyzed for six dialkylphosphate (DAP) compounds. After adjustment for demographic and exposure variables, the odds of being a case were greater (Odds Ratio=1.30, 95% Confidence Interval 1.02-1.65) in men with higher urinary concentrations of dimethylphosphate (DMP) compared to men with lower levels. No significant differences between cases and controls were found among the other DAP concentrations. DMP exposure and sperm concentration and motility should be explored further in environmental exposure studies. PMID:20850521

  4. Mammalian Sperm Motility: Observation and Theory

    NASA Astrophysics Data System (ADS)

    Gaffney, E. A.; Gadêlha, H.; Smith, D. J.; Blake, J. R.; Kirkman-Brown, J. C.

    2011-01-01

    Mammalian spermatozoa motility is a subject of growing importance because of rising human infertility and the possibility of improving animal breeding. We highlight opportunities for fluid and continuum dynamics to provide novel insights concerning the mechanics of these specialized cells, especially during their remarkable journey to the egg. The biological structure of the motile sperm appendage, the flagellum, is described and placed in the context of the mechanics underlying the migration of mammalian sperm through the numerous environments of the female reproductive tract. This process demands certain specific changes to flagellar movement and motility for which further mechanical insight would be valuable, although this requires improved modeling capabilities, particularly to increase our understanding of sperm progression in vivo. We summarize current theoretical studies, highlighting the synergistic combination of imaging and theory in exploring sperm motility, and discuss the challenges for future observational and theoretical studies in understanding the underlying mechanics.

  5. Comparative evolutionary psychology of sperm competition.

    PubMed

    Shackelford, Todd K; Goetz, Aaron T

    2006-05-01

    A comparative evolutionary psychological perspective predicts that species that recurrently faced similar adaptive problems may have evolved similar psychological mechanisms to solve these problems. Sperm competition provides an arena in which to assess the heuristic value of such a comparative evolutionary perspective. The sperm competition that results from female infidelity and polyandry presents a similar class of adaptive problems for individuals across many species. The authors first describe mechanisms of sperm competition in insects and in birds. They suggest that the adaptive problems and evolved solutions in these species provide insight into human anatomy, physiology, psychology, and behavior. The authors then review recent theoretical and empirical arguments for the existence of sperm competition in humans and discuss proposed adaptations in humans that have analogs in insects or birds. The authors conclude by highlighting the heuristic value of a comparative evolutionary psychological approach in this field.

  6. Sperm macromolecules associated with bull fertility.

    PubMed

    Kaya, Abdullah; Memili, Erdoğan

    2016-06-01

    Bull fertility, ability of the sperm to fertilize and activate the egg that sustain embryo development, is vitally important for effective and efficient production of cattle. Fertility is a complex trait with low heritability. Despite recent advances in genomic selection and possibility of enormous paternal benefits to profitable cattle production, there exist no reliable tests for evaluating semen quality and predicting bull fertility. This review focuses on sperm macromolecules such as transcripts, proteins and the epigenome, i.e., the functional genome that are associated with bull fertility. Generating new information in these systems is important beyond agriculture because such progress advances the fundamental science of the mammalian male gamete while at the same time introduces biotechnology into livestock production. Sperm macromolecules and epigenome markers associated with bull fertility can be used alone or in combination with the current SNP microarrays to determine sperm quality and to indicate bull fertility.

  7. Rat sperm motility analysis: methodologic considerations

    EPA Science Inventory

    The objective of these studies was to optimize conditions for computer-assisted sperm analysis (CASA) of rat epididymal spermatozoa. Methodologic issues addressed include sample collection technique, sampling region within the epididymis, type of diluent medium used, and sample c...

  8. Membrane hyperpolarization during human sperm capacitation

    PubMed Central

    López-González, I.; Torres-Rodríguez, P.; Sánchez-Carranza, O.; Solís-López, A.; Santi, C.M.; Darszon, A.; Treviño, C.L.

    2014-01-01

    Sperm capacitation is a complex and indispensable physiological process that spermatozoa must undergo in order to acquire fertilization capability. Spermatozoa from several mammalian species, including mice, exhibit a capacitation-associated plasma membrane hyperpolarization, which is necessary for the acrosome reaction to occur. Despite its importance, this hyperpolarization event has not been adequately examined in human sperm. In this report we used flow cytometry to show that a subpopulation of human sperm indeed undergo a plasma membrane hyperpolarization upon in vitro capacitation. This hyperpolarization correlated with two other well-characterized capacitation parameters, namely an increase in intracellular pH and Ca2+ concentration, measured also by flow cytometry. We found that sperm membrane hyperpolarization was completely abolished in the presence of a high external K+ concentration (60 mM), indicating the participation of K+ channels. In order to identify, which of the potential K+ channels were involved in this hyperpolarization, we used different K+ channel inhibitors including charybdotoxin, slotoxin and iberiotoxin (which target Slo1) and clofilium (a more specific blocker for Slo3). All these K+ channel antagonists inhibited membrane hyperpolarization to a similar extent, suggesting that both members of the Slo family may potentially participate. Two very recent papers recorded K+ currents in human sperm electrophysiologically, with some contradictory results. In the present work, we show through immunoblotting that Slo3 channels are present in the human sperm membrane. In addition, we found that human Slo3 channels expressed in CHO cells were sensitive to clofilium (50 μM). Considered altogether, our data indicate that Slo1 and Slo3 could share the preponderant role in the capacitation-associated hyperpolarization of human sperm in contrast to what has been previously reported for mouse sperm, where Slo3 channels are the main contributors to the

  9. Sperm DNA oxidative damage and DNA adducts

    PubMed Central

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-01-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps = 0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps = 0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps = 0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on

  10. Phagosome maturation: aging gracefully.

    PubMed Central

    Vieira, Otilia V; Botelho, Roberto J; Grinstein, Sergio

    2002-01-01

    Foreign particles and apoptotic bodies are eliminated from the body by phagocytic leucocytes. The initial stage of the elimination process is the internalization of the particles into a plasma membrane-derived vacuole known as the phagosome. Such nascent phagosomes, however, lack the ability to kill pathogens or to degrade the ingested targets. These properties are acquired during the course of phagosomal maturation, a complex sequence of reactions that result in drastic remodelling of the phagosomal membrane and contents. The determinants and consequences of the fusion and fission reactions that underlie phagosomal maturation are the topic of this review. PMID:12061891

  11. Influence of staining and sampling procedures on goat sperm morphometry using the Sperm Class Analyzer.

    PubMed

    Hidalgo, Manuel; Rodríguez, Inmaculada; Dorado, Jesús

    2006-09-01

    Computer-assisted sperm morphometry analysis (ASMA) has improved the assessment of sperm morphology, but the results depend on the use of adequate sampling and staining procedures of spermatozoa from individual species. In this study, the Sperm Class Analyzer ASMA system was used for the morphometric analysis of goat sperm heads. Semen samples, obtained from four bucks, were used to evaluate the influence of three staining procedures (Diff-Quik, Hemacolor and Harris' Haematoxylin) on the accuracy of image processing and sperm morphometry, the effect of the number of cells analysed and the repeatability of the method. These experiments were performed to obtain objective, accurate and reliable sperm morphometric measurements of goat spermatozoa. Diff-Quik and Harris' Haematoxylin were significantly (p<0.05) more accurate than Hemacolor. However, Diff-Quik obtained the highest proportion of correctly analysed sperm heads (86.06%) and the lowest coefficients of variation on the image processing and morphometric measurements. The staining methods affected significantly the sperm dimensions (p<0.001) with increased values from Diff-Quik than Hemacolor and Harris' Haematoxylin, respectively (Diff-Quik>Hemacolor>Harris' Haematoxylin). No differences in morphometric parameters were found when 100, 150, 175 or 200 spermatozoa were analysed. The repeatability of results obtained was very high since no differences were found when measuring the same sperm on multiple attempts. In conclusion, to obtain objective, accurate and repeatable sperm morphometric measurements by the Sperm Class Analyzer system in goats, the analysis of 100 spermatozoa from slides which have been previously stained with Diff-Quik is recommended.

  12. CD9-positive microvesicles mediate the transfer of molecules to Bovine Spermatozoa during epididymal maturation.

    PubMed

    Caballero, Julieta N; Frenette, Gilles; Belleannée, Clémence; Sullivan, Robert

    2013-01-01

    Acquisition of fertilization ability by spermatozoa during epididymal transit occurs in part by the transfer of molecules from membranous vesicles called epididymosomes. Epididymosomes are heterogeneous in terms of both size and molecular composition. Exosomes and other related small membranous vesicles (30-120 nm) containing tetraspanin proteins on their surface are found in many biological fluids. In this study, we demonstrate that these vesicles are present in bovine cauda epididymal fluid as a subpopulation of epididymosomes. They contain tetraspanin CD9 in addition to other proteins involved in sperm maturation such as P25b, GliPr1L1, and MIF. In order to study the mechanism of protein transfer to sperm, DilC12-labeled unfractionated epididymosomes or CD9-positive microvesicles were coincubated with epididymal spermatozoa, and their transfer was evaluated by flow cytometry. CD9-positive microvesicles from epididymal fluid specifically transferred molecules to spermatozoa, whereas those prepared from blood were unable to do so. The CD9-positive microvesicles transferred molecules to the same sperm regions (acrosome and midpiece) as epididymosomes, with the same kinetics; however, the molecules were preferentially transferred to live sperm and, in contrast to epididymosomes, Zn(2+) did not demonstrate potentiated transfer. Tetraspanin CD9 was associated with other proteins on the membrane surface of CD9-positive microvesicles according to coimmunoprecipitation experiments. CD26 cooperated with CD9 in the molecular transfer to sperm since the amount of molecules transferred was significantly reduced in the presence of specific antibodies. In conclusion, CD9-positive microvesicles are present in bovine cauda epididymal fluid and transfer molecules to live maturing sperm in a tissue-specific manner that involves CD9 and CD26.

  13. Protein kinase C activity in boar sperm.

    PubMed

    Teijeiro, J M; Marini, P E; Bragado, M J; Garcia-Marin, L J

    2017-03-01

    Male germ cells undergo different processes within the female reproductive tract to successfully fertilize the oocyte. These processes are triggered by different extracellular stimuli leading to activation of protein phosphorylation. Protein kinase C (PKC) is a key regulatory enzyme in signal transduction mechanisms involved in many cellular processes. Studies in boar sperm demonstrated a role for PKC in the intracellular signaling involved in motility and cellular volume regulation. Experiments using phorbol 12-myristate 13-acetate (PMA) showed increases in the Serine/Threonine phosphorylation of substrates downstream of PKC in boar sperm. In order to gain knowledge about those cellular processes regulated by PKC, we evaluate the effects of PMA on boar sperm motility, lipid organization of plasma membrane, integrity of acrosome membrane and sperm agglutination. Also, we investigate the crosstalk between PKA and PKC intracellular pathways in spermatozoa from this species. The results presented here reveal a participation of PKC in sperm motility regulation and membrane fluidity changes, which is probably associated to acrosome reaction and to agglutination. Also, we show the existence of a hierarchy in the kinases pathway. Previous works on boar sperm suggest a pathway in which PKA is positioned upstream to PKC and this new results support such model.

  14. Major regulatory mechanisms involved in sperm motility.

    PubMed

    Pereira, Rute; Sá, Rosália; Barros, Alberto; Sousa, Mário

    2017-01-01

    The genetic bases and molecular mechanisms involved in the assembly and function of the flagellum components as well as in the regulation of the flagellar movement are not fully understood, especially in humans. There are several causes for sperm immotility, of which some can be avoided and corrected, whereas other are related to genetic defects and deserve full investigation to give a diagnosis to patients. This review was performed after an extensive literature search on the online databases PubMed, ScienceDirect, and Web of Science. Here, we review the involvement of regulatory pathways responsible for sperm motility, indicating possible causes for sperm immotility. These included the calcium pathway, the cAMP-dependent protein kinase pathway, the importance of kinases and phosphatases, the function of reactive oxygen species, and how the regulation of cell volume and osmolarity are also fundamental components. We then discuss main gene defects associated with specific morphological abnormalities. Finally, we slightly discuss some preventive and treatments approaches to avoid development of conditions that are associated with unspecified sperm immotility. We believe that in the near future, with the development of more powerful techniques, the genetic causes of sperm immotility and the regulatory mechanisms of sperm motility will be better understand, thus enabling to perform a full diagnosis and uncover new therapies.

  15. Major regulatory mechanisms involved in sperm motility

    PubMed Central

    Pereira, Rute; Sá, Rosália; Barros, Alberto; Sousa, Mário

    2017-01-01

    The genetic bases and molecular mechanisms involved in the assembly and function of the flagellum components as well as in the regulation of the flagellar movement are not fully understood, especially in humans. There are several causes for sperm immotility, of which some can be avoided and corrected, whereas other are related to genetic defects and deserve full investigation to give a diagnosis to patients. This review was performed after an extensive literature search on the online databases PubMed, ScienceDirect, and Web of Science. Here, we review the involvement of regulatory pathways responsible for sperm motility, indicating possible causes for sperm immotility. These included the calcium pathway, the cAMP-dependent protein kinase pathway, the importance of kinases and phosphatases, the function of reactive oxygen species, and how the regulation of cell volume and osmolarity are also fundamental components. We then discuss main gene defects associated with specific morphological abnormalities. Finally, we slightly discuss some preventive and treatments approaches to avoid development of conditions that are associated with unspecified sperm immotility. We believe that in the near future, with the development of more powerful techniques, the genetic causes of sperm immotility and the regulatory mechanisms of sperm motility will be better understand, thus enabling to perform a full diagnosis and uncover new therapies. PMID:26680031

  16. Oocyte Maturation and Development

    PubMed Central

    Verlhac, Marie-Hélène; Terret, Marie-Emilie

    2016-01-01

    Sexual reproduction is essential for many organisms to propagate themselves. It requires the formation of haploid female and male gametes: oocytes and sperms. These specialized cells are generated through meiosis, a particular type of cell division that produces cells with recombined genomes that differ from their parental origin. In this review, we highlight the end process of female meiosis, the divisions per se, and how they can give rise to a functional female gamete preparing itself for the ensuing zygotic development. In particular, we discuss why such an essential process in the propagation of species is so poorly controlled, producing a strong percentage of abnormal female gametes in the end. Eventually, we examine aspects related to the lack of centrosomes in female oocytes, the asymmetry in size of the mammalian oocyte upon division, and in mammals the direct consequences of these long-lived cells in the ovary. PMID:26998245

  17. Sperm motility of externally fertilizing fish and amphibians.

    PubMed

    Browne, R K; Kaurova, S A; Uteshev, V K; Shishova, N V; McGinnity, D; Figiel, C R; Mansour, N; Agney, D; Wu, M; Gakhova, E N; Dzyuba, B; Cosson, J

    2015-01-01

    We review the phylogeny, sperm competition, morphology, physiology, and fertilization environments of the sperm of externally fertilizing fish and amphibians. Increased sperm competition in both fish and anurans generally increases sperm numbers, sperm length, and energy reserves. The difference between the internal osmolarity and iconicity of sperm cells and those of the aquatic medium control the activation, longevity, and velocity of sperm motility. Hypo-osmolarity of the aquatic medium activates the motility of freshwater fish and amphibian sperm and hyperosmolarity activates the motility of marine fish sperm. The average longevity of the motility of marine fish sperm (~550 seconds) was significantly (P < 0.05) greater than that of freshwater fish sperm (~150 seconds), with the longevities of both marine and freshwater fish being significantly (P < 0.05) lower than that of anuran sperm (~4100 seconds). The average velocity of anuran sperm (25 μm/s) was significantly (P < 0.05) lower than that of marine fish (140 μm/s) or freshwater fish (135 μm/s) sperm. The longevity of the sperm of giant salamanders (Cryptobranchoidea) of approximately 600 seconds was greater than that of freshwater fish sperm but much lower than anuran sperm. Our research and information from the literature showed that higher osmolarities promote greater longevity in anuran sperm, and some freshwater fish sperm, and that anuran and cryptobranchid sperm maintained membrane integrity long after the cessation of motility, demonstrating a preferential sharing of energy reserves toward the maintenance of membrane integrity. The maintenance of the membrane integrity of anuran sperm in fresh water for up to 6 hours showed an extremely high osmotic tolerance relative to fish sperm. The very high longevity and osmotic tolerance of anuran sperm and high longevity of cryptobranchid sperm, relative to those of freshwater fish, may reflect the complex fertilization history of amphibian sperm in

  18. One-shot genitalia are not an evolutionary dead end - Regained male polygamy in a sperm limited spider species

    PubMed Central

    2011-01-01

    Background Monogynous mating systems with extremely low male mating rates have several independent evolutionary origins and are associated with drastic adaptations involving self-sacrifice, one-shot genitalia, genital damage, and termination of spermatogenesis immediately after maturation. The combination of such extreme traits likely restricts evolutionary potential perhaps up to the point of making low male mating rates irreversible and hence may constitute an evolutionary dead end. Here, we explore the case of a reversion to multiple mating from monogynous ancestry in golden orb-web spiders, Nephila senegalensis. Results Male multiple mating is regained by the loss of genital damage and sexual cannibalism but spermatogenesis is terminated with maturation, restricting males to a single loading of their secondary mating organs and a fixed supply of sperm. However, males re-use their mating organs and by experimentally mating males to many females, we show that the sperm supply is divided between copulations without reloading the pedipalps. Conclusion By portioning their precious sperm supply, males achieve an average mating rate of four females which effectively doubles the maximal mating rate of their ancestors. A heritage of one-shot genitalia does not completely restrict the potential to increase mating rates in Nephila although an upper limit is defined by the available sperm load. Future studies should now investigate how males use this potential in the field and identify selection pressures responsible for a reversal from monogynous to polygynous mating strategies. PMID:21740561

  19. Sperm-attractant peptide influences the spermatozoa swimming behavior in internal fertilization in Octopus vulgaris.

    PubMed

    De Lisa, Emilia; Salzano, Anna Maria; Moccia, Francesco; Scaloni, Andrea; Di Cosmo, Anna

    2013-06-15

    Marine invertebrates exhibit both chemokinesis and chemotaxis phenomena, induced in most cases by the release of water-borne peptides or pheromones. In mollusks, several peptides released during egg-laying improve both male attraction and mating. Unlike other cephalopods, Octopus vulgaris adopts an indirect internal fertilization strategy. We here report on the identification and characterization of a chemoattractant peptide isolated from mature eggs of octopus females. Using two-chamber and time-lapse microscopy assays, we demonstrate that this bioactive peptide is able to increase sperm motility and induce chemotaxis by changing the octopus spermatozoa swimming behavior in a dose-dependent manner. We also provide evidence that chemotaxis in the octopus requires the presence of extracellular calcium and membrane protein phophorylation at tyrosine. This study is the first report on a sperm-activating factor in a non-free-spawning marine animal.

  20. The genetic basis and fitness consequences of sperm midpiece size in deer mice

    PubMed Central

    Fisher, Heidi S.; Jacobs-Palmer, Emily; Lassance, Jean-Marc; Hoekstra, Hopi E.

    2016-01-01

    An extensive array of reproductive traits varies among species, yet the genetic mechanisms that enable divergence, often over short evolutionary timescales, remain elusive. Here we examine two sister-species of Peromyscus mice with divergent mating systems. We find that the promiscuous species produces sperm with longer midpiece than the monogamous species, and midpiece size correlates positively with competitive ability and swimming performance. Using forward genetics, we identify a gene associated with midpiece length: Prkar1a, which encodes the R1α regulatory subunit of PKA. R1α localizes to midpiece in Peromyscus and is differentially expressed in mature sperm of the two species yet is similarly abundant in the testis. We also show that genetic variation at this locus accurately predicts male reproductive success. Our findings suggest that rapid evolution of reproductive traits can occur through cell type-specific changes to ubiquitously expressed genes and have an important effect on fitness. PMID:27910854

  1. Royal jelly supplementation in semen extender enhances post-thaw quality and fertility of Nili-Ravi buffalo bull sperm.

    PubMed

    Shahzad, Qaisar; Mehmood, Muhammad Usman; Khan, Hamayun; ul Husna, Asma; Qadeer, Saima; Azam, Asima; Naseer, Zahid; Ahmad, Ejaz; Safdar, Muhammad; Ahmad, Mushtaq

    2016-04-01

    Two experiments were conducted to evaluate the effect of royal jelly (RJ) on post-thaw sperm quality, in vitro and in vivo fertility rate of cryopreserved buffalo bull sperm. The semen was collected from three mature regular donor buffalo bulls, ejaculates were pooled and semen evaluated initially. In Experiment 1, the ejaculates were extended in tris-citric acid diluter supplemented with different RJ concentrations (0, 0.05, 0.1, 0.2, 0.3 or 0.4%). The diluted semen was cooled to 4°C, packaged into 0.5 mL straws and frozen using standard procedure. The straws were thawed and assessed for sperm progressive motility, viability, plasma membrane, acrosome, and chromatin integrity. The results indicated that sperm progressive motility was significantly greater (P<0.05) in 0.05, 0.1, 0.2 and 0.3% RJ than 0.4% RJ supplemented and control groups. The sperm viability, plasma membrane and acrosome integrity were significantly improved (P<0.05) in 0.1% RJ supplemented group the compared to other treatment groups. In Experiment 2, cryopreserved sperm with 0.1% RJ supplementation and control (without RJ supplementation) were used to observe the in vitro fertilizing potential and in vivo fertility. In vitro fertilization method was applied to assess the cleavage rate; whereas, AI was performed in buffalo during in vivo fertility trial. The buffaloes were inseminated 12h after standing estrus and pregnancy diagnosis was performed through ultrasonography. The results revealed that the cleavage rate was higher (P<0.05) in 0.1% RJ as compared to control group. However, the pregnancy rate was similar (P>0.05) between 0.1% RJ supplemented and control groups. It is concluded that supplementation of RJ in freezing extender can improve the cryosurvival rate and in vitro fertilizing capacity of buffalo bull sperm.

  2. Field evidence of reproduction impairment through sperm DNA damage in the fish nase (Chondrostoma nasus) in anthropized hydrosystems.

    PubMed

    Devaux, Alain; Bony, Sylvie; Plenet, Sandrine; Sagnes, Pierre; Segura, Samuel; Suaire, Rémi; Novak, Morgane; Gilles, André; Olivier, Jean-Michel

    2015-12-01

    This work aims to explore in the field the relationship between the integrity of sperm DNA and the quality of offspring as a possible cause of the decline of a feral fish population through reproduction impairment. Mature nase (Chondrostoma nasus) were caught during the breeding season in three locations (A-C) of the Rhône River basin and gametes collected by stripping. Sampling locations were chosen according to the following gradient of contamination due to human activities on the watershed: A≤Bsperm of individual males and then incubated individually back in the lab to study embryo-larval development as well as using sperm samples to assess DNA integrity. Genetic analysis clearly showed the absence of a difference in genetic structure between the three studied C. nasus populations from the Rhône basin. Sperm DNA integrity was significantly lower in males from station C compared to other ones while sperm biochemical characteristics and fertilization rate remained almost unchanged whatever the station. Mortality and abnormality rates measured at both hatching and at the end of yolk sac resorption stages followed the same trend as the sperm DNA damage, demonstrating an impact of river water quality on nase fitness through a loss of sperm DNA integrity. Since the level of both abnormalities and mortality measured in offspring of fish caught in the most contaminated area reached high values up to 15% and 80%, respectively, the hypothesis that the observed nase decline in Rhône River stemming through selection forces can be put forward.

  3. Exposure to 17α-ethinylestradiol decreases motility and ATP in sperm of male fighting fish Betta splendens.

    PubMed

    Montgomery, Tracy M; Brown, Alexandria C; Gendelman, Hannah K; Ota, Mizuho; Clotfelter, Ethan D

    2014-03-01

    The synthetic estrogen 17α-ethinylestradiol (EE2) is an endocrine-disrupting chemical released into aquatic environments from sewage treatment facilities. We tested the effects of two environmentally relevant concentrations of waterborne EE2, 10 and 100 ng L(-1) , on reproductive endpoints in the teleost fish Betta splendens. In the first experiment, testes were removed from males and sperm were exposed to EE2 directly through the activation water. Direct exposure to EE2 had no effect on any measure of sperm swimming performance. In the second experiment, we exposed sexually mature male B. splendens to EE2 using a semi-static exposure protocol for 4 weeks. There were no significant treatment effects in the 10 ng L(-1) treatment group, but at the 100 ng L(-1) dose we found that fish had smaller gonads and reduced sperm swimming velocity. When allowed to interact freely with female conspecifics, males exposed to 100 ng L(-1) EE2 built smaller nests and showed a nonsignificant decrease in fertilization success. To investigate further the potential mechanism underlying the decrease in sperm quality, we repeated the chronic exposure experiment and analyzed the ATP content of sperm from fish in each treatment group. We found that males exposed to 100 ng L(-1) of EE2 had fewer moles of ATP per sperm than did fish in the other two treatment groups, suggesting that a decrease in intracellular ATP caused a reduction in sperm swimming velocity. The current study adds to the growing body of literature that indicates the risks to aquatic organisms of exposure to environmentally relevant concentrations of EE2.

  4. Epididymal hypo-osmolality induces abnormal sperm morphology and function in the estrogen receptor alpha knockout mouse.

    PubMed

    Joseph, Avenel; Shur, Barry D; Ko, CheMyong; Chambon, Pierre; Hess, Rex A

    2010-05-01

    Estrogen receptor-alpha (ESR1) is highly expressed in the efferent ductules of all species studied as well as in the epididymal epithelium in mice and other select species. Male mice lacking ESR1 (Esr1KO) are infertile, but transplantation studies demonstrated that Esr1KO germ cells are capable of fertilization when placed in a wild-type reproductive tract. These results suggest that extratesticular regions, such as the efferent ductules and epididymis, are the major source of pathological changes in Esr1KO males. Previous studies have shown alterations in ion and fluid transporters in the efferent duct and epididymal epithelia of Esr1KO males, leading to misregulation of luminal fluid pH. To determine the effect of an altered epididymal milieu on Esr1KO sperm, we assayed sperm morphology in the different regions of the epididymis. Sperm recovered from the epididymis exhibited abnormal flagellar coiling and increased incidence of spontaneous acrosome reactions, both of which are consistent with exposure to abnormal epididymal fluid. Analysis of the epididymal fluid revealed that the osmolality of the Esr1KO fluid was reduced relative to wild type, consistent with prior reports of inappropriate fluid absorption from the efferent ductules. This, along with the finding that morphological defects increased with transit through the epididymal duct, suggests that the anomalies in sperm are a consequence of the abnormal luminal environment. Consistent with this, incubating Esr1KO sperm in a more wild-type-like osmotic environment significantly rescued the abnormal flagellar coiling. This work demonstrates that Esr1KO mice exhibit an abnormal fluid environment in the lumen of the efferent ducts and epididymis, precluding normal sperm maturation and instead resulting in progressive deterioration of sperm that contributes to infertility.

  5. Absence of effects on the rat sperm quality after subacute exposure to low doses of fungicide prochloraz.

    PubMed

    Sanabria, Marciana; Pessin, Alessandra; Zanutto, Mirella Rossitto; Perobelli, Juliana Elaine; Guerra, Marina Trevizan; Banzato, Thais Petrochelli; Borges, Cibele dos Santos; Kempinas, Wilma De Grava

    2015-01-01

    Prochloraz (PCZ) is a fungicide and androgen-receptor antagonist used worldwide in horticulture and agriculture. Pre- and perinatal exposure to this pesticide during sexual differentiation is deleterious for male offspring. Since data on the effects of PCZ on epididymal functions are scarce, and because sperm maturation occurs in this organ, the present investigation aimed to determine whether low PCZ doses administered to rats during the phase of sperm transit through the epididymis might affect the morphophysiology of this organ and sperm quality. Adult male Wistar rats were assigned to 4 different groups: 0 (control, vehicle) or 10, 15, or 30 mg/kg bw/d PCZ diluted in corn oil administered orally for 4 consecutive days. Morphofunctional parameters of the male reproductive tract, hormone concentrations, sperm evaluations, and fertility and histopathologic analysis of testis and epididymis were assessed. There were no statistically significant differences between treated and control groups in relation to all evaluated parameters. Data demonstrated show that PCZ exposure for a brief 4-d exposure and low doses did not produce reproductive toxicity or compromise sperm quality in adult rats.

  6. Brain maturation and epilepsy.

    PubMed

    Dulac, Olivier; Milh, Mathieu; Holmes, Gregory L

    2013-01-01

    At full term, both glutamate and gamma-amino-butyric acid (GABA) are excitatory; cortical synapses are beginning to appear, there is little myelin in the cerebral hemispheres, and long tracts hardly start to develop. Neonatal myoclonic encephalopathy can result from premature activation of N-methyl-D-aspartate (NMDA) transmission. Benign neonatal seizures and migrating partial seizures in infancy could involve excessive or premature excitability of deep cortical layers. Benign rolandic epilepsy and continuous spike waves in slow sleep are consistent with an excess of both excitatory and inhibitory cortical synapses. West and Lennox-Gastaut syndromes express age-related diffuse cortical hyperexcitability, the pattern depending on the age of occurrence; synchronization of spikes is becoming possible with maturation of the myelin. Idiopathic generalized epilepsy is itself modulated by maturation that causes frontal hyperexcitability generating myoclonic-astatic seizures, between the ages of infantile and juvenile myoclonic epilepsies. Physiological delay of hippocampo-neocortical pathways maturation could account for the delayed occurrence of mesial temporal epilepsy following infantile damage, whereas premature maturation could contribute to fronto-temporal damage characteristic of fever-induced epileptic encephalopathy in school-age children, a dramatic school-age epileptic encephalopathy.

  7. Maturation in Larch 1

    PubMed Central

    Greenwood, Michael S.; Hopper, Catherine A.; Hutchison, Keith W.

    1989-01-01

    The time course of maturation in eastern larch (Larix laricina [Du Roi] K. Koch) was examined by grafting scions from trees of different ages onto 2-year-old root stock and following scion development for several years. Height, diameter, foliar chlorophyll content, and rooting ability of scion-derived cuttings all varied linearly as a function of log10 age. Chlorophyll content (milligrams per gram of dry weight) increased while height, diameter, and ability to root decreased with age (P < 0.01). The tendency toward orthotropic growth and branch formation per centimeter of main stem decreased abruptly between age 1 and 5 years (P < 0.01). Total chlorophyll content of both long and short shoot foliage increased by 30 to 50% with increasing age, but the chlorophyll a/b ratio did not change. Also, juvenile long shoot needles were significantly longer than mature (P < 0.01). Surprisingly, the juvenile scions produced more total strobili over two successive years, but the mature scions produced a significantly higher proportion of male strobili (P < 0.001 year 1; P < 0.02 year 2). The age-related changes in foliar traits were not associated with changes in DNA methylation between juvenile and mature scions. Using HPLC, we found that 20% of foliar DNA cytosine residues were methylated in both scion types. Images Figure 1 PMID:16666785

  8. Mature Students Studying Mathematics.

    ERIC Educational Resources Information Center

    Hirst, Keith

    1999-01-01

    Discusses mature students in the single subject area of mathematics in a single institution and makes comparisons with traditional universities. Reviews some features of the age distribution, entry qualifications, degree-class distribution, non-completion rates and gender distribution. (Author/ASK)

  9. Is There a Link Between Expression Levels of Histone Deacetylase/Acetyltransferase in Mouse Sperm and Subsequent Blastocyst Development?

    PubMed

    Kim, Jayeon; Kim, Ji-Hee; Jee, Byung-Chul; Suh, Chang-Suk; Kim, Seok-Hyun

    2015-11-01

    Histone acetylation has been known to be significant in spermatogenesis. Histone acetylation is regulated by the act of histone deacetylases (HDACs) and histone acetyltransferases (HATs). We investigated the link between expression levels of HDACs and HATs in mouse sperm and subsequent blastocyst formation rate. In the univariate analysis, expression levels of HDAC1 and HAT were generally not associated with the blastocyst formation rate. When divided by the mature oocyte number category, a significant positive association was observed between the expression levels of HDAC1 and the blastocyst-forming rate in the highest (> 75th) percentile group (a group with ≥34 mature oocytes). In conclusion, expression of sperm HDAC1 could be considered as a possible predictor of embryo development in mice with high ovarian response.

  10. Sperm-egg interaction and functional assessment of springbok, impala and blesbok cauda epididymal spermatozoa using a domestic cattle in vitro fertilization system.

    PubMed

    Chatiza, F P; Bartels, P; Nedambale, T L; Wagenaar, G M

    2013-12-01

    The study assesses the possibility to estimate the potential fertility of post-thawed antelope (Antidorcas marsupialis), impala (Aepyceros melampus) and blesbok (Damaliscus dorcus phillipsi) epididymal sperm using homologous and heterologous IVF and the functioning of cattle IVF system to produce antelope embryos. Cauda epididymal sperm were collected from the antelope and cryopreserved under field conditions. In vitro matured domestic cow, blesbok and springbok oocytes were co-incubated in modified-Tyrode Lactate (m-TL) IVF media with springbok, impala and blesbok sperm for heterologous IVF and springbok and blesbok sperm for homologous IVF. A group of presumptive zygotes from each treatment were examined for sperm penetration and male pronuclear formation after 18h and the remainder were cultured and evaluated for embryo cleavage 22h later. The study shows that Modified Tyrode Lactate in vitro fertilization media supports survivability, capacitation and hyperactivation of springbok, impala and blesbok sperm. Springbok, impala and blesbok post-thawed epididymal spermatozoa are capable of fertilizing domestic cow oocytes under conditions that support domestic cattle IVF. Penetration, male pronuclear formation and embryo cleavage did not differ (p>0.05) between cow oocytes inseminated with sperm from springbok, impala or blesbok however these parameters were higher (p<0.05) for oocytes inseminated with bull sperm. Modified Tyrode Lactate IVF media supported homologous fertilization and embryo development in springbok and blesbok however did not support blastocyst development. These findings suggest that cattle provide a useful model for evaluating springbok, impala and blesbok post-thawed cauda epididymal sperm functionality. Domestic cattle embryo culture conditions need to be modified to promote blastosyst development in these antelope species. Such research provides an important tool in assisted reproductive technology development when high biological value

  11. Characterization of epididymal sperm motion and its correlation with stages of target cells in rats given alpha-chlorohydrin, cyclophosphamide or nitrazepam.

    PubMed

    Kaneto, M; Kanamori, S; Hara, K; Kishi, K

    1999-08-01

    Epididymal sperm motion in rats was characterized by computer-aided sperm motion analysis (CASA) with its correlation to testicular lesions in the 2-week treatment study, using three compounds which are known to affect different stages of germ cells. Mature male rats were treated daily for 2 weeks with alpha-chlorohydrin (alpha-CH, 5 mg/kg), cyclophosphamide (CP, 20 mg/kg) or nitrazepam (NZ, 20, 40, 60 mg/kg). Changes in sperm motion were detected only in the alpha-CH and 60-mg/kg NZ-treated groups. Of the sperm motion parameters, velocity and amplitude of lateral head displacement (ALH) were concomitantly reduced in these two groups with good correlation. With respect to the distribution of the values in parameters, however, alpha-CH shifted the values down within a small range with high percentages of motile sperm, while NZ distributed them over a wide range with low percentages of motile sperm. CP treatment showed no histopathological changes in advanced germ cells, though it showed a decrease in the number of early germ cells. NZ treatment affected round and elongating spermatids (approximately step 14) at doses of 20 and 40 mg/kg, and affected also more advanced spermatids (approximately step 19) at the dose of 60 mg/kg. alpha-CH treatment did not affect testicular histopathology. These findings indicate that 60-mg/kg NZ treatment reduced sperm motion as a result of lesions affected in elongated spermatids and alpha-CH reduced it by direct effects on epididymal spermatozoa. The present study indicates that in addition to percentage of motile sperm, the velocity and ALH can be useful to detect the changes in sperm motion caused by different actions of NZ and alpha-CH, though each compound showed a distinct distribution pattern of these parameters.

  12. Ocean acidification impacts on sperm mitochondrial membrane potential bring sperm swimming behaviour near its tipping point.

    PubMed

    Schlegel, Peter; Binet, Monique T; Havenhand, Jonathan N; Doyle, Christopher J; Williamson, Jane E

    2015-04-01

    Broadcast spawning marine invertebrates are susceptible to environmental stressors such as climate change, as their reproduction depends on the successful meeting and fertilization of gametes in the water column. Under near-future scenarios of ocean acidification, the swimming behaviour of marine invertebrate sperm is altered. We tested whether this was due to changes in sperm mitochondrial activity by investigating the effects of ocean acidification on sperm metabolism and swimming behaviour in the sea urchin Centrostephanus rodgersii. We used a fluorescent molecular probe (JC-1) and flow cytometry to visualize mitochondrial activity (measured as change in mitochondrial membrane potential, MMP). Sperm MMP was significantly reduced in ΔpH -0.3 (35% reduction) and ΔpH -0.5 (48% reduction) treatments, whereas sperm swimming behaviour was less sensitive with only slight changes (up to 11% decrease) observed overall. There was significant inter-individual variability in responses of sperm swimming behaviour and MMP to acidified seawater. We suggest it is likely that sperm exposed to these changes in pH are close to their tipping point in terms of physiological tolerance to acidity. Importantly, substantial inter-individual variation in responses of sperm swimming to ocean acidification may increase the scope for selection of resilient phenotypes, which, if heritable, could provide a basis for adaptation to future ocean acidification.

  13. Human sperm chromosome analysis after subzonal sperm insemination of hamster oocytes

    SciTech Connect

    Cozzi, J.

    1994-09-01

    Sperm microinjection techniques, subzonal sperm insemination (SUZI) and intracytoplasmic sperm injection (ICSI), have achieved a wide spread clinical application for the treatment of male infertility. To date, only one study has focused on sperm karyotypes after microinjection. Martin et al. reported a very high incidence of abnormal human sperm complements after ICSI into hamster oocytes. In the present study, are reported the first human sperm karyotypes after SUZI of hamster oocytes. Spermatozoa from two control donors were treated by calcium ionophore A23187 and injected under the zona of hamster eggs. The microinjected eggs were then cultured for cytogenetic analysis of the pronuclei. Out of 47 analyzed sperm chromosome metaphases, 5 (10.6%) were abnormal, 4 (8.5%) were hypohaploid and 1 (2.1%) had a structural abnormality. The sex ratio was not significantly different from the expected 1:1 ratio. Rates of chromosomal abnormalities in microinjected spermatozoa were similar to those observed in spermatozoa inseminated with zona free eggs, suggesting that SUZI procedure per se does not increase sperm chromosomal abnormalities.

  14. SMART USE OF COMPUTER-AIDED SPERM ANALYSIS (CASA) TO CHARACTERIZE SPERM MOTION

    EPA Science Inventory

    Computer-aided sperm analysis (CASA) has evolved over the past fifteen years to provide an objective, practical means of measuring and characterizing the velocity and parttern of sperm motion. CASA instruments use video frame-grabber boards to capture multiple images of spermato...

  15. Effect of sex-sorting and cryopreservation on the post-thaw sperm quality of Iberian red deer spermatozoa.

    PubMed

    Anel-López, L; García-Álvarez, O; Parrilla, I; Del Olmo, D; Maroto-Morales, A; Fernandez-Santos, M R; Ortiz, J A; Soler, A J; Martínez, E M; Vazquez, J M; Garde, J J

    2017-02-01

    This study investigated the effect of sex-sorting and cryopreservation on post-thaw characteristics and fertility of red deer (Cervus elaphus) sperm for the first time. Semen was collected by electroejaculation from 10 mature stags during the breeding season, and each ejaculate split into four experimental groups: Bulk sorted spermatozoa, sorted but not sexed (BSS); sorted high purity X-spermatozoa (XSS); sorted high purity Y-spermatozoa (YSS); and, control non-sorted spermatozoa (NS). Following, all samples were frozen over liquid nitrogen. Two straws per stag and sample type were analyzed immediately post-thaw and following a 2-h incubation period at 37 °C. Post-thaw total motility (TM) as assessed by CASA was not different (P < 0.05) among NS, BSS and YSS sperm. For XSS, post-thaw TM was lower (39%, P < 0.05) than that for NS (54%) or BSS (50%), but similar (P > 0.05) to that of YSS (47%) sperm. The percentage of apoptotic spermatozoa as assessed by PI/YO-PRO-1 and flow cytometry analysis, was higher (17%, P ≤ 0.05) for XSS sperm than NS (12%), BSS (13%) and YSS (14%) sperm. Following incubation there were no differences (P > 0.05) in TM or percent apoptosis among treatments. Post-thaw chromatin stability calculated as the DNA fragmentation index (%DFI) was similar among treatments; following incubation %DFI increased in all except YSS, which displayed the lowest value (P < 0.05). Artificial insemination of synchronized hinds yielded 44, 52 and 62% delivery rates for YSS, NS and standard frozen-thawed sperm, respectively (P < 0.05). Notably, 93 and 55% of fawns born were males for the YSS and NS spermatozoa, respectively (P < 0.05). In summary, Y-sorted sperm displayed acceptable post-thaw sperm evaluation parameters and the expected offspring sex ratio. More studies are needed to understand the source of sperm damage that may compromise the fertility of Y-sorted red deer sperm.

  16. Proteomics of ionomycin-induced ascidian sperm reaction: Released and exposed sperm proteins in the ascidian Ciona intestinalis.

    PubMed

    Nakazawa, Shiori; Shirae-Kurabayashi, Maki; Otsuka, Kei; Sawada, Hitoshi

    2015-12-01

    Sperm proteins mediating sperm-egg interaction should be exhibited on the sperm surface, or exposed or released when sperm approach an egg. In ascidians (protochordates), sperm undergo a sperm reaction, characterized by enhanced sperm motility and mitochondrial swelling and shedding on contact with the vitelline coat (VC) or by treatment with Ca(2+) ionophore. Here, proteomic analysis was conducted on sperm exudates and sperm surface proteins using ionomycin-induced sperm reaction and cell-impermeable labeling in Ciona intestinalis type A (C. robusta). In the exudate from sperm treated with ionomycin, membrane proteins including a possible VC receptor CiUrabin were abundant, indicating the release of membranous compartments during sperm reaction. Among the surface proteins XP_009859314.1 (uncharacterized protein exhibiting homology to HrTTSP-1) was most abundant before the sperm reaction, but XP_004227079.1 (unknown Ig superfamily protein) appears to be most abundantly exposed by the sperm reaction. Moreover, proteins containing a notable set of domains, astacin-like metalloprotease domain and thrombospondin type 1 repeat(s), were found in this fraction. Possible roles in fertilization as well as localizations and behaviors of these proteins are discussed.

  17. Sperm cryopreservation in fish and shellfish.

    PubMed

    Tiersch, Terrence R; Yang, Huiping; Jenkins, Jill A; Dong, Qiaoxiang

    2007-01-01

    Initial success in sperm cryopreservation came at about the same time for aquatic species and livestock. However, in the 50-plus years since then cryopreserved sperm of livestock has grown into a billion-dollar global industry, while despite work in some 200 species with well over 200 published reports, cryopreservation of aquatic species sperm remains essentially a research activity with little commercial application. Most research has focused on large-bodied culture and sport fishes, such as salmonids, carps, and catfishes, and mollusks such as commercially important oyster and abalone species. However, only a handful of studies have addressed sperm cryopreservation in small fishes, such as zebrafish, and in endangered species. Overall, this work has yielded techniques that are being applied with varying levels of success around the world. Barriers to expanded application include a diverse and widely distributed literature base, technical problems, small sperm volumes, variable results, a general lack of access to the technology, and most importantly, the lack of standardization in practices and reporting. The benefits of cryopreservation include at least five levels of improvements for existing industries and for creation of new industries. First, cryopreservation can be used to improve existing hatchery operations by providing sperm on demand and simplifying the timing of induced spawning. Second, frozen sperm can enhance efficient use of facilities and create new opportunities in the hatchery by eliminating the need to maintain live males, potentially freeing resources for use with females and larvae. Third, valuable genetic lineages such as endangered species, research models, or improved farmed strains can be protected by storage of frozen sperm. Fourth, cryopreservation opens the door for rapid genetic improvement. Frozen sperm can be used in breeding programs to create improved lines and shape the genetic resources available for aquaculture. Finally

  18. Genes for embryo development are packaged in blocks of multivalent chromatin in zebrafish sperm.

    PubMed

    Wu, Shan-Fu; Zhang, Haiying; Cairns, Bradley R

    2011-04-01

    In mature human sperm, genes of importance for embryo development (i.e., transcription factors) lack DNA methylation and bear nucleosomes with distinctive histone modifications, suggesting the specialized packaging of these developmental genes in the germline. Here, we explored the tractable zebrafish model and found conceptual conservation as well as several new features. Biochemical and mass spectrometric approaches reveal the zebrafish sperm genome packaged in nucleosomes and histone variants (and not protamine), and we find linker histones high and H4K16ac absent, key factors that may contribute to genome condensation. We examined several activating (H3K4me2/3, H3K14ac, H2AFV) and repressing (H3K27me3, H3K36me3, H3K9me3, hypoacetylation) modifications/compositions genome-wide and find developmental genes packaged in large blocks of chromatin with coincident activating and repressing marks and DNA hypomethylation, revealing complex "multivalent" chromatin. Notably, genes that acquire DNA methylation in the soma (muscle) are enriched in transcription factors for alternative cell fates. Remarkably, whereas H3K36me3 is located in the 3' coding region of heavily transcribed genes in somatic cells, H3K36me3 is present in the promoters of "silent" developmental regulators in sperm, suggesting different rules for H3K36me3 in the germline and soma. We also reveal the chromatin patterns of transposons, rDNA, and tDNAs. Finally, high levels of H3K4me3 and H3K14ac in sperm are correlated with genes activated in embryos prior to the mid-blastula transition (MBT), whereas multivalent genes are correlated with activation at or after MBT. Taken together, gene sets with particular functions in the embryo are packaged by distinctive types of complex and often atypical chromatin in sperm.

  19. Heterogenous turnover of sperm and seminal vesicle proteins in the mouse revealed by dynamic metabolic labeling.

    PubMed

    Claydon, Amy J; Ramm, Steven A; Pennington, Andrea; Hurst, Jane L; Stockley, Paula; Beynon, Robert

    2012-06-01

    Plasticity in ejaculate composition is predicted as an adaptive response to the evolutionary selective pressure of sperm competition. However, to respond rapidly to local competitive conditions requires dynamic modulation in the production of functionally relevant ejaculate proteins. Here we combine metabolic labeling of proteins with proteomics to explore the opportunity for such modulation within mammalian ejaculates. We assessed the rate at which proteins are synthesized and incorporated in the seminal vesicles of male house mice (Mus musculus domesticus), where major seminal fluid proteins with potential roles in sperm competition are produced. We compared rates of protein turnover in the seminal vesicle with those during spermatogenesis, the timing of which is well known in mice. The subjects were fed a diet containing deuterated valine ([(2)H(8)]valine) for up to 35 days, and the incorporation of dietary-labeled amino acid into seminal vesicle- or sperm-specific proteins was assessed by liquid chromatography-mass spectrometry of samples recovered from the seminal vesicle lumen and cauda epididymis, respectively. Analyses of epididymal contents were consistent with the known duration of spermatogenesis and sperm maturation in this species and in addition revealed evidence for a subset of epididymal proteins subject to rapid turnover. For seminal vesicle proteins, incorporation of the stable isotope was evident from day 2 of labeling, reaching a plateau of labeling by day 24. Hence, even in the absence of copulation, the seminal vesicle proteins and certain epididymal proteins demonstrate considerable turnover, a response that is consonant with the capacity to rapidly modulate protein production. These techniques can now be used to assess the extent of phenotypic plasticity in mammalian ejaculate production and allocation according to social and environmental cues of sperm competition.

  20. Inhibition of viral reverse transcriptase and human sperm DNA polymerase by anti-sperm antibodies.

    PubMed Central

    Witkin, S S; Higgins, P J; Bendich, A

    1978-01-01

    The IgG fraction of serum from a rabbit immunized with detergent-prepared human sperm nuclei inhibited the DNA polymerase activities in human sperm and seminal fluid as well as the partially purified reverse transcriptase of the baboon endogenous type-C retrovirus (BEV). The analogous enzymes from lysates of oncogenic type-C viruses was unaffected. IgG from the serum of individual partners from infertile marriages similarly inhibited both purified BEV reverse transcriptase and human sperm DNA polymerase, but not a DNA polymerase isolated from human prostatic fluid. The data suggest that BEV reverse transcriptase and the human sperm DNA polymerase are antigenically related. Furthermore, the sperm appears to be auto-antigenic and the antibodies thus formed may be capable of interfering with reproductive success. PMID:82498

  1. Fluid flow and sperm guidance: a simulation study of hydrodynamic sperm rheotaxis

    PubMed Central

    Ishimoto, Kenta; Gaffney, Eamonn A.

    2015-01-01

    How does a sperm find its way? The study of guidance cues has fascinated sperm biologists and in particular the prospect of rheotaxis, that is a fluid flow orienting the direction of sperm swimming, has been the subject of extensive recent study, as readily motivated by the prospect that such guidance may be active in the mammalian female reproductive tract. For instance, it has been hypothesized that helical sperm flagellar beating is necessary for such guidance, whereas there is an extensive diversity of flagellar beating patterns, with planar sperm beating readily observed in human cells for example. In particular, such cells will not be guided by fluid flow according to hypothesized mechanisms for rheotaxis presented thus far. Here, using simulation methods, we investigate rheotaxis for a wide range of flagellar beat patterns. Providing the virtual sperm firstly does not possess a tightly circling trajectory in the absence of a background flow and secondly, remains within a region of low shear to prevent being washed away by the background flow, rheotaxis is generally observed with the sperm swimming into the flow together with a possible transverse velocity. Tight circling sperm motility, as observed in select hyperactivated sperm and CatSper mutants, is predicted to disrupt the rheotactic response, whereas confinement to low shear regions generally requires boundary accumulation, thus introducing subtleties in the relationship between rheotactic behaviours and the flagellar waveform and sperm characteristics. Nonetheless, such predictions suggest such rheotactic guidance may be more common and robust than previously thought, and we document simple criteria for the presence of rheotaxis that are consistent with our simulations and understanding, as well as reported observations to date. PMID:25878133

  2. Moonlighting proteins in sperm-egg interactions.

    PubMed

    Petit, François M; Serres, Catherine; Auer, Jana

    2014-12-01

    Sperm-egg interaction is a highly species-specific step during the fertilization process. The first steps consist of recognition between proteins on the sperm head and zona pellucida (ZP) glycoproteins, the acellular coat that protects the oocyte. We aimed to determine which sperm head proteins interact with ZP2, ZP3 and ZP4 in humans. Two approaches were combined to identify these proteins: immunoblotting human spermatozoa targeted by antisperm antibodies (ASAs) from infertile men and far-Western blotting of human sperm proteins overlaid by each of the human recombinant ZP (hrZP) proteins. We used a proteomic approach with 2D electrophoretic separation of sperm protein revealed using either ASAs eluted from infertile patients or recombinant human ZP glycoproteins expressed in Chinese-hamster ovary (CHO) cells. Only spots highlighted by both methods were analysed by MALDI-MS/MS for identification. We identified proteins already described in human spermatozoa, but implicated in different metabolic pathways such as glycolytic enzymes [phosphokinase type 3 (PK3), enolase 1 (ENO1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase A (ALDOA) and triose phosphate isomerase (TPI)], detoxification enzymes [GST Mu (GSTM) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) 4], ion channels [voltage-dependent anion channel 2 (VDAC2)] or structural proteins (outer dense fibre 2). Several proteins were localized on the sperm head by indirect immunofluorescence, and their interaction with ZP proteins was confirmed by co-precipitation experiments. These results confirm the complexity of the sperm-ZP recognition process in humans with the implication of different proteins interacting with the main three ZP glycoproteins. The multiple roles of these proteins suggest that they are multifaceted or moonlighting proteins.

  3. Thyroxin Is Useful to Improve Sperm Motility

    PubMed Central

    Mendeluk, Gabriela Ruth; Rosales, Mónica

    2016-01-01

    Background The aim of this study was to evaluate the non-genomic action of thyroxin on sperm kinetic and its probable use to improve sperm recovery after applying an en- richment method like “swim-up” in comparison with the available one, pentoxifylline. Materials and Methods This is an experimental study. A total of 50 patients were re- cruited, followed by infertility consultation. Conventional sperm assays were performed according to World Health Organization criteria-2010 (WHO-2010). A Computer Aided Semen Analysis System was employed to assess kinetic parameters and concentrations. Number of the motile sperm recovered after preparation technique was calculated. Results Addition of T4 (0.002 µg/ml) to semen samples increased hypermotility at 20 minutes (control: 14.18 ± 5.1% vs. 17.66 ± 8.88%, P<0.03, data expressed as mean ± SD) and remained unchanged after 40 minutes. Significant differences were found in the motile sperm recovered after swim-up (control: 8.93×106 ± 9.52× 06vs. 17.20×106 ± 21.16×106, P<0.03), achieving all of the tested samples a desirable threshold value for artificial insemination outcome, while adding pentoxifylline increased the number of recovered sperm after swim-up in 60% of the studied cases. No synergism between two treatments could be determined. Conclusion We propose a new physiological tool to artificially improve insemination. The discussion opens windows to investigate unknown pathways involved in sperm ca- pacitation and gives innovative arguments to better understand infertility mechanisms. PMID:27441054

  4. Experimental evolution of sperm competitiveness in a mammal

    PubMed Central

    2011-01-01

    Background When females mate with multiple partners, sperm from rival males compete to fertilise the ova. Studies of experimental evolution have proven the selective action of sperm competition on male reproductive traits. However, while reproductive traits may evolve in response to sperm competition, this does not necessarily provide evidence that sperm competitive ability responds to selection. Indeed, a study of Drosophila failed to observe divergence in sperm competitive ability of males in lines selected for enhanced sperm offence and defence. Results Adopting the naturally polygamous house mouse (Mus domesticus) as our vertebrate model, we performed an experimental evolution study and observed genetic divergence in sperm quality; males from the polygamous selection lines produced ejaculates with increased sperm numbers and greater sperm motility compared to males from the monogamous lines. Here, after 12 generations of experimental evolution, we conducted competitive matings between males from lineages evolving under sperm competition and males from lineages subject to relaxed selection. We reduced variation in paternity arising from embryo mortality by genotyping embryos in utero at 14 days gestation. Our microsatellite data revealed a significant paternity bias toward males that evolved under the selective regime of sperm competition. Conclusion We provide evidence that the sperm competitiveness phenotype can respond to selection, and show that improved sperm quality translates to greater competitive fertilisation success in house mice. PMID:21251249

  5. Female choice of young sperm in a genetically monogamous bird.

    PubMed Central

    Wagner, Richard H; Helfenstein, Fabrice; Danchin, Etienne

    2004-01-01

    When females copulate with multiple males the potential exists for female sperm choice. Females may increase the probability of being fertilized by preferred males by selectively retaining their sperm while ejecting the sperm of unfavoured males. An alternative criterion to male quality for female sperm choice may be sperm age because old sperm degrade and can lead to zygote death or unhealthy offspring. Here, we report that in a genetically monogamous bird, the black-legged kittiwake Rissa tridactyla, females eject their mates' sperm according to when the copulations were performed. Following copulations that were performed approximately two weeks before egg laying, females ejected inseminations at high frequencies while retaining inseminations that occurred soon before laying. Females that suffered hatching failure had ejected sperm from early copulations less than half as frequently as females whose entire clutches hatched. Furthermore, chicks that hatched from eggs fertilized by old sperm were in poor condition relative to those fertilized by young sperm. These findings support the 'young sperm' hypothesis, which predicts that females choose fresh sperm to avoid reproductive failure and are the first to show intra-male sperm choice by females. PMID:15252964

  6. DNA fragmentation and sperm head morphometry in cat epididymal spermatozoa.

    PubMed

    Vernocchi, Valentina; Morselli, Maria Giorgia; Lange Consiglio, Anna; Faustini, Massimo; Luvoni, Gaia Cecilia

    2014-10-15

    Sperm DNA fragmentation is an important parameter to assess sperm quality and can be a putative fertility predictor. Because the sperm head consists almost entirely of DNA, subtle differences in sperm head morphometry might be related to DNA status. Several techniques are available to analyze sperm DNA fragmentation, but they are labor-intensive and require expensive instrumentations. Recently, a kit (Sperm-Halomax) based on the sperm chromatin dispersion test and developed for spermatozoa of different species, but not for cat spermatozoa, became commercially available. The first aim of the present study was to verify the suitability of Sperm-Halomax assay, specifically developed for canine semen, for the evaluation of DNA fragmentation of epididymal cat spermatozoa. For this purpose, DNA fragmentation indexes (DFIs) obtained with Sperm-Halomax and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) were compared. The second aim was to investigate whether a correlation between DNA status, sperm head morphology, and morphometry assessed by computer-assisted semen analysis exists in cat epididymal spermatozoa. No differences were observed in DFIs obtained with Sperm-Halomax and TUNEL. This result indicates that Sperm-Halomax assay provides a reliable evaluation of DNA fragmentation of epididymal feline spermatozoa. The DFI seems to be independent from all the measured variables of sperm head morphology and morphometry. Thus, the evaluation of the DNA status of spermatozoa could effectively contribute to the completion of the standard analysis of fresh or frozen semen used in assisted reproductive technologies.

  7. The development of cat testicular sperm cryopreservation protocols: Effects of tissue fragments or sperm cell suspension.

    PubMed

    Chatdarong, Kaywalee; Thuwanut, Paweena; Morrell, Jane M

    2016-01-15

    In endangered animals that have been found dead or sterilized for medical reasons, testis is the ultimate source of haploid DNA or sperm. Thus, preservation of testicular sperm may be performed to rescue their genetics. The aim of this study was to evaluate protocols for testicular sperm freezing: as tissue fragments or cell suspension in domestic cats as a model. A pair of testes from each cat (n = 9) were cut into eight equal pieces. Four randomly selected pieces were cryopreserved as: (1) tissue pieces using two-step freezing; (2) tissue pieces using a slow passive cooling device (CoolCell); (3) sperm suspension after single-layer centrifugation (SLC) through colloids; and (4) sperm suspension without being processed through SLC. A testicular piece from each cat served as fresh control. Testicular sperm membrane and DNA integrity were evaluated before, and after, the cryopreservation process. In addition, spermatogenic cell types (testicular sperm, spermatogonia, spermatocyte, and spermatid) present in the suspension samples were counted before and after SLC. The results found that testicular sperm membrane integrity in the suspension after SLC process was higher than that in the fragment form neither using the two-step nor CoolCell freezing, both before and after freezing (before freezing: 92.3 ± 3.4 vs. 81 ± 4.5 and 80.0 ± 7.0; after freezing: 84.5 ± 4.6 vs. 71.2 ± 12 and 76.2 ± 4.6; P ≤ 0.05). Testicular sperm DNA integrity was, however, not different among groups. Furthermore, the samples processed through the SLC had higher ration of sperm cells: other spermatogenic cells than those were not processed through the SLC (88.9 ± 3.8 vs. 30 ± 7.9; P ≤ 0.05). In summary, testicular sperm cryopreserved as a minced suspension is considered suitable in terms of preventing sperm membrane integrity, and SLC is considered a selection tool for enriching haploid sperm cells from castrated or postmortem cats.

  8. Delayed visual maturation.

    PubMed Central

    Cole, G F; Hungerford, J; Jones, R B

    1984-01-01

    Sixteen blind babies who were considered to be showing the characteristics of delayed visual maturation were studied prospectively. The diagnosis was made on clinical grounds, and the criteria for this are discussed. All of these infants developed visual responses between 4 and 6 months of age and had normal or near normal visual acuities by 1 year of age. Long term follow up, however, has shown neurological abnormalities in some of these children. PMID:6200080

  9. Proteomic identification of rainbow trout sperm proteins.

    PubMed

    Nynca, Joanna; Arnold, Georg J; Fröhlich, Thomas; Otte, Kathrin; Ciereszko, Andrzej

    2014-06-01

    Proteomics represents a powerful tool for the analysis of fish spermatozoa, since these cells are transcriptionally inactive. The aim of the present study was to generate an inventory of the most prominent rainbow trout sperm proteins by SDS-PAGE prefractionation combined with nano-LC-MS/MS based identification. This study provides the first in-depth analysis of the rainbow trout sperm proteome, with a total of 206 identified proteins. We found that rainbow trout spermatozoa are equipped with functionally diverse proteins related to energetic metabolism, signal transduction, protein turnover, transport, cytoskeleton, oxidative injuries, and stress and reproduction. The availability of a catalog of rainbow trout sperm proteins provides a crucial tool for the understanding of fundamental molecular processes in fish spermatozoa, for the ongoing development of novel markers of sperm quality and for the optimization of short- and long-term sperm preservation procedures. The MS data are available at ProteomeXchange with the dataset identifier PXD000355 and DOI 10.6019/PXD000355.

  10. Toxicants and human sperm chromatin integrity.

    PubMed

    Delbès, Geraldine; Hales, Barbara F; Robaire, Bernard

    2010-01-01

    The integrity of the paternal genome is essential as the spermatozoon can bring genetic damage into the oocyte at fertilization and contribute to the development of abnormal pregnancy outcome. During the past two decades, many assays have been developed to measure sperm DNA strand breaks, chromatin structure and compaction and assess the proteins associated with the DNA, as well as epigenetic modifications. Using these assays, it has been shown that exposure to physical agents or chemicals, including therapeutic drugs and environmental toxicants, can affect the integrity of sperm chromatin, inducing structural, genetic and/or epigenetic abnormalities. The mechanisms by which such damage is triggered are still largely unresolved and the susceptibility of each individual will depend on their genetic background, lifestyle and exposure to various insults. Depending on the nature of the chemicals, they may directly target the DNA, induce an oxidative stress, or modify the epigenetic elements. The significance of measuring the sperm chromatin integrity comes from the fact that this end-point correlates well with the low IVF and ICSI outcomes, and idiopathic infertility. Nevertheless, it is hard to establish a direct link between the paternal sperm chromatin integrity and the health of the future generations. Thus, it seems essential to undertake studies that will resolve the impact of chemical and environmental factors on chromatin structure and epigenetic components of human spermatozoa and to elucidate what sperm nuclear end-points are predictors of the quality of progeny outcome.

  11. Sperm Cells of a Primitive Strepsipteran

    PubMed Central

    Nardi, James B.; Delgado, Juan A.; Collantes, Francisco; Miller, Lou Ann; Bee, Charles M.; Kathirithamby, Jeyaraney

    2013-01-01

    The unusual life style of Strepsiptera has presented a long-standing puzzle in establishing its affinity to other insects. Although Strepsiptera share few structural similarities with other insect orders, all members of this order share a parasitic life style with members of two distinctive families in the Coleoptera—the order now considered the most closely related to Strepsiptera based on recent genomic evidence. Among the structural features of several strepsipteran families and other insect families that have been surveyed are the organization of testes and ultrastructure of sperm cells. For comparison with existing information on insect sperm structure, this manuscript presents a description of testes and sperm of a representative of the most primitive extant strepsipteran family Mengenillidae, Eoxenos laboulbenei. We compare sperm structure of E. laboulbenei from this family with that of the three other families of Strepsiptera in the other strepsipteran suborder Stylopidia that have been studied as well as with members of the beetle families Meloidae and Rhipiphoridae that share similar life histories with Strepsiptera. Meloids, Rhipiphorids and Strepsipterans all begin larval life as active and viviparous first instar larvae. This study examines global features of these insects’ sperm cells along with specific ultrastructural features of their organelles. PMID:26462430

  12. Sperm Cells of a Primitive Strepsipteran.

    PubMed

    Nardi, James B; Delgado, Juan A; Collantes, Francisco; Miller, Lou Ann; Bee, Charles M; Kathirithamby, Jeyaraney

    2013-09-04

    The unusual life style of Strepsiptera has presented a long-standing puzzle in establishing its affinity to other insects. Although Strepsiptera share few structural similarities with other insect orders, all members of this order share a parasitic life style with members of two distinctive families in the Coleoptera-the order now considered the most closely related to Strepsiptera based on recent genomic evidence. Among the structural features of several strepsipteran families and other insect families that have been surveyed are the organization of testes and ultrastructure of sperm cells. For comparison with existing information on insect sperm structure, this manuscript presents a description of testes and sperm of a representative of the most primitive extant strepsipteran family Mengenillidae, Eoxenos laboulbenei. We compare sperm structure of E. laboulbenei from this family with that of the three other families of Strepsiptera in the other strepsipteran suborder Stylopidia that have been studied as well as with members of the beetle families Meloidae and Rhipiphoridae that share similar life histories with Strepsiptera. Meloids, Rhipiphorids and Strepsipterans all begin larval life as active and viviparous first instar larvae. This study examines global features of these insects' sperm cells along with specific ultrastructural features of their organelles.

  13. Sperm Membrane Behaviour during Cooling and Cryopreservation.

    PubMed

    Sieme, H; Oldenhof, H; Wolkers, W F

    2015-09-01

    Native sperm is only marginally stable after collection. Cryopreservation of semen facilitates transport and storage for later use in artificial reproduction technologies, but cryopreservation processing may result in cellular damage compromising sperm function. Membranes are thought to be the primary site of cryopreservation injury. Therefore, insights into the effects of cooling, ice formation and protective agents on sperm membranes may help to rationally design cryopreservation protocols. In this review, we describe membrane phase behaviour of sperm at supra- and subzero temperatures. In addition, factors affecting membrane phase transitions and stability, sperm osmotic tolerance limits and mode of action of cryoprotective agents are discussed. It is shown how cooling only results in minor thermotropic non-cooperative phase transitions, whereas freezing causes sharp lyotropic fluid-to-gel phase transitions. Membrane cholesterol content affects suprazero membrane phase behaviour and osmotic tolerance. The rate and extent of cellular dehydration coinciding with freezing-induced membrane phase transitions are affected by the cooling rate and ice nucleation temperature and can be modulated by cryoprotective agents. Permeating agents such as glycerol can move across cellular membranes, whereas non-permeating agents such as sucrose cannot. Both, permeating and non-permeating protectants preserve biomolecular and cellular structures by forming a protective glassy state during freezing.

  14. Living Kinship Trouble: Danish Sperm Donors' Narratives of Relatedness.

    PubMed

    Mohr, Sebastian

    2015-01-01

    Danish sperm donors face a particular kind of kinship trouble: they find themselves in a cultural and organizational context that offers different and contrary ways of how to make connections to donor-conceived individuals meaningful. Whereas Danish sperm banks and Danish law want sperm donors to regard these connections as contractual issues, the dominant kinship narrative in Denmark asks sperm donors to also consider them as family and kinship relations. Based on interviews with Danish sperm donors and participant observation at Danish sperm banks, I argue that Danish sperm donors make sense of connections to donor-conceived individuals as a particular kind of relatedness that cannot be reduced to either contractual or kinship relations. Making sense of these connections, sperm donors negotiate their social significance and thereby participate in opening a space which offers avenues for new kinds of sociality.

  15. Protective effect of Rhodiola rosea polysaccharides on cryopreserved boar sperm.

    PubMed

    Yang, Shen-Min; Wang, Ting; Wen, Duan-Gai; Hou, Jian-Quan; Li, Hai-Bo

    2016-01-01

    Cryopreservation brings sublethal damage to sperm, resulting in reduced fertile life of sperm. Rhodiola rosea polysaccharides (RPs) have antiviral, antioxidant and antitumor activities. In the present study, the cryoprotective effect of RPs on boar sperm quality parameters after frozen-thawed process was investigated. Boar sperm was cryopreserved in the extender with RPs added at concentrations of 0 (used as control), 2, 4, 6, 8 and 10mg/L and their effects on the quality of frozen-thawed boar sperm were assessed. Addition of RPs significantly improved sperm motility, mitochondrial activity, acrosomal integrity, plasma membrane integrity, superoxide dismutase and glutathione peroxidase activity and decreased sperm malonaldehyde level (p<0.05). The results indicated that the addition of RPs to the freezing extender decreased the cryodamage to the boar sperm.

  16. The postnatal maturation of efferent tubules in the rat: a light and electron microscopy study.

    PubMed

    Francavilla, S; Moscardelli, S; Bruno, B; Barcellona, P S; De Martino, C

    1986-07-01

    The postnatal maturation of the epithelium and tubule wall of efferent tubules in the rat was investigated by light and transmission electron microscopy, from birth to 50 days of age, when sperms were released from the seminiferous tubules and appeared in the genital duct. At the end of the first week of life, an endocytotic apparatus is differentiated in the epithelial cells. During the third week of life, efferent tubules developed specializations for the transport of sperms and fluids, namely the appearance of ciliated elements interspersed among the principal cells of the epithelium, and differentiation of myoid elements in the tubule wall. The appearance of specializations related to endocytosis and fluid transport across the epithelium preceded the canalization of the seminiferous cords which, in fact, is reported to appear at the end of the second week of life in the rat, along with the initial secretion of testicular fluid. This suggested that the maturation of efferent tubules is not triggered by the passage of testicular fluid, as surmised for the postnatal differentiation of caput epididymis. The postnatal maturation of efferent tubules was almost complete 35 days after birth. The appearance of sperms in the genital duct of 50-day-old animals was not associated with any remarkable structural change.

  17. Spermatogenesis and sperm ultrastructure in the polychaete genus Ophryotrocha (Dorvilleidae)

    NASA Astrophysics Data System (ADS)

    Pfannenstiel, Hans-Dieter; Grünig, Charlotte

    1990-06-01

    The details of spermatogenesis and spermiogenesis are described for Ophryotrocha puerilis. The ultrastructure of mature sperm is shown for O. puerilis, O. hartmanni, O. gracilis, O. diadema, O. labronica, and O. notoglandulata. Clusters of sixteen cells each are proliferated by two stem cells in each setigerous segment of O. puerilis representing the very early stages of both oogenesis and spermatogenesis. In each spermatocyte-I cluster, the cells are interconnected by cytoplasmic bridges. Early, clusters are enveloped by peritoneal sheath cells. These transient gonad walls break down prior to meiosis. The meiotic processes may start in the clusters with the cells still interconnected, or during breakdown of the original cluster, giving rise to smaller subclusters of both spermatocytes I and spermatocytes II with various numbers of cells. Finally, spermatid tetrads are present. As spermiogenesis progresses, the tetrads disintegrate. Golgi vesicles in both spermatocytes and spermatids contain electron-dense material, presumably preacrosomal. The acrosome is formed by such vesicles. In the six species studied here, the acrosomes appear to be of a similar overall structure but are of different shape. Centrioles are usually located beneath the acrosome. The distal centriole forms the basal body of a flagellum-like cytoplasmic process. The microtubules of these flagellar equivalents do not show a normal ciliar arrangement. The flagellar equivalent appears to be non-motile. In O. hartmanni and in O. notoglandulata, a flagellar equivalent is missing. Microtubules originating from the proximal end of the distal centriole stretch to the nuclear envelope. This feature appears to be especially conspicuous in O. puerilis and in O. labronica. In O. labronica and in O. notoglandulata, bundles of microtubules paralleling the cell perimeter appear to stabilise the sperm. Various numbers of mitochondria are either randomly distributed around the nucleus or accumulate on one side

  18. Control of Oocyte Growth and Meiotic Maturation in C. elegans

    PubMed Central

    Kim, Seongseop; Spike, Caroline; Greenstein, David

    2013-01-01

    In sexually reproducing animals, oocytes arrest at diplotene or diakinesis and resume meiosis (meiotic maturation) in response to hormones. Chromosome segregation errors in female meiosis I are the leading cause of human birth defects, and age-related changes in the hormonal environment of the ovary are a suggested cause. C. elegans is emerging as a genetic paradigm for studying hormonal control of meiotic maturation. The meiotic maturation processes in C. elegans and mammals share a number of biological and molecular similarities. Major sperm protein (MSP) and luteinizing hormone (LH), though unrelated in sequence, both trigger meiotic resumption using somatic Gαs-adenylate cyclase pathways and soma-germline gap-junctional communication. At a molecular level, the oocyte responses apparently involve the control of conserved protein kinase pathways and post-transcriptional gene regulation in the oocyte. At a cellular level, the responses include cortical cytoskeletal rearrangement, nuclear envelope breakdown, assembly of the acentriolar meiotic spindle, chromosome segregation, and likely changes important for fertilization and the oocyte-to-embryo transition. This chapter focuses on signaling mechanisms required for oocyte growth and meiotic maturation in C. elegans and discusses how these mechanisms coordinate the completion of meiosis and the oocyte-to-embryo transition. PMID:22872481

  19. Speract, a sea urchin egg peptide that regulates sperm motility, also stimulates sperm mitochondrial metabolism.

    PubMed

    García-Rincón, Juan; Darszon, Alberto; Beltrán, Carmen

    2016-04-01

    Sea urchin sperm have only one mitochondrion, that in addition to being the main source of energy, may modulate intracellular Ca(2+) concentration ([Ca(2+)]i) to regulate their motility and possibly the acrosome reaction. Speract is a decapeptide from the outer jelly layer of the Strongylocentrotus purpuratus egg that upon binding to its receptor in the sperm, stimulates sperm motility, respiration and ion fluxes, among other physiological events. Altering the sea urchin sperm mitochondrial function with specific inhibitors of this organelle, increases [Ca(2+)]i in an external Ca(2+) concentration ([Ca(2+)]ext)-dependent manner (Ardón, et al., 2009. BBActa 1787: 15), suggesting that the mitochondrion is involved in sperm [Ca(2+)]i homeostasis. To further understand the interrelationship between the mitochondrion and the speract responses, we measured mitochondrial membrane potential (ΔΨ) and NADH levels. We found that the stimulation of sperm with speract depolarizes the mitochondrion and increases the levels of NADH. Surprisingly, these responses are independent of external Ca(2+) and are due to the increase in intracellular pH (pHi) induced by speract. Our findings indicate that speract, by regulating pHi, in addition to [Ca(2+)]i, may finely modulate mitochondrial metabolism to control motility and ensure that sperm reach the egg and fertilize it.

  20. Sperm traits in farmed and wild Atlantic salmon Salmo salar.

    PubMed

    Camarillo-Sepulveda, N; Hamoutene, D; Lush, L; Burt, K; Volkoff, H; Fleming, I A

    2016-02-01

    Differences in sperm metabolism and morphology between wild and non-local farmed Atlantic salmon Salmo salar were assessed by measuring metabolic enzyme activities and length of sperm flagella. No differences were observed between wild and farmed S. salar sperm with regards to cell counts or any of the biochemical variables assessed. Flagella of sperm cells were significantly longer in wild than farmed S. salar; however, this did not result in higher energy levels or different fertilization rates.

  1. Oocyte recovery, maturation and fertilization in vitro in the puma (Felis concolor).

    PubMed

    Miller, A M; Roelke, M E; Goodrowe, K L; Howard, J G; Wildt, D E

    1990-01-01

    Eight female pumas were treated i.m. with 1000 (N = 5) or 2000 (N = 3) i.u. PMSG followed 84 h later by 800 i.u. hCG. Eggs were recovered 24-26 h after hCG from ovarian follicles by using laparoscopy and transabdominal aspiration. Mature eggs were inseminated in vitro 4-6 h later whereas immature eggs were cultured for 24 h and then inseminated. Electroejaculates from 3 pumas were diluted with mKRB before insemination to evaluate the influence of sperm concentration on fertilization. Seven of 8 pumas responded with follicle development, and 140 eggs were recovered from 145 follicles (96.6%; 77 mature, 43 immature, 20 degenerate eggs; mean +/- s.e.m., 20.0 +/- 5.9 eggs/female). Overall fertilization rate was 43.5% (total eggs fertilized = 40) despite using inseminates containing 82-99% pleiomorphic spermatozoa. Of the 36 immature oocytes matured in vitro and inseminated, 12 were fertilized even though 50% of the inseminating spermatozoa contained an acrosomal defect. Fertilization rate of mature oocytes collected from follicles appeared unrelated (P greater than 0.05) to PMSG dose or number of spermatozoa/inseminate. This study demonstrates that a high proportion of follicular eggs can be recovered laparoscopically from adult pumas treated with PMSG and hCG. These gametes are capable of being fertilized in vitro (immediately or after maturation in vitro) even with low quality semen with a high incidence of sperm pleiomorphisms.

  2. Protective effect of royal jelly on the sperm parameters and testosterone level and lipid peroxidation in adult mice treated with oxymetholone

    PubMed Central

    Zahmatkesh, Ensieh; Najafi, Gholamreza; Nejati, Vahid; Heidari, Reza

    2014-01-01

    Objectives : The aim of the present study was to evaluate protective effect of royal jelly on sperm parameters, testosterone level, and malondialdehyde (MDA) production in mice. Materials and Methods: Thirty-two adult male NMRI mice weighing 30±2 g were used. All the animals were divided into 4 groups. Control group: received saline 0.1 ml/mouse/day orally for 30 days. Royal jelly group (RJ): received royal jelly at dose of 100 mg/kg daily for 30 days orally. Oxymetholone group: the received Oxymetholone (OX) at dose of 5 mg/kg daily for 30 days orally. Royal jelly+Oxymetholone group: received royal jelly at dose of 100 mg/kg/day orally concomitant with OX administration. Sperm count, sperm motility, viability, maturity, and DNA integrity were analyzed. Furthermore, serum testosterone and MDA concentrations were determined. Results: In Oxymetholone group, sperm count, motility as well as testosterone concentration reduced significantly (p<0.05), while significant (p<0.05) increases in immature sperm, sperm with DNA damaged, and MDA concentration were announced in Oxymetholone group in comparison with control group and Royal jelly+Oxymetholone group. RJ caused partially amelioration in all of the above- mentioned parameters in Royal Jelly+Oxymetholone group. Conclusion: In conclusion, RJ may be used in combination with OX to improve OX-induced oxidative stress and male infertility. PMID:25050300

  3. Sperm dilution ratio affects post-thaw motility rate and velocity of Prochilodus lineatus (Characiformes) sperm.

    PubMed

    Viveiros, Ana T M; Leal, Marcelo C

    2016-10-01

    There is a lack of standardization in sperm cryopreservation of aquatic organisms and, thus, a necessity of more accurate investigations in all steps of this process. In this study, the effects of sperm dilution ratio on post-thaw sperm quality of Prochilodus lineatus were evaluated. Sperm was diluted in a standard freezing medium (glucose and methyl glycol) at four different ratios (sperm to final volume = 1:5, 1:10, 1:50 or 1:100), frozen in a nitrogen vapour vessel at -170°C and then stored in liquid nitrogen vessel at -196°C. Post-thaw motility rate and velocities (curvilinear = VCL; average path = VAP; straight line = VSL) were determined using a Computer-Assisted Sperm Analyzer (CASA) at 10 and 40 s post-activation. The highest motility rates were observed when sperm was frozen at a ratio of 1:5 (76%) and 1:10 (75%). The highest VCL (225 μm/s) and VAP (203 μm/s) were observed at a ratio of 1:10, while VSL was similar among samples frozen at 1:5, 1:10 and 1:50 (97-124 μm/s). When those parameters were evaluated again 30 s later, motility decreased significantly in samples frozen at a ratio of 1:5 (57%) and 1:10 (61%), while velocities decreased significantly in all samples regardless of dilution ratio (75-85 μm/s of VCL, 38-53 μm/s of VAP and 25-39 μm/s of VSL). P. lineatus sperm should be frozen at a ratio of 1:10, where both the number of loaded sperm per straw and the post-thaw quality are maximized.

  4. Sperm retrieval outcomes with microdissection testicular sperm extraction (micro-TESE) in men with cryptozoospermia.

    PubMed

    Alrabeeah, K; Wachter, A; Phillips, S; Cohen, B; Al-Hathal, N; Zini, A

    2015-05-01

    Several studies support of the use of testicular rather than ejaculated spermatozoa for intracytoplasmic sperm injection (ICSI) in couples with virtual azoospermia or cryptozoospermia, although this approach remains controversial. We sought to evaluate sperm retrieval outcomes with microdissection testicular sperm extraction (micro-TESE) in men with cryptozoospermia. We conducted a retrospective study of 24 consecutive micro-TESEs in men with cryptozoospermia. We also evaluated the outcomes of seven consecutive TESAs (testicular sperm aspiration) in cryptozoospermic men during the same time period (January 2007 and September 2014). Micro-TESE and TESA were performed on the day prior to ICSI. Final assessment of sperm recovery (reported on the day of ICSI) was recorded as (i) successful (available spermatozoa for ICSI) or (ii) unsuccessful (no spermatozoa for ICSI). The decision to perform a unilateral or bilateral micro-TESE was guided by the intra-operative evaluation of sperm recovery from the first testicle. A unilateral procedure was performed in 87.5% (21/24) and 57% (4/7) of the micro-TESE and TESA cohorts, respectively. Sperm recovery was successful in 96% (23/24) of the men who underwent micro-TESE and 43% (3/7) of the men who underwent TESA (p < 0.01). The ICSI pregnancy rates (per embryo transfer) in the micro-TESE and TESA groups were comparable [33% (6/18) and 50% (1/2), respectively]. The data indicate that micro-TESE is a highly successful sperm retrieval technique for men with cryptozoospermia and few of these men will require a bilateral procedure. Moreover, sperm retrieval rates are higher with micro-TESE than TESA in this group of men.

  5. Sperm acrosin levels in semen: comparison between ACCU-SPERM and Kennedy's methods.

    PubMed

    Ikemoto, I; Fanning, L; Loughlin, K R; Agarwal, A

    1994-01-01

    The purpose of this experiment was to evaluate the accuracy, specificity, and sensitivity of a new acrosin activity assay, ACCU-SPERM, and to correlate these results with the original Kennedy method. Thirty-nine specimens (26 patients and 13 donors) of 54 (72%) were found to be in the normal range (> 25 microIU acrosin/10(6) sperm) by the Kennedy method; the other 15 specimens were in either the indeterminate or subfertile range (< 14 microIU). However, according to the ACCU-SPERM method, (normal: 6.6-27 AAI; infertile: < 3.6), 90% of specimens (49 of 54) whose acrosin activity was measured were in the subfertile or infertile range. Similarly, only 28% (4 of 14) of donors in the ACCU-SPERM method were in the normal range in contrast to the 93% (13 of 14) in Kennedy. After calculating the ACCU-SPERM normal range in our laboratory using the linear regression curve between the acrosin values generated by the Kennedy and ACCU-SPERM methods, we again compared results of the two methods. The new normal range of > 1.82 AAI in ACCU-SPERM corresponded to > 25 microIU in the Kennedy method; similarly a value of < 1.35 AAI in ACCU-SPERM corresponded to < 14 microIU in the Kennedy technique. Analysis of the results generated by the two methods revealed a poor correlation with a positive concordance of 51% and a negative concordance of 50% in both assays. These results strongly suggest that the ACCU-SPERM method for measurement of acrosin activity is not a reliable assay.

  6. Sound production in neonate sperm whales (L)

    NASA Astrophysics Data System (ADS)

    Madsen, P. T.; Carder, D. A.; Au, W. W. L.; Nachtigall, P. E.; Møhl, B.; Ridgway, S. H.

    2003-06-01

    Acoustic data from two sperm whale neonates (Physeter macrocephalus) in rehabilitation are presented and implications for sound production and function are discussed. The clicks of neonate sperm whale are very different from usual clicks of adult specimens in that neonate clicks are of low directionality [SL anomaly (0°-90°) <8 dB], long duration (2-12 ms), and low frequency (centroid frequency between 300 and 1700 Hz) with estimated SLs between 140 and 162 dB//1 μPa (rms). Such neonate clicks are unsuited for biosonar, but can potentially convey homing information between calves and submerged conspecifics in open ocean waters at ranges of some 2 km. Moreover, it is demonstrated that sperm whale clicks are produced at the anterior placed monkey lips, thereby substantiating a key point in the modified Norris and Harvey theory and supporting the unifying theory of sound production in odontocetes.

  7. Evolution of sperm structure and energetics in passerine birds.

    PubMed

    Rowe, Melissah; Laskemoen, Terje; Johnsen, Arild; Lifjeld, Jan T

    2013-02-22

    Spermatozoa exhibit considerable interspecific variability in size and shape. Our understanding of the adaptive significance of this diversity, however, remains limited. Determining how variation in sperm structure translates into variation in sperm performance will contribute to our understanding of the evolutionary diversification of sperm form. Here, using data from passerine birds, we test the hypothesis that longer sperm swim faster because they have more available energy. We found that sperm with longer midpieces have higher levels of intracellular adenosine triphosphate (ATP), but that greater energy reserves do not translate into faster-swimming sperm. Additionally, we found that interspecific variation in sperm ATP concentration is not associated with the level of sperm competition faced by males. Finally, using Bayesian methods, we compared the evolutionary trajectories of sperm morphology and ATP content, and show that both traits have undergone directional evolutionary change. However, in contrast to recent suggestions in other taxa, we show that changes in ATP are unlikely to have preceded changes in morphology in passerine sperm. These results suggest that variable selective pressures are likely to have driven the evolution of sperm traits in different taxa, and highlight fundamental biological differences between taxa with internal and external fertilization, as well as those with and without sperm storage.

  8. Evolution: sperm, cryptic choice, and the origin of species.

    PubMed

    Chippindale, Adam K

    2013-10-07

    In two fruit fly species, in vivo observations of competing sperm reveal how differences in sperm size, female behavior and reproductive architecture promote retention of same-species sperm. Sexual selection continues after mating and may play an important role in speciation.

  9. LOCALIZATION OF SP22 ON HUMAN SPERM OF DIFFERING QUALITY

    EPA Science Inventory

    LOCALIZATION OF SP22 ON HUMAN SPERM OF DIFFERING QUALITY. AE Lavers*1, GR Klinefelter2, DW Hamilton1, KP Roberts1, 1University of Minnesota, Minneapolis, MN and 2US EPA, Research Triangle Park, NC.
    SP22 is a sperm membrane protein that has been implicated in sperm function d...

  10. Drosophila Sperm Motility in the Reproductive Tract1

    PubMed Central

    Yang, Yong; Lu, Xiangyi

    2011-01-01

    Motile cilia and flagella exhibit many waveforms as outputs of dynein activation sequences on the highly conserved axoneme. Motility change of sperm in the reproductive tract is difficult to study and remains an important area of investigation. Sperm typically execute a sinusoidal waveform. Increased viscosity in the medium induces somewhat unusual arc-line and helical waveforms in some sperm. However, whether the latter two waveforms occur in vivo is not known. Using green fluorescence protein imaging, we show that Drosophila sperm in the uterus move in circular foci via arc-line waves, predominantly in a tail-leading orientation. From the uterus, a small fraction of the sperm enters the seminal receptacle (SR) in parallel formations. After sperm storage and coincident with fertilization of the egg, the sperm exit the SR via head-leading helical waves. Consistent with the observed bidirectional movements, the sperm show the ability to propagate both base-to-tip and tip-to-base flagellar waves. Numerous studies have shown that sperm motility is regulated by intraflagellar calcium concentrations; in particular, the Pkd2 calcium channel has been shown to affect sperm storage. Our analyses here suggest that Pkd2 is required for the sperm to adopt the correct waveform and movement orientation during SR entry. A working model for the sperm's SR entry movement is proposed. PMID:21293028

  11. Vocational Maturity and Self Concepts.

    ERIC Educational Resources Information Center

    Helbing, Hans

    The relationship between separate dimensions of vocational maturity and different self-concept and identity variables were examined. Subjects were Dutch students, age 14-18 years. The vocational maturity dimensions were measured by Dutch adaptations of American vocational maturity scales. Instruments for self-concept and identity measurement were…

  12. Cryopreservation of Iberian pig spermatozoa. Comparison of different freezing extenders based on post-thaw sperm quality.

    PubMed

    De Mercado, Eduardo; Rodríguez, Ana; Gómez, Emilio; Sanz, Elena

    2010-03-01

    The aim of this study was to evaluate the cryoprotective effect of different freezing extenders against cryopreservation injuries on Iberian boar sperm. The sperm-rich fraction was collected and pooled from six sexually mature Iberian boars, and was frozen in different extenders containing glucose, lactose or fructose as sugar source and including Orvus ES Paste only in the freezing extender-2 (Glucose; Lactose and Fructose) or in both freezing extenders (Glucose2; Lactose2 and Fructose2). During the cryopreservation process, the supernatant was removed after the centrifugation step, then was extended with freezing extender-1 for the equilibration period and with freezing extender-2 immediately before freezing. Post-thaw sperm characteristics, such as plasma membrane integrity (SYBR-14/PI), mitochondrial function (Rhodamine 123) and acrosome integrity (NAR), were monitored. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer [SCA]) were recorded in the different experimental treatments. Measurements were taken at 30 and 150 min post-thaw. The state of the acrosome after thawing did not show significant differences between the freezing extenders studied. Freezing-thawing caused a significant decrease (P<0.001) in plasma membrane integrity and in mitochondrial activity in the spermatozoa frozen with Orvus ES Paste in both freezing extenders. Furthermore, spermatozoa frozen with Orvus ES Paste in both freezing extenders exhibited lower (P<0.05) motility and kinematic parameters than those frozen in the absence of Orvus ES Paste in the first freezing extender. The spermatozoa frozen with the Lactose extender and with Orvus ES Paste only in the second freezing extender showed a better evolution of the motility and kinematic characteristics (P<0.05) over time. The deterioration in post-thaw sperm motility and kinematic parameters were concurrent with reduced sperm

  13. Dissociation of motor maturation.

    PubMed

    DiMario, Francis J

    2003-06-01

    We prospectively acquired clinical data regarding the presentation, evaluation, and developmental progress of all patients identified with dissociated motor maturation to define their clinical outcomes. Children (N = 8) referred for evaluation of suspected cerebral palsy because of delayed sitting or walking and identified to have dissociated motor maturation were followed with serial clinical examination. All displayed the characteristic "sitting on air" posture while held in vertical suspension and had otherwise normal developmental assessments. This posture is composed of the hips held in flexion and abduction with the knees extended and feet plantar or dorsiflexed. Three children were initially evaluated at 10 months of age owing to absence of sitting and five other children were evaluated at a mean of 14 months (range 12-19 months) owing to inability to stand. Follow-up evaluations were conducted over a mean of 10.5 months (range 5-34 months). Five children were born prematurely at 34 to 36 weeks gestation. Denver Developmental Screening Test and general and neurologic examinations were normal except to note hypotonia in six children and the "sitting on air" posture in all of the children. Four children have older siblings or parents who "walked late" (after 15 months). On average, the children attained sitting by 8 months (range 7-10 months). One child did not crawl prior to independent walking, two children scooted rather than crawled, and five children crawled at an average of 13.5 months (range 10-16 months). All children cruised by a mean of 18 months (range 16-21.5 months) and attained independent walking by 20.1 months (range 18-25 months). Neuroimaging and serum creatine kinase enzyme testing were normal in two children who were tested. These eight children conform to the syndrome of dissociated motor maturation. The "sitting on air" posture serves as a diagnostic sign and anticipated excellent prognosis, but follow-up is required to ensure a normal

  14. Maturation in Larch 1

    PubMed Central

    Hutchison, Keith W.; Sherman, Christopher D.; Weber, Jill; Smith, Sandra Schiller; Singer, Patricia B.; Greenwood, Michael S.

    1990-01-01

    The effect of maturation on the morphological and photosynthetic characteristics, as well as the expression of two genes involved in photosynthesis in the developing, current year foliage of Eastern larch (Larix laricina [Du Roi]) is described. These effects were observed on foliage during the third growing season after grafting of scions from trees of different ages onto 2 year old rootstock. Specific leaf weight (gram dry weight per square meter), leaf cross-sectional area (per square millimeter), and chlorophyll content (milligram per gram dry weight) all increase with increasing age in long shoot foliage from both indoor- and outdoor-grown trees. Net photosynthesis (NPS) (mole of CO2 per square millimeter per second) increases with age on indoor- but not outdoor-grown trees. NPS also increases with increased chlorophyll content, but outdoor-grown scions of all ages had higher chlorophyll content, and chlorophyll does not appear to be limiting for NPS outdoors. To extend these studies of maturation-related differences in foliar morphology and physiology to the molecular genetic level, sequences were cloned from the cab and rbsS gene families of larch. Both cab and rbcS gene families are expressed in foliage but not in roots, and they are expressed in light-grown seedlings of larch but only at very low levels in dark-grown seedlings (~2% of light-grown seedlings). Steady-state cab mRNA levels are relatively higher (~40%) in newly expanding short shoot foliage from juvenile plants compared to mature plants. Unlike cab, the expression of the rbcS gene family did not seem to vary with age. These data show that the maturation-related changes in morphological and physiological phenotypes are associated with changes in gene expression. No causal relationship has been established, however. Indeed, we conclude that the faster growth of juvenile scions reported previously (MS Greenwood, CA Hopper, KW Hutchison [1989] Plant Physiol 90: 406-412) is not due to increased NPS

  15. Boar spermatozoa and prostaglandin F2alpha. Quality of boar sperm after the addition of prostaglandin F2alpha to the short-term extender over cooling time.

    PubMed

    Yeste, M; Briz, M; Pinart, E; Sancho, S; Garcia-Gil, N; Badia, E; Bassols, J; Pruneda, A; Bussalleu, E; Casas, I; Bonet, S

    2008-10-01

    Prostaglandin F2alpha (PGF2alpha) has been used to improve reproductive performance in swine. The goal of the present work was to determine how the addition of PGF2alpha affects boar sperm quality. Eleven different treatments were evaluated: eight with only PGF2alpha (0.625, 1.25, 2.50, 5, 10, 12.50, 25 and 50mg PGF2alpha/100ml) and three binary treatments (0.625mg PGF2alpha/100ml+200microg/ml hyaluronic acid (HA), 1.25mg PGF2alpha/100ml+200microg/ml HA, 0.625mg PGF2alpha/100ml+7.5microM caffeine (Caf)). All these substances were added to 16 ejaculates from 16 healthy and sexually mature boars (n=16), and each ejaculate was considered as a replicate. Our study also assessed the effects of these 11 treatments over different periods of preservation. Sperm quality was tested immediately after the addition of treatments (time 0), and after 1, 3, 6 and 10 days of cooling at 15 degrees C. To evaluate sperm quality, five parameters were analysed: (1) sperm viability, acrosome and mitochondrial sheath integrity (using a multiple fluorochrome-staining test), (2) sperm motility, (3) sperm morphology and (4) agglutination (using a computer assisted system) and (5) osmotic resistance (using the ORT). Parametric (analysis of variance for repeated measures) and non-parametric tests (Friedman test) were used as statistical analyses. Treatments with PGF2alpha concentrations higher than 12.5mg/100ml were cytotoxic while the others did not damage boar spermatozoa. Thus, the other treatments may be used to produce profitable effects without adverse effects. Moreover, the addition of PGF2alpha at 5mg/100ml to sperm diluted in BTS may maintain sperm viability and motility better after 6 days of cooling, because significant differences were observed (P<0.05) compared with control at the same time.

  16. Src Kinase Is the Connecting Player between Protein Kinase A (PKA) Activation and Hyperpolarization through SLO3 Potassium Channel Regulation in Mouse Sperm.

    PubMed

    Stival, Cintia; La Spina, Florenza A; Baró Graf, Carolina; Arcelay, Enid; Arranz, Silvia E; Ferreira, Juan J; Le Grand, Sibylle; Dzikunu, Victor A; Santi, Celia M; Visconti, Pablo E; Buffone, Mariano G; Krapf, Dario

    2015-07-24

    Plasma membrane hyperpolarization is crucial for mammalian sperm to acquire acrosomal responsiveness during capacitation. Among the signaling events leading to mammalian sperm capacitation, the immediate activation of protein kinase A plays a pivotal role, promoting the subsequent stimulation of protein tyrosine phosphorylation that associates with fertilizing capacity. We have shown previously that mice deficient in the tyrosine kinase cSrc are infertile and exhibit improper cauda epididymis development. It is therefore not clear whether lack of sperm functionality is due to problems in epididymal maturation or to the absence of cSrc in sperm. To further address this problem, we investigated the kinetics of cSrc activation using anti-Tyr(P)-416-cSrc antibodies that only recognize active cSrc. Our results provide evidence that cSrc is activated downstream of PKA and that inhibition of its activity blocks the capacitation-induced hyperpolarization of the sperm plasma membrane without blocking the increase in tyrosine phosphorylation that accompanies capacitation. In addition, we show that cSrc inhibition also blocks the agonist-induced acrosome reaction and that this inhibition is overcome by pharmacological hyperpolarization. Considering that capacitation-induced hyperpolarization is mediated by SLO3, we evaluated the action of cSrc inhibitors on the heterologously expressed SLO3 channel. Our results indicate that, similar to SLO1 K(+) channels, cSrc blockers significantly decreased SLO3-mediated currents. Together, these results are consistent with findings showing that hyperpolarization of the sperm plasma membrane is necessary and sufficient to prepare the sperm for the acrosome reaction and suggest that changes in sperm membrane potential are mediated by cSrc activation.

  17. Missense mutations in SLC26A8, encoding a sperm-specific activator of CFTR, are associated with human asthenozoospermia.

    PubMed

    Dirami, Thassadite; Rode, Baptiste; Jollivet, Mathilde; Da Silva, Nathalie; Escalier, Denise; Gaitch, Natacha; Norez, Caroline; Tuffery, Pierre; Wolf, Jean-Philippe; Becq, Frédéric; Ray, Pierre F; Dulioust, Emmanuel; Gacon, Gérard; Bienvenu, Thierry; Touré, Aminata

    2013-05-02

    The cystic fibrosis transmembrane conductance regulator (CFTR) is present in mature sperm and is required for sperm motility and capacitation. Both these processes are controlled by ions fluxes and are essential for fertilization. We have shown that SLC26A8, a sperm-specific member of the SLC26 family of anion exchangers, associates with the CFTR channel and strongly stimulates its activity. This suggests that the two proteins cooperate to regulate the anion fluxes required for correct sperm motility and capacitation. Here, we report on three heterozygous SLC26A8 missense mutations identified in a cohort of 146 men presenting with asthenozoospermia: c.260G>A (p.Arg87Gln), c.2434G>A (p.Glu812Lys), and c.2860C>T (p.Arg954Cys). These mutations were not present in 121 controls matched for ethnicity, and statistical analysis on a control population of 8,600 individuals (from dbSNP and 1000 Genomes) showed them to be associated with asthenozoospermia with a power > 95%. By cotransfecting Chinese hamster ovary (CHO)-K1 cells with SLC26A8 variants and CFTR, we showed that the physical interaction between the two proteins was partly conserved but that the capacity to activate CFTR-dependent anion transport was completely abolished for all mutants. Biochemical studies revealed the presence of much smaller amounts of protein for all variants, but these amounts were restored to wild-type levels upon treatment with the proteasome inhibitor MG132. Immunocytochemistry also showed the amounts of SLC26A8 in sperm to be abnormally small in individuals carrying the mutations. These mutations might therefore impair formation of the SLC26A8-CFTR complex, principally by affecting SLC26A8 stability, consistent with an impairment of CFTR-dependent sperm-activation events in affected individuals.

  18. Missense Mutations in SLC26A8, Encoding a Sperm-Specific Activator of CFTR, Are Associated with Human Asthenozoospermia

    PubMed Central

    Dirami, Thassadite; Rode, Baptiste; Jollivet, Mathilde; Da Silva, Nathalie; Escalier, Denise; Gaitch, Natacha; Norez, Caroline; Tuffery, Pierre; Wolf, Jean-Philippe; Becq, Frédéric; Ray, Pierre F.; Dulioust, Emmanuel; Gacon, Gérard; Bienvenu, Thierry; Touré, Aminata

    2013-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is present in mature sperm and is required for sperm motility and capacitation. Both these processes are controlled by ions fluxes and are essential for fertilization. We have shown that SLC26A8, a sperm-specific member of the SLC26 family of anion exchangers, associates with the CFTR channel and strongly stimulates its activity. This suggests that the two proteins cooperate to regulate the anion fluxes required for correct sperm motility and capacitation. Here, we report on three heterozygous SLC26A8 missense mutations identified in a cohort of 146 men presenting with asthenozoospermia: c.260G>A (p.Arg87Gln), c.2434G>A (p.Glu812Lys), and c.2860C>T (p.Arg954Cys). These mutations were not present in 121 controls matched for ethnicity, and statistical analysis on a control population of 8,600 individuals (from dbSNP and 1000 Genomes) showed them to be associated with asthenozoospermia with a power > 95%. By cotransfecting Chinese hamster ovary (CHO)-K1 cells with SLC26A8 variants and CFTR, we showed that the physical interaction between the two proteins was partly conserved but that the capacity to activate CFTR-dependent anion transport was completely abolished for all mutants. Biochemical studies revealed the presence of much smaller amounts of protein for all variants, but these amounts were restored to wild-type levels upon treatment with the proteasome inhibitor MG132. Immunocytochemistry also showed the amounts of SLC26A8 in sperm to be abnormally small in individuals carrying the mutations. These mutations might therefore impair formation of the SLC26A8-CFTR complex, principally by affecting SLC26A8 stability, consistent with an impairment of CFTR-dependent sperm-activation events in affected individuals. PMID:23582645

  19. An evaluation of soybean lecithin as an alternative to avian egg yolk in the cryopreservation of fish sperm.

    PubMed

    Yildiz, Cengiz; Bozkurt, Yusuf; Yavas, Ilker

    2013-08-01

    Plant-derived lecithin has been used as a more sanitary alternative to avian egg yolk in livestock sperm cryopreservation protocols but its efficacy for cryopreserving fish sperm has not previously been tested comparatively. Here various concentrations of soybean lecithin were evaluated for the cryopreservation of carp (Cyprinus carpio) sperm. Sexually mature fish were induced to spermiation and ovulation with ovopel. The extenders were prepared by using 300 mM glucose, 10% DMSO, supplemented with different ratios of lecithin (5%, 10%, 15%, and 20%) and 10% egg yolk (control I). Negative control was made without egg yolk and soybean lecithin (control II). The pooled semen was diluted separately at ratio of 1:3 (v/v) by using egg yolk and soybean-based extenders. Diluted semen placed into 0.25 ml straws were equilibrated at 4 °C for 15 min and frozen in liquid nitrogen vapor. Fertilization was conducted using a ratio of 1 × 10(5)spermatozoa/egg. Supplementation of 10% lecithin to extender showed the best cryoprotective effect for sperm motility and duration of motility against freezing damage compared to 15%, 20% and control II groups (p<0.05). Cryopreserved sperm with extender containing 10% lecithin provided a greater result in terms of fertilization success when compared to extenders containing 20% lecithin or control II (p<0.05). It is concluded that the animal protein-free extender containing 10% soybean lecithin has a similar cryoprotective actions with conventional egg yolk-based extender against freezing damages and fertilization. Therefore, soybean lecithin is a suitable alternative to avian egg yolk for the cryopreservation of fish sperm.

  20. Methamphetamine induces abnormal sperm morphology, low sperm concentration and apoptosis in the testis of male rats.

    PubMed

    Nudmamud-Thanoi, S; Thanoi, S

    2011-08-01

    Methamphetamine has been reported to be an important drug in the field of reproductive toxicology. The aim of this study was to investigate the effects of methamphetamine administrations on sperm morphology, sperm concentration and apoptotic activity inside seminiferous tubule in male rats. Rats were administered a dose of 8 mg kg(-1) , intraperitoneally (IP), for acute group and a dose of 4 mg kg(-1) , IP, once daily for 14 days for sub-acute group. Percentage of normal sperm morphology was decreased in acute group when compared with control. Total numbers of sperm count were significantly decreased in acute and sub-acute groups. Apoptotic activities were most abundant in the seminiferous tubules of acute treated animals with a highly significant increase in the number of apoptotic cells per tubule. Those effects of methamphetamine seem to be dose-dependent. The results suggest that methamphetamine not only works as drug of abuse in central nervous system, but also in gametogenesis of males.

  1. Cytometry of deoxyribonuclei acid content and morphology of mammalian sperm

    SciTech Connect

    Gledhill, B.L.

    1983-01-01

    Because spermatogenesis is exquisitely sensitive to external influences, sperm can serve as a biological dosimeter. Advances in interpreting induced sperm abnormalities require a better understanding of sperm characteristics. This report reviews the application of several methods for automated, quantitative detection of shape changes, methods that are faster and more sensitive than conventional subjective technqiues. Variability of sperm deoxyribonucleic acid content as a bioassay of genetic damage is explored, and limitations of the bioassay are discussed. New flow cytometric techniques that could lead to sexing mammalian sperm are examined.

  2. Cytogenetics of human sperm: Structural aberrations and DNA replication

    SciTech Connect

    Brandriff, B.F.; Gordon, L.A.; Carrano, A.V.

    1989-07-11

    The human sperm-hamster egg system, first introduced in 1978 (Rudak et al), has yielded some important insights into questions on chromosomal integrity of human sperm. In this system, human sperm are co-incubated with eggs from the golden hamster. After the gametes fuse, eggs are cultured overnight and approximately 15 hours after fusion, display the haploid chromosomal complement of individual human sperm cells. These chromosomes can be analyzed by standard banding techniques to identify and quantify structural and numerical abnormalities in single sperm. 32 refs., 1 fig.

  3. The quest for the sea urchin egg receptor for sperm.

    PubMed

    Vacquier, Victor D

    2012-08-31

    This review discusses identification, isolation and characterization of proteins mediating species-selective sperm-to-egg adhesion during sea urchin fertilization. Bindin is the only sea urchin sperm protein known to mediate species-selective sperm attachment to eggs. Two completely different egg surface proteins, 350-kDa and EBR1, have affinity for bindin and each one meets all the criteria to be a species-selective sperm receptor. Experiments suggest that sperm bindin recognizes both the sulfated O-linked oligosaccharides on the egg 350-kDa glycoprotein, and also the repeated protein sequence modules of EBR1.

  4. Sperm whale assessment in the Western Ionian Sea using acoustic data from deep sea observatories

    NASA Astrophysics Data System (ADS)

    Caruso, Francesco; Bellia, Giorgio; Beranzoli, Laura; De Domenico, Emilio; Larosa, Giuseppina; Marinaro, Giuditta; Papale, Elena; Pavan, Gianni; Pellegrino, Carmelo; Pulvirenti, Sara; Riccobene, Giorgio; Scandura, Danila; Sciacca, Virginia; Viola, Salvatore

    2015-04-01

    The Italian National Institute of Nuclear Physics (INFN) operates two deep sea infrastructures: Capo Passero, Western Ionian Sea 3,600 meters of depth, and Catania Wester Ionian Sea 2,100 m depth. At the two sites, several research observatories have been run: OnDE, NEMO-SN1, SMO, KM3NeT-Italia most of them jointly operated between INFN and INGV. In all these observatories, passive acoustic sensors (hydrophones) have been installed. Passive Acoustics Monitoring (PAM) is nowadays the main tool of the bioacoustics to study marine mammals. In particular, receiving the sounds emitted by cetaceans from a multi-hydrophones array installed in a cabled seafloor observatory, a research about the ecological dynamics of the species may be performed. Data acquired with the hydrophones installed aboard the OnDE, SMO and KM3NeT-Italia observatories will be reported. Thanks to acquired data, the acoustic presence of the sperm whales was assessed and studied for several years (2005:2013). An "ad hoc" algorithm was also developed to allow the automatic identification of the "clicks" emitted by the sperm whales and measure the size of detected animals. According to the results obtained, the sperm whale population in the area is well-distributed in size, sex and sexual maturity. Although specimens more than 14 meters of length (old males) seem to be absent.

  5. First steps in the development of a functional assay for human sperm using pig oocytes.

    PubMed

    Canovas, Sebastian; Coy, Pilar; Gomez, Emilio

    2007-01-01

    The use of mammalian oocytes to assess human sperm functionality could be a helpful tool with potential applications in clinical and research programs. In an attempt to develop the pig model, the aim of the present work was to study the interaction between human spermatozoa and pig oocytes at the zona pellucida (ZP), the oolemma, and the ooplasm levels. In vitro matured pig oocytes and human spermatozoa from fertile and low-fertility donors were employed. The induction of the acrosome reaction by the ZP, the ability of the sperm to penetrate the oocyte after coincubation, and the male pronuclear formation after ICSI were evaluated. Human spermatozoa can bind to pig ZP and undergo the acrosome reaction (15% to 58%, depending on the individual); they are not able to fuse with the oolemma but they can decondense and form a male pronucleus (40%-100%) when injected into pig oocytes. In conclusion, this study shows that pig oocytes can be a useful model to assess human sperm functionality.

  6. Porcine circovirus type 2 detection in in vitro produced porcine blastocysts after virus sperm exposure.

    PubMed

    Galeati, Giovanna; Zannoni, Augusta; Spinaci, Marcella; Bucci, Diego; Ostanello, Fabio; Panarese, Serena; Tamanini, Carlo; Sarli, Giuseppe

    2016-04-01

    This study was aimed at assessing the capability of semen experimentally infected with porcine circovirus type 2 (PCV2) to produce porcine blastocysts PCR positive for PCV2. Embryos were obtained from in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes or by parthenogenesis. Sperm suspension was exposed to PCV2b and utilized for IVF. PCV2 spiked semen did not reveal any reduction in sperm viability or motility but its ability to produce infected blastocysts was irrelevant as only one out of 15 blastocysts obtained by IVF were PCV2b; however two blastocysts were PCV2a positive. Furthermore, the presence of PCV2 was demonstrated also in embryos obtained by parthenogenesis (one out of 17 was PCV2b and one PCV2a positive). Even if PCV2 firmly attaches to the surface of spermatozoa, experimentally spiked sperm were not effective in infecting oocytes during IVF and in producing PCR positive embryos. The infected blastocysts we obtained derived most probably from infected oocytes recovered at the abattoir.

  7. LYZL6, an acidic, bacteriolytic, human sperm-related protein, plays a role in fertilization

    PubMed Central

    Huang, Peng; Li, Wenshu; Yang, Zhifang; Zhang, Ning; Xu, Yixin; Bao, Jianying; Jiang, Deke; Dong, Xianping

    2017-01-01

    Lysozyme-like proteins (LYZLs) belong to the c-type lysozyme/α-lactalbumin family and are selectively expressed in the mammalian male reproductive tract. Two members, human sperm lysozyme-like protein (SLLP) -1 and mouse LYZL4, have been reported to contribute to fertilization but show no bacteriolytic activity. Here, we focused on the possible contribution of LYZL6 to immunity and fertilization. In humans, LYZL6 was selectively expressed by the testis and epididymis and became concentrated on spermatozoa. Native LYZL6 isolated from sperm extracts exhibited bacteriolytic activity against Micrococcus lysodeikticus. Recombinant LYZL6 (rLYZL6) reached its peak activity at pH 5.6 and 15 mM of Na+, and could inhibit the growth of Gram-positive, but not Gram-negative bacteria. Nevertheless, the bacteriolytic activity of rLYZL6 proved to be much lower than that of human lysozyme under physiological conditions. Immunodetection with a specific antiserum localized the LYZL6 protein on the postacrosomal membrane of mature spermatozoa. Immunoneutralization of LYZL6 significantly decreased the numbers of human spermatozoa fused with zona-free hamster eggs in a dose-dependent manner in vitro. Thus, we report here for the first time that LYZL6, an acidic, bacteriolytic and human sperm-related protein, is likely important for fertilization but not for the innate immunity of the male reproductive tract. PMID:28182716

  8. Galactosylceramidase deficiency causes sperm abnormalities in the mouse model of globoid cell leukodystrophy

    SciTech Connect

    Luddi, A.; Strazza, M.; Carbone, M.; Moretti, E.; Costantino-Ceccarini, E. . E-mail: costantino@unisi.it

    2005-03-10

    The classical recessive mouse mutant, 'the twitcher,' is one of the several animal models of the human globoid cell leukodystrophy (Krabbe disease) caused by a deficiency in the gene encoding the lysosomal enzyme galactosylceramidase (GALC). The failure to hydrolyze galactosylceramide (gal-cer) and galactosylsphingosine (psychosine) leads to degeneration of oligodendrocytes and severe demyelination. Substrate for GALC is also the galactosyl-alkyl-acyl-glycerol (GalAAG), precursor of the seminolipid, the most abundant glycolipid in spermatozoa of mammals. In this paper, we report the pathobiology of the testis and sperm in the twitcher mouse and demonstrate the importance of GALC for normal sperm maturation and function. The GALC deficit results in accumulation of GalAAG in the testis of the twitcher mouse. Morphological studies revealed that affected spermatozoa have abnormally swollen acrosomes and angulation of the flagellum mainly at midpiece-principal piece junction. Multiple folding of the principal piece was also observed. Electron microscopy analysis showed that in the twitcher sperm, acrosomal membrane is redundant, detached from the nucleus and folded over. Disorganization and abnormal arrangements of the axoneme components were also detected. These results provide in vivo evidence that GALC plays a critical role in spermiogenesis.

  9. Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization.

    PubMed

    Zimmerman, Shawn W; Manandhar, Gaurishankar; Yi, Young-Joo; Gupta, Satish K; Sutovsky, Miriam; Odhiambo, John F; Powell, Michael D; Miller, David J; Sutovsky, Peter

    2011-02-23

    Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced

  10. Effect of donkey seminal plasma on sperm movement and sperm-polymorphonuclear neutrophils attachment in vitro.

    PubMed

    Miró, Jordi; Vilés, Karina; García, Wilber; Jordana, Jordi; Yeste, Marc

    2013-08-01

    To evaluate the effect of seminal plasma in endometrial inflammation in donkeys, samples from fresh pure, fresh diluted and frozen-thawed semen of three different jackasses were co-incubated in water bath at 37°C with uterine Jennie's secretions collected 6h after artificial insemination with frozen-thawed donkey semen. Individual sperm movement parameters using the computerised sperm analysis system (CASA) and sperm-polymorphonuclear neutrophils (sperm-PMN) attachment observed in Diff-Quick stained smears were evaluated at 0, 1, 2, 3 and 4h of co-incubation. Controls consisted of incubating diluted or frozen-thawed sperm in the absence of uterine secretions. For data analyses, a repeated measures ANOVA was performed with incubation time as intra-subject factor and with treatment and donkey as inter-subject factor, followed by a post-hoc Bonferroni's test. Greater values (P<0.05) of sperm-PMN percentages and a loss of progressive motility were observed in frozen-thawed semen compared with pure and diluted fresh semen samples throughout the incubation time. In addition, the presence of seminal plasma in fresh and diluted semen samples reduced the inflammatory response of polymorphonuclear neutrophils produced after insemination by suppressing the sperm-PMN attachment in vitro. Motility sperm parameters analysed by CASA were also less affected than those in frozen-thawed semen samples. In conclusion, seminal plasma in jennies appears to have a modulation on the endometrial response after artificial insemination with frozen-thawed donkey semen. As a result, spermatozoa with the greater motility characteristics are selected.

  11. The dynamics of sperm cooperation in a competitive environment

    PubMed Central

    Fisher, Heidi S.; Giomi, Luca; Hoekstra, Hopi E.; Mahadevan, L.

    2014-01-01

    Sperm cooperation has evolved in a variety of taxa and is often considered a response to sperm competition, yet the benefit of this form of collective movement remains unclear. Here, we use fine-scale imaging and a minimal mathematical model to study sperm aggregation in the rodent genus Peromyscus. We demonstrate that as the number of sperm cells in an aggregate increase, the group moves with more persistent linearity but without increasing speed. This benefit, however, is offset in larger aggregates as the geometry of the group forces sperm to swim against one another. The result is a non-monotonic relationship between aggregate size and average velocity with both a theoretically predicted and empirically observed optimum of six to seven sperm per aggregate. To understand the role of sexual selection in driving these sperm group dynamics, we compared two sister-species with divergent mating systems. We find that sperm of Peromyscus maniculatus (highly promiscuous), which have evolved under intense competition, form optimal-sized aggregates more often than sperm of Peromyscus polionotus (strictly monogamous), which lack competition. Our combined mathematical and experimental study of coordinated sperm movement reveals the importance of geometry, motion and group size on sperm velocity and suggests how these physical variables interact with evolutionary selective pressures to regulate cooperation in competitive environments. PMID:25056618

  12. Sodium affects the sperm motility in the European eel.

    PubMed

    Vílchez, M Carmen; Morini, Marina; Peñaranda, David S; Gallego, Víctor; Asturiano, Juan F; Pérez, Luz

    2016-08-01

    The role of seminal plasma sodium and activation media sodium on sperm motility was examined by selectively removing the element from these two media, in European eel sperm. Sperm size (sperm head area) was also measured using an ASMA (Automated Sperm Morphometry Analyses) system, in the different conditions. Intracellular sodium [Na(+)]i was quantitatively analyzed by first time in the spermatozoa from a marine fish species. Measurement of [Na(+)]i was done before and after motility activation, by Flow Cytometry, using CoroNa Green AM as a dye. Sperm motility activation induced an increase in [Na(+)]i, from 96.72mM in quiescent stage to 152.21mM post-activation in seawater. A significant decrease in sperm head area was observed post-activation in seawater. There was a notable reduction in sperm motility when sodium was removed from the seminal plasma, but not when it was removed from the activation media. Sodium removal was also linked to a significant reduction in sperm head area in comparison to the controls. Our results indicate that the presence of the ion Na(+) in the seminal plasma (or in the extender medium) is necessary for the preservation of sperm motility in European eel, probably because it plays a role in maintaining an appropriate sperm cell volume in the quiescent stage of the spermatozoa.

  13. Microscopic analysis of MTT stained boar sperm cells.

    PubMed

    van den Berg, B M

    2015-01-01

    The ability of sperm cells to develop colored formazan by reduction of MTT was used earlier to develop a spectrophotometric assay to determine the viability of sperm cells for several mammalian species. It was the objective of the present study to visualize microscopically the location of the formazan in boar sperm cells. The MTT staining process of boar sperm cells can be divided into a series of morphological events. Incubation of the sperm cells in the presence of MTT resulted after a few min in a diffuse staining of the midpiece of the sperm cells. Upon further incubation the staining of the midpiece became more intense, and gradually the formation of packed formazan granules became more visible. At the same time, a small formazan stained granule appeared medially on the sperm head, which increased in size during further incubation. After incubation for about 1 h the midpiece granules were intensely stained and more clearly distinct as granules, while aggregation of sperm cells occurred. Around 90% of the sperm cells showed these staining events. At the end of the staining the formazan granules have disappeared from both the sperm cells and medium, whereas formazan crystals appeared as thin crystal threads, that became heavily aggregated in the incubation medium. It was concluded that formazan is taken up by lipid droplets in the cytoplasm. Further, the use of the MTT assay to test for sperm viability should be regarded as a qualitative assay, whereas its practical use at artificial insemination (AI) Stations is limited.

  14. Detecting coevolution in mammalian sperm-egg fusion proteins.

    PubMed

    Claw, Katrina G; George, Renee D; Swanson, Willie J

    2014-06-01

    Interactions between sperm and egg proteins can occur physically between gamete surface-binding proteins, and genetically between gamete proteins that work in complementary pathways in which they may not physically interact. Physically interacting sperm-egg proteins have been functionally identified in only a few species, and none have been verified within mammals. Candidate genes on both the sperm and egg surfaces exist, but gene deletion studies do not support functional interactions between these sperm-egg proteins; interacting sperm-egg proteins thus remain elusive. Cooperative gamete proteins undergo rapid evolution, and it is predicted that these sperm-egg proteins will also have correlated evolutionary rates due to compensatory changes on both the sperm and egg. To explore potential physical and genetic interactions in sperm-egg proteins, we sequenced four candidate genes from diverse primate species, and used regression and likelihood methods to test for signatures of coevolution between sperm-egg gene pairs. With both methods, we found that the egg protein CD9 coevolves with the sperm protein IZUMO1, suggesting a physical or genetic interaction occurs between them. With regression analysis, we found that CD9 and CRISP2 have correlated rates of evolution, and with likelihood analysis, that CD9 and CRISP1 have correlated rates. This suggests that the different tests may reflect different levels of interaction, be it physical or genetic. Coevolution tests thus provide an exploratory method for detecting potentially interacting sperm-egg protein pairs.

  15. Increased sperm ubiquitination correlates with abnormal chromatin integrity.

    PubMed

    Hodjat, M; Akhondi, M A; Al-Hasani, S; Mobaraki, M; Sadeghi, M R

    2008-09-01

    Ubiquitin, a 8.5 kDa peptide that marks other proteins for proteasomal degradation, tags defective spermatozoa during epididymal passage and is proposed as a biomarker for sperm quality. The present study was designed to evaluate the relationships between sperm ubiquitination, sperm chromatin integrity and semen parameters. Semen samples from 63 couples were collected and analysed according to World Health Organization criteria. Each sample was evaluated for sperm ubiquitination by the direct immunofluorescence method, using anti-ubiquitin antibodies. Chromatin integrity of the same samples was analysed using acridine orange (AO) and toluidine blue (TB) tests. A positive correlation was found between ubiquitinated spermatozoa and the percentage of spermatozoa with abnormal chromatin (AO: r = 0.58, P < 0.001 and TB: r = 0.48, P < 0.001). Negative correlations were obtained between sperm ubiquitination and: sperm count (r = -0.2, P = 0.048), sperm morphology (r = -0.36, P = 0.003), rapidly progressive motility (r = -0.25, P = 0.044) and slow progressive motility (r = -0.28, P = 0.022). Sperm ubiquitination was positively correlated with the percentage of immotile spermatozoa. These results show that among semen parameters, chromatin abnormality is more closely associated with sperm ubiquitination and further validate sperm ubiquitination as a suitable marker for sperm quality.

  16. Sperm processing for advanced reproductive technologies: Where are we today?

    PubMed

    Rappa, Kari L; Rodriguez, Harold F; Hakkarainen, Gloria C; Anchan, Raymond M; Mutter, George L; Asghar, Waseem

    2016-01-01

    Assisted reproductive technologies (ARTs) utilize sperm sorting methods to select viable sperm from the semen samples. Conventional sperm sorting techniques in current use are density gradient centrifugation, direct swim-up, and conventional swim-up. These methods use multiple centrifugation steps, which have been shown to generate reactive oxygen species (ROS) that decrease DNA integrity and damage sperm. Newer technologies, such as microfluidics, electrophoresis, motile sperm organelle morphology examination (MSOME), and birefringence eliminate the centrifugation steps and can improve the selection of sperm with higher DNA integrity, normal morphology, and motility as well as improved artificial insemination outcomes. In this review, we discuss some recent research in centrifugation and non-centrifugation based techniques and their effect on sperm quality and ART outcomes.

  17. Sperm cells as vectors in the production of transgenic animals

    SciTech Connect

    Prince, R.M.

    1993-04-28

    Transgenic animals are used in industry and in biomedical research in order to provide in vivo experimental model systems. Sperm cells have been reported used as vectors in the production of transgenic animals before, however no approach has of yet proven to be successful. Fertilizing eggs with genetically modified sperm would be advantageous in that sperm are readily accessible and stable, and eggs can be fertilized by modified sperm cells in vivo. Recent elucidations regarding the unique manner of DNA packaging in sperm chromatin by protamines has provided us with the insight for developing a method of introducing foreign DNA into sperm which is likely to succeed where others have failed. We have developed a method for mimicking the in vivo system of sperm chromatin toroid subunits in vitro, concentrating these toroids, and fluorescent visualization. Our present work concerns development of a method to successfully deliver DNA across the cell membranes and into the nucleus.

  18. COMP-1 promotes competitive advantage of nematode sperm.

    PubMed

    Hansen, Jody M; Chavez, Daniela R; Stanfield, Gillian M

    2015-03-19

    Competition among sperm to fertilize oocytes is a ubiquitous feature of sexual reproduction as well as a profoundly important aspect of sexual selection. However, little is known about the cellular mechanisms sperm use to gain competitive advantage or how these mechanisms are regulated genetically. In this study, we utilize a forward genetic screen in Caenorhabditis elegans to identify a gene, comp-1, whose function is specifically required in competitive contexts. We show that comp-1 functions in sperm to modulate their migration through and localization within the reproductive tract, thereby promoting their access to oocytes. Contrary to previously described models, comp-1 mutant sperm show no defects in size or velocity, thereby defining a novel pathway for preferential usage. Our results indicate not only that sperm functional traits can influence the outcome of sperm competition, but also that these traits can be modulated in a context-dependent manner depending on the presence of competing sperm.

  19. Queen reproductive tract secretions enhance sperm motility in ants

    PubMed Central

    Baer, Boris; Boomsma, Jacobus J.

    2016-01-01

    Queens of Acromyrmex leaf-cutting ants store sperm of multiple males after