Assembly of the fluorescent acrosomal matrix and its fate in fertilization in the water strider, Aquarius remigis

PubMed Central

Miyata, Haruhiko; Noda, Naoki; Fairbairn, Daphne J.; Oldenbourg, Rudolf; Cardullo, Richard A.

2011-01-01

Animal sperm show remarkable diversity in both morphology and molecular composition. Here we provide the first report of intense intrinsic fluorescence in an animal sperm. The sperm from a semi-aquatic insect, the water strider, Aquarius remigis, contains an intrinsically fluorescent molecule with properties consistent with those of Flavin Adenine Dinucleotid (FAD) which appears first in the acrosomal vesicle of round spermatids and persists in the acrosome throughout spermiogenesis. Fluorescence recovery after photobleaching reveals that the fluorescent molecule exhibits unrestricted mobility in the acrosomal vesicle of round spermatids, but is completely immobile in the acrosome of mature sperm. Fluorescence polarization microscopy shows a net alignment of the fluorescent molecules in the acrosome of the mature sperm but not in the acrosomal vesicle of round spermatids. These results suggest that acrosomal molecules are rearranged in the elongating acrosome and FAD is incorporated into the acrosomal matrix during its formation. Further, we followed the fate of the acrosomal matrix in fertilization utilizing the intrinsic fluorescence. The fluorescent acrosomal matrix was observed inside the fertilized egg and remained structurally intact even after gastrulation started. This observation suggests that FAD is not released from the acrosomal matrix during the fertilization process or early development and supports an idea that FAD is involved in the formation of the acrosomal matrix. The intrinsic fluorescence of the A. remigis acrosome will be a useful marker for following spermatogenesis and fertilization. PMID:20857404

Toward an integrative and predictive sperm quality analysis in Bos taurus.

PubMed

Yániz, J L; Soler, C; Alquézar-Baeta, C; Santolaria, P

2017-06-01

There is a need to develop more integrative sperm quality analysis methods, enabling researchers to evaluate different parameters simultaneously cell by cell. In this work, we present a new multi-parametric fluorescent test able to discriminate different sperm subpopulations based on their labeling pattern and motility characteristics. Cryopreserved semen samples from 20 Holstein bulls were used in the study. Analyses of sperm motility using computer-assisted sperm analysis (CASA-mot), membrane integrity by acridine orange-propidium iodide combination and multi-parametric by the ISAS ® 3Fun kit, were performed. The new method allows a clear discrimination of sperm subpopulations based on membrane and acrosomal integrity, motility and morphology. It was also possible to observe live spermatozoa showing signs of capacitation such as hyperactivated motility and changes in acrosomal structure. Sperm subpopulation with intact plasma membrane and acrosome showed a higher proportion of motile sperm than those with damaged acrosome or increased fluorescence intensity. Spermatozoa with intact plasmalemma and damaged acrosome were static or exhibit weak movement. Significant correlations among the different sperm quality parameters evaluated were also described. We concluded that the ISAS ® 3Fun is an integrated method that represents an advance in sperm quality analysis with the potential to improve fertility predictions. Copyright © 2017 Elsevier B.V. All rights reserved.

Presence and function of dopamine transporter (DAT) in stallion sperm: dopamine modulates sperm motility and acrosomal integrity.

PubMed

Urra, Javier A; Villaroel-Espíndola, Franz; Covarrubias, Alejandra A; Rodríguez-Gil, Joan Enric; Ramírez-Reveco, Alfredo; Concha, Ilona I

2014-01-01

Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility. In the present study, using immunodetection methods, we revealed the presence of several proteins important for the dopamine uptake and signalling in mammalian sperm, specifically monoamine transporters as dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in equine sperm. We also demonstrated for the first time in equine sperm a functional dopamine transporter using 4-[4-(Dimethylamino)styryl]-N-methylpyridinium iodide (ASP(+)), as substrate. In addition, we also showed that dopamine (1 mM) treatment in vitro, does not affect sperm viability but decreases total and progressive sperm motility. This effect is reversed by blocking the dopamine transporter with the selective inhibitor vanoxerine (GBR12909) and non-selective inhibitors of dopamine reuptake such as nomifensine and bupropion. The effect of dopamine in sperm physiology was evaluated and we demonstrated that acrosome integrity and thyrosine phosphorylation in equine sperm is significantly reduced at high concentrations of this catecholamine. In summary, our results revealed the presence of monoamine transporter DAT, NET and SERT in equine sperm, and that the dopamine uptake by DAT can regulate sperm function, specifically acrosomal integrity and sperm motility.

Presence and Function of Dopamine Transporter (DAT) in Stallion Sperm: Dopamine Modulates Sperm Motility and Acrosomal Integrity

PubMed Central

Covarrubias, Alejandra A.; Rodríguez-Gil, Joan Enric; Ramírez-Reveco, Alfredo; Concha, Ilona I.

2014-01-01

Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility. In the present study, using immunodetection methods, we revealed the presence of several proteins important for the dopamine uptake and signalling in mammalian sperm, specifically monoamine transporters as dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in equine sperm. We also demonstrated for the first time in equine sperm a functional dopamine transporter using 4-[4-(Dimethylamino)styryl]-N-methylpyridinium iodide (ASP+), as substrate. In addition, we also showed that dopamine (1 mM) treatment in vitro, does not affect sperm viability but decreases total and progressive sperm motility. This effect is reversed by blocking the dopamine transporter with the selective inhibitor vanoxerine (GBR12909) and non-selective inhibitors of dopamine reuptake such as nomifensine and bupropion. The effect of dopamine in sperm physiology was evaluated and we demonstrated that acrosome integrity and thyrosine phosphorylation in equine sperm is significantly reduced at high concentrations of this catecholamine. In summary, our results revealed the presence of monoamine transporter DAT, NET and SERT in equine sperm, and that the dopamine uptake by DAT can regulate sperm function, specifically acrosomal integrity and sperm motility. PMID:25402186

Equatorin is not essential for acrosome biogenesis but is required for the acrosome reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hao, Jianxiu; Graduate School of the Chinese Academy of Sciences, Beijing 100049; Chen, Min

Highlights: • Eqtn knockout mice were used for these experiments. • In vivo and in vitro fertilization analyses were performed. • Eqtn-deficient sperm were evaluated by transmission electron microscopy (TEM) and an A23187-induced acrosome reaction (AR) assay. • Co-immunoprecipitation (Co-IP) was performed to assess the interaction between Eqtn and the SNARE complex. - Abstract: The acrosome is a specialized organelle that covers the anterior part of the sperm nucleus and plays an essential role in mammalian fertilization. However, the regulatory mechanisms controlling acrosome biogenesis and acrosome exocytosis during fertilization are largely unknown. Equatorin (Eqtn) is a membrane protein that ismore » specifically localized to the acrosomal membrane. In the present study, the physiological functions of Eqtn were investigated using a gene knockout mouse model. We found that Eqtn{sup −/−} males were subfertile. Only approximately 50% of plugged females were pregnant after mating with Eqtn{sup −/−} males, whereas more than 90% of plugged females were pregnant after mating with control males. Sperm and acrosomes from Eqtn{sup −/−} mice presented normal motility and morphology. However, the fertilization and induced acrosome exocytosis rates of Eqtn-deficient sperm were dramatically reduced. Further studies revealed that the Eqtn protein might interact with Syntaxin1a and SNAP25, but loss of Eqtn did not affect the protein levels of these genes. Therefore, our study demonstrates that Eqtn is not essential for acrosome biogenesis but is required for the acrosome reaction. Eqtn is involved in the fusion of the outer acrosomal membrane and the sperm plasma membrane during the acrosome reaction, most likely via an interaction with the SNARE complex.« less

Characterization of functional variables in epididymal alpaca (Vicugna pacos) sperm using imaging flow cytometry.

PubMed

Santiani, Alexei; Ugarelli, Alejandra; Evangelista-Vargas, Shirley

2016-10-01

Epididymal alpaca sperm represent an alternative model for the study of alpaca semen. The objective of this study was to characterize the normal values of some functional variables in epididymal alpaca sperm using imaging flow cytometry. Alpaca testicles (n=150) were processed and sperm were recovered from the cauda epididymides. Only 76 samples with acceptable motility and sperm count were considered for assessment by imaging flow cytometry. Acrosome integrity and integrity/viability were assessed by FITC-PSA/PI and FITC-PNA/PI. Mitochondrial membrane potential (MMP) was assessed by MitoTracker CMXRos and MitoTracker Deep Red FM. Lipid peroxidation was evaluated using BODIPY 581/591 C11. Results show that the mean values for acrosome-intact sperm were 95.03±6.39% and 93.34±7.96%, using FITC-PSA and FITC-PNA, respectively. The mean values for acrosome-intact viable sperm were 60.58±12.12% with FITC-PSA/PI and 58.81±12.94% with FITC-PNA/PI. Greater MMP was detected in 65.03±15.92% and 59.52±19.19%, using MitoTracker CMXRos and MitoTracker Deep Red FM, respectively. Lipid peroxidation was 0.84±0.95%. Evaluation of acrosome-intact and acrosome-intact viable sperm with FITC-PSA/PI compared with. FITC-PNA/PI or MMP with MitoTracker CMXRos compared with MitoTracker Deep Red FM were correlated (P<0.05). The MMP using MitoTracker CMXRos was the only variable correlated (P<0.05) with sperm motility (r=0.3979). This report provides a basis for future research related to alpaca semen using the epididymal sperm model. Copyright © 2016 Elsevier B.V. All rights reserved.

Intact Cell MALDI-TOF MS on Sperm: A Molecular Test For Male Fertility Diagnosis.

PubMed

Soler, Laura; Labas, Valérie; Thélie, Aurore; Grasseau, Isabelle; Teixeira-Gomes, Ana-Paula; Blesbois, Elisabeth

2016-06-01

Currently, evaluation of sperm quality is primarily based on in vitro measures of sperm function such as motility, viability and/or acrosome reaction. However, results are often poorly correlated with fertility, and alternative diagnostic tools are therefore needed both in veterinary and human medicine. In a recent pilot study, we demonstrated that MS profiles from intact chicken sperm using MALDI-TOF profiles could detect significant differences between fertile/subfertile spermatozoa showing that such profiles could be useful for in vitro male fertility testing. In the present study, we performed larger standardized experimental procedures designed for the development of fertility- predictive mathematical models based on sperm cell MALDI-TOF MS profiles acquired through a fast, automated method. This intact cell MALDI-TOF MS-based method showed high diagnostic accuracy in identifying fertile/subfertile males in a large male population of known fertility from two distinct genetic lineages (meat and egg laying lines). We additionally identified 40% of the m/z peaks observed in sperm MS profiles through a top-down high-resolution protein identification analysis. This revealed that the MALDI-TOF MS spectra obtained from intact sperm cells contained a large proportion of protein degradation products, many implicated in important functional pathways in sperm such as energy metabolism, structure and movement. Proteins identified by our predictive model included diverse and important functional classes providing new insights into sperm function as it relates to fertility differences in this experimental system. Thus, in addition to the chicken model system developed here, with the use of appropriate models these methods should effectively translate to other animal taxa where similar tests for fertility are warranted. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Egg water from the amphibian Bufo arenarum modulates the ability of homologous sperm to undergo the acrosome reaction in the presence of the vitelline envelope.

PubMed

Krapf, Darío; O'Brien, Emma D; Cabada, Marcelo O; Visconti, Pablo E; Arranz, Silvia E

2009-02-01

Sperm from the toad Bufo arenarum must penetrate the egg jelly before reaching the vitelline envelope (VE), where the acrosome reaction is triggered. When the jelly coat is removed, sperm still bind to the VE, but acrosomal exocytosis is not promoted. Our previous work demonstrated that diffusible substances of the jelly coat, termed "egg water" (EW), triggered capacitation-like changes in B. arenarum sperm, promoting the acquisition of a transient fertilizing capacity. In the present work, we correlated this fertilizing capacity with the ability of the sperm to undergo the acrosome reaction, further substantiating the role of the jelly coat in fertilization. When sperm were exposed to the VE, only those preincubated in EW for 5 or 8 min underwent an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)), which led to acrosomal exocytosis. Responsiveness to the VE was not acquired on preincubation in EW for 2 or 15 min or in Ringer solution regardless of the preincubation time. In contrast, depletion of intracellular Ca(2+) stores (induced by thapsigargin) promoted [Ca(2+)](i) rise and the acrosome reaction even in sperm that were not exposed to EW. Acrosomal exocytosis was blocked by the presence of Ca(2+) chelators independent of whether a physiological or pharmacological stimulus was used. However, Ni(2+) and mibefradil prevented [Ca(2+)](i) rise and the acrosome reaction of sperm exposed to the VE but not of sperm exposed to thapsigargin. These data suggest that the acrosomal responsiveness of B. arenarum sperm, present during a narrow period, is acquired during EW incubation and involves the modulation of a voltage-dependent Ca(2+) channel.

Egg Water from the Amphibian Bufo arenarum Modulates the Ability of Homologous Sperm to Undergo the Acrosome Reaction in the Presence of the Vitelline Envelope1

PubMed Central

Krapf, Darío; O'Brien, Emma D.; Cabada, Marcelo O.; Visconti, Pablo E.; Arranz, Silvia E.

2008-01-01

Sperm from the toad Bufo arenarum must penetrate the egg jelly before reaching the vitelline envelope (VE), where the acrosome reaction is triggered. When the jelly coat is removed, sperm still bind to the VE, but acrosomal exocytosis is not promoted. Our previous work demonstrated that diffusible substances of the jelly coat, termed “egg water” (EW), triggered capacitation-like changes in B. arenarum sperm, promoting the acquisition of a transient fertilizing capacity. In the present work, we correlated this fertilizing capacity with the ability of the sperm to undergo the acrosome reaction, further substantiating the role of the jelly coat in fertilization. When sperm were exposed to the VE, only those preincubated in EW for 5 or 8 min underwent an increase in the intracellular Ca2+ concentration ([Ca2+]i), which led to acrosomal exocytosis. Responsiveness to the VE was not acquired on preincubation in EW for 2 or 15 min or in Ringer solution regardless of the preincubation time. In contrast, depletion of intracellular Ca2+ stores (induced by thapsigargin) promoted [Ca2+]i rise and the acrosome reaction even in sperm that were not exposed to EW. Acrosomal exocytosis was blocked by the presence of Ca2+ chelators independent of whether a physiological or pharmacological stimulus was used. However, Ni2+ and mibefradil prevented [Ca2+]i rise and the acrosome reaction of sperm exposed to the VE but not of sperm exposed to thapsigargin. These data suggest that the acrosomal responsiveness of B. arenarum sperm, present during a narrow period, is acquired during EW incubation and involves the modulation of a voltage-dependent Ca2+ channel. PMID:18923159

Dose-response effects of estrogenic mycotoxins (zearalenone, alpha- and beta-zearalenol) on motility, hyperactivation and the acrosome reaction of stallion sperm

PubMed Central

2011-01-01

Background The aim of this study was to investigate the in vitro effects of the Fusarium fungus-derived mycotoxin, zearalenone and its derivatives alpha-zearalenol and beta-zearalenol on motility parameters and the acrosome reaction of stallion sperm. Since the toxic effects of zearalenone and its derivatives are thought to result from their structural similarity to 17beta-estradiol, 17beta-estradiol was used as a positive control for 'estrogen-like' effects. Methods Stallion spermatozoa were exposed in vitro to zearalenone, alpha-zearalenol, beta-zearalenol or 17beta-estradiol at concentrations ranging from 1 pM - 0.1 mM. After 2 hours exposure, motility parameters were evaluated by computer-assisted analysis, and acrosome integrity was examined by flow cytometry after staining with fluoroscein-conjugated peanut agglutinin. Results Mycotoxins affected sperm parameters only at the highest concentration tested (0.1 mM) after 2 hours exposure. In this respect, all of the compounds reduced the average path velocity, but only alpha-zearalenol reduced percentages of motile and progressively motile sperm. Induction of motility patterns consistent with hyperactivation was stimulated according to the following rank of potency: alpha-zearalenol >17beta-estradiol > zearalenone = beta-zearalenol. The hyperactivity-associated changes observed included reductions in straight-line velocity and linearity of movement, and an increase in the amplitude of lateral head displacement, while curvilinear velocity was unchanged. In addition, whereas alpha- and beta- zearalenol increased the percentages of live acrosome-reacted sperm, zearalenone and 17beta-estradiol had no apparent effect on acrosome status. In short, alpha-zearalenol inhibited normal sperm motility, but stimulated hyperactive motility in the remaining motile cells and simultaneously induced the acrosome reaction. Beta-zearalenol induced the acrosome reaction without altering motility. Conversely, zearalenone and 17beta

The Acrosome Reaction: A Historical Perspective.

PubMed

Okabe, Masaru

2016-01-01

Acrosome reaction is often referred to as acrosomal exocytosis, but it differs significantly from normal exocytosis. While the vesicle membrane initially holding excreting molecules remains on the cell surface during exocytosis, the outer acrosomal membrane and plasma membrane are lost by forming vesicles during acrosome reaction. In this context, the latter process resembles a release of exosome. However, recent experimental data indicate that the most important roles of acrosome reaction lie not in the release of acrosomal contents (or "vesiculated" plasma and outer acrosomal membrane complexes) but rather in changes in sperm membrane. This review describes the mechanism of fertilization vis-a-vis sperm membrane change, with a brief historical overview of the half-century study of acrosome reaction.

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. Copyright © 2014 Elsevier Inc. All rights reserved.

CaMKII prevents spontaneous acrosomal exocytosis in sperm through induction of actin polymerization.

PubMed

Shabtay, Ortal; Breitbart, Haim

2016-07-01

In order to interact with the egg and undergo acrosomal exocytosis or the acrosome reaction (AR), mammalian spermatozoa must undergo a series of biochemical changes in the female reproductive tract, collectively called capacitation. We showed that F-actin is formed during sperm capacitation and fast depolymerization occurs prior to the AR. We hypothesized that F-actin protects the sperm from undergoing spontaneous-AR (sAR) which decreases fertilization rate. We show that activation of the actin-severing protein gelsolin induces a significant increase in sAR. Moreover, inhibition of CaMKII or PLD during sperm capacitation, caused an increase in sAR and inhibition of F-actin formation. Spermine, which leads to PLD activation, was able to reverse the effects of CaMKII inhibition on sAR-increase and F-actin-decrease. Furthermore, the increase in sAR and the decrease in F-actin caused by the inactivation of the PLD-pathway, were reversed by activation of CaMKII using H2O2 or by inhibiting protein phosphatase 1 which enhance the phosphorylation and oxidation states of CaMKII. These results indicate that two distinct pathways lead to F-actin formation in the sperm capacitation process which prevents the occurrence of sAR. Copyright © 2016 Elsevier Inc. All rights reserved.

Remodeling of the plasma membrane in preparation for sperm–egg recognition: roles of acrosomal proteins

PubMed Central

Tanphaichitr, Nongnuj; Kongmanas, Kessiri; Kruevaisayawan, Hathairat; Saewu, Arpornrad; Sugeng, Clarissa; Fernandes, Jason; Souda, Puneet; Angel, Jonathan B; Faull, Kym F; Aitken, R John; Whitelegge, Julian; Hardy, Daniel; Berger, Trish; Baker, Mark

2015-01-01

The interaction of sperm with the egg's extracellular matrix, the zona pellucida (ZP) is the first step of the union between male and female gametes. The molecular mechanisms of this process have been studied for the past six decades with the results obtained being both interesting and confusing. In this article, we describe our recent work, which attempts to address two lines of questions from previous studies. First, because there are numerous ZP binding proteins reported by various researchers, how do these proteins act together in sperm–ZP interaction? Second, why do a number of acrosomal proteins have ZP affinity? Are they involved mainly in the initial sperm–ZP binding or rather in anchoring acrosome reacting/reacted spermatozoa to the ZP? Our studies reveal that a number of ZP binding proteins and chaperones, extracted from the anterior sperm head plasma membrane, coexist as high molecular weight (HMW) complexes, and that these complexes in capacitated spermatozoa have preferential ability to bind to the ZP. Zonadhesin (ZAN), known as an acrosomal protein with ZP affinity, is one of these proteins in the HMW complexes. Immunoprecipitation indicates that ZAN interacts with other acrosomal proteins, proacrosin/acrosin and sp32 (ACRBP), also present in the HMW complexes. Immunodetection of ZAN and proacrosin/acrosin on spermatozoa further indicates that both proteins traffic to the sperm head surface during capacitation where the sperm acrosomal matrix is still intact, and therefore they are likely involved in the initial sperm–ZP binding step. PMID:25994642

Sperm Mitochondrial Integrity Is Not Required for Hyperactivated Motility, Zona Binding, or Acrosome Reaction in the Rhesus Macaque1

PubMed Central

Hung, Pei-hsuan; Miller, Marion G.; Meyers, Stuart A.; VandeVoort, Catherine A.

2008-01-01

Whether the main energy source for sperm motility is from oxidative phosphorylation or glycolysis has been long-debated in the field of reproductive biology. Using the rhesus monkey as a model, we examined the role of glycolysis and oxidative phosphorylation in sperm function by using alpha-chlorohydrin (ACH), a glycolysis inhibitor, and pentachlorophenol (PCP), an oxidative phosphorylation uncoupler. Sperm treated with ACH showed no change in percentage of motile sperm, although sperm motion was impaired. The ACH-treated sperm did not display either hyperactivity- or hyperactivation-associated changes in protein tyrosine phosphorylation. When treated with PCP, sperm motion parameters were affected by the highest level of PCP (200 μM); however, PCP did not cause motility impairments even after chemical activation. Sperm treated with PCP were able to display hyperactivity and tyrosine phosphorylation after chemical activation. In contrast with motility measurements, treatment with either the glycolytic inhibitor or the oxidative phosphorylation inhibitor did not affect sperm-zona binding and zona-induced acrosome reaction. The results suggest glycolysis is essential to support sperm motility, hyperactivity, and protein tyrosine phosphorylation, while energy from oxidative phosphorylation is not necessary for hyperactivated sperm motility, tyrosine phosphorylation, sperm-zona binding, and acrosome reaction in the rhesus macaque. PMID:18480469

Identification of an anti-sperm auto-monoclonal antibody (Ts4)-recognized molecule in the mouse sperm acrosomal region and its inhibitory effect on fertilization in vitro.

PubMed

Yoshitake, Hiroshi; Oda, Risako; Yanagida, Mitsuaki; Kawasaki, Yu; Sakuraba, Mayumi; Takamori, Kenji; Hasegawa, Akiko; Fujiwara, Hiroshi; Araki, Yoshihiko

2016-06-01

We previously established an anti-mouse sperm auto-monoclonal antibody, Ts4, which shows immunoreactivity against several kinds of glycoproteins in the acrosomal region of epididymal spermatozoa, testicular germ cells, and early embryo, via binding to an epitope containing a common N-linked oligosaccharide (OS) chain on the molecules. In mice, we have already demonstrated that the OS chain in the epitope for Ts4 is a fucosylated agalacto-complex-type biantennary glycan carrying bisecting N-acetylglucosamine. In the testis, one of the specific OS chain-conjugated molecules is TEX101, a germ cell-marker glycoprotein, which is expressed in spermatocytes, spermatids, and testicular spermatozoa, but not in epididymal spermatozoa. In this study, we identified a Ts4-reactive glycoprotein in mouse cauda epididymal sperm. An immunoprecipitation method together with liquid chromatography-tandem mass spectrometry showed that alpha-N-acetylglucosaminidase (Naglu; a degradation enzyme of heparan sulfate) is one of the glycoproteins recognized by Ts4 in the epididymal spermatozoa. Western blot and immunohistochemical analyses revealed that mouse Naglu exists in two forms (82 and 77kDa) and is expressed in the acrosomal region and the flagellum of cauda epididymal sperm. Of the two Naglu-forms expressed in sperm, Ts4 immunoreacted against only the 82-kDa form located on the acrosomal region. The Ts4 mAb and anti-Naglu pAb negatively affected mouse fertilization in vitro. In addition, Ts4 inhibited sperm acrosome reaction induced by heparan sulfate. The Ts4-recognized fucosylated agalactobiantennary complex-type glycan with bisecting N-acetylglucosamine and Naglu on cauda epididymal spermatozoa may play a role in the process of fertilization. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Sperm Pretreatment with Dithiothreitol Increases Male Pronucleus Formation Rates After Intracytoplasmic Sperm Injection (ICSI) in Swamp Buffalo Oocytes

PubMed Central

CHANKITISAKUL, Vibuntita; AM-IN, Nutthee; THARASANIT, Theerawat; SOMFAI, Tamas; NAGAI, Takashi; TECHAKUMPHU, Mongkol

2012-01-01

Abstract Failure of male pronucleus formation has hampered the success of intracytoplasmic sperm injection (ICSI) in swamp buffalo. The aim of the present study was to improve male pronucleus formation by pretreating sperm with various chemicals before ICSI. In Experiments1 and 2, sperm were treated according to one of the following protocols: (1) 0.1% Triton-X 100 (TX) for 1 min, (2) 10 µM calcium ionophore (CaI) for 20 min, (3) freezing and thawing (FT) without any cryoprotectant, or (4) no treatment (control). These sperm treatment groups then either did or did not receive additional sperm treatment with 5 mM dithiothreitol (DTT) for 20 min. Acrosomal integrity (Experiment 1) and DNA fragmentation (Experiment 2) were evaluated in the sperm before ICSI. In Experiment 3, oocytes matured in vitro were subjected to ICSI using pretreated sperm as described above and then were cultured either with or without activation. The TX- and CaI-treated sperm caused an increase in the number of acrosome-loss sperm, whereas the FT treatment and control increased the proportion of acrosome-reacted sperm (P<0.05). The DNA fragmentation did not differ among treatments (P>0.05). At 18 h post-ICSI, pronucleus (PN) formation was found only in activated oocytes. The majority of the activated ICSI oocytes contained intact sperm heads. Normal fertilization was observed in the CaI and FT treatment groups and control group when sperm were treated with DTT before ICSI. In conclusion, DTT treatment of sperm with reacted acrosomes before ICSI together with activation of the ICSI oocytes is important for successful male pronucleus formation. PMID:23132520

Sperm competition, sperm numbers and sperm quality in muroid rodents.

PubMed

Gómez Montoto, Laura; Magaña, Concepción; Tourmente, Maximiliano; Martín-Coello, Juan; Crespo, Cristina; Luque-Larena, Juan José; Gomendio, Montserrat; Roldan, Eduardo R S

2011-03-25

Sperm competition favors increases in relative testes mass and production efficiency, and changes in sperm phenotype that result in faster swimming speeds. However, little is known about its effects on traits that contribute to determine the quality of a whole ejaculate (i.e., proportion of motile, viable, morphologically normal and acrosome intact sperm) and that are key determinants of fertilization success. Two competing hypotheses lead to alternative predictions: (a) sperm quantity and quality traits co-evolve under sperm competition because they play complementary roles in determining ejaculate's competitive ability, or (b) energetic constraints force trade-offs between traits depending on their relevance in providing a competitive advantage. We examined relationships between sperm competition levels, sperm quantity, and traits that determine ejaculate quality, in a comparative study of 18 rodent species using phylogenetically controlled analyses. Total sperm numbers were positively correlated to proportions of normal sperm, acrosome integrity and motile sperm; the latter three were also significantly related among themselves, suggesting no trade-offs between traits. In addition, testes mass corrected for body mass (i.e., relative testes mass), showed a strong association with sperm numbers, and positive significant associations with all sperm traits that determine ejaculate quality with the exception of live sperm. An "overall sperm quality" parameter obtained by principal component analysis (which explained 85% of the variance) was more strongly associated with relative testes mass than any individual quality trait. Overall sperm quality was as strongly associated with relative testes mass as sperm numbers. Thus, sperm quality traits improve under sperm competition in an integrated manner suggesting that a combination of all traits is what makes ejaculates more competitive. In evolutionary terms this implies that a complex network of genetic and

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. © 2011 Blackwell Verlag GmbH.

Actin filaments in the acrosomal reaction of Limulus sperm. Motion generated by alterations in the packing of the filaments.

PubMed

Tilney, L G

1975-02-01

When Limulus sperm are induced to undergo the acrosomal reaction, a process, 50 mum in length, is generated in a few seconds. This process rotates as it elongates; thus the acrosomal process literally screws through the jelly of the egg. Within the process is a bundle of filaments which before induction are coiled up inside the sperm. The filament bundle exists in three stable states in the sperm. One of the states can be isolated in pure form. It is composed of only three proteins whose molecular weights (mol wt) are 43,000, 55,000, and 95,000. The 43,000 mol wt protein is actin, based on its molecular weight, net charge, morphology, G-F transformation, and heavy meromyosin (HMM) binding. The 55,000 mol wt protein is in equimolar ratio to actin and is not tubulin, binds tenaciously to actin, and inhibits HMM binding. Evidence is presented that both the 55,000 mol wt protein and the 95,000 mol wt protein (possibly alpha-actinin) are also present in Limulus muscle. Presumably these proteins function in the sperm in holding the actin filaments together. Before the acrosomal reaction, the actin filaments are twisted over one another in a supercoil; when the reaction is completed, the filaments lie parallel to each other and form an actin paracrystal. This change in their packing appears to give rise to the motion of the acrosomal process and is under the control of the 55,000 mol wt protein and the 95,000 mol wt protein.

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.

Human sperm Toll-like receptor 4 (TLR4) mediates acrosome reaction, oxidative stress markers, and sperm parameters in response to bacterial lipopolysaccharide in infertile men.

PubMed

Sahnoun, Sana; Sellami, Afifa; Chakroun, Nozha; Mseddi, Malek; Attia, Hammadi; Rebai, Tarek; Lassoued, Saloua

2017-08-01

To study the role of Toll-like receptor 4 (TLR4) in human spermatozoa and to assess sperm parameters, oxidative stress markers, and acrosome reaction in response to the stimulation of TLR4 by its ligand, the lipopolysaccharide (LPS), as a major endotoxin of Gram-negative bacteria. Our study was carried out in 73 sperm samples from patients undergoing semen analysis for couple infertility investigations. The studied patients were divided into three groups: normozoospermic fertile patients (n = 13), patients with abnormal and leukospermic semen (n = 13), and patients with abnormal and non-leukospermic semen (n = 47). TLR4 expression in human spermatozoa was initially analyzed by western blot. Sperm samples were incubated in the presence of LPS (200 ng/ml) for 18 h. Then, sperm motility and vitality were evaluated by microscopic observation and oxidative stress markers as malondialdehyde (MDA) and carbonyl groups (CG) were spectrophotometrically assessed in neat and selected sperm. A triple-stain technique was also performed to evaluate acrosome reaction in 15 sperm samples from infertile patients. TLR4 expression was confirmed in human spermatozoa with a molecular weight of 69 kDa. In the normozoospermic group, no significant differences in sperm parameters and oxidative stress markers were shown after incubation with LPS in neat and selected sperms. Regarding samples from the non-leukospermic group, LPS reduced spermatozoa motility and vitality rates in selected sperm (P = 0.003; P = 0.004, respectively). A significant increase of MDA and CG levels was also detected (P = 0.01; P = 0.02, respectively). However, only the MDA levels were significantly increased (P = 0.01) in neat LPS-stimulated sperm. The same results were shown within the leukospermic group. The comparison between the two groups, leukospermic and non-leukospermic, in selected sperms showed a more important LPS effect in the leukospermic group significantly on motility and MDA rates

Mouse SLLP1, a sperm lysozyme-like protein involved in sperm-egg binding and fertilization.

PubMed

Herrero, María Belén; Mandal, Arabinda; Digilio, Laura C; Coonrod, Scott A; Maier, Bernhard; Herr, John C

2005-08-01

This study demonstrates the retention of mouse sperm lysozyme-like protein (mSLLP1) in the equatorial segment of spermatozoa following the acrosome reaction and a role for mSLLP1 in sperm-egg binding and fertilization. Treatment of cumulus intact oocytes with either recmSLLP1 or its antiserum resulted in a significant (P < or = 0.05) inhibition of fertilization. Co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of anti-recmSLLP1 serum or recmSLLP1 also inhibited sperm-oolemma binding. A complete inhibition of binding and fusion of spermatozoa to the oocyte occurred at 12.5 muM concentration of recmSLLP1, while conventional chicken and human lysozymes did not block sperm-egg binding. mSLLP1 showed receptor sites in the perivitelline space as well as on the microvillar region of the egg plasma membrane. The retention of mSLLP1 in the equatorial segment of acrosome-reacted sperm, the inhibitory effects of both recmSLLP1 and antibodies to SLLP1 on in vitro fertilization with both cumulus intact and zona-free eggs, and the definition of complementary SLLP1-binding sites on the egg plasma membrane together support the hypothesis that a c lysozyme-like protein is involved in the binding of spermatozoa to the egg plasma membrane during fertilization.

Effect of nitric oxide on boar sperm motility, membrane integrity, and acrosomal status during semen storage.

PubMed

Jovicić, M; Pintus, E; Fenclova, T; Simonik, O; Chmelikova, E; Ros-Santaella, J L; Sedmikova, M

2018-03-01

Nitric oxide (NO) is a major gasotransmitter involved in several physiological processes of male reproduction. There is, nevertheless, little information concerning the role of NO during semen storage. The aim of this study was to evaluate the effect of NO on boar semen stored at 17oC for 72 h. For this purporse, sperm samples were treated with 0.625, 1.25, 2.5, 5, and 10 mM aminoguanidine (AG) or Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), a selective and non-selective NO synthase (NOS) inhibitor, respectively. Moreover, sodium nitroprusside (SNP), a NO donor, was used at the dose of 18.75, 37.5, 75, and 150 μM. Sperm motility, membrane integrity, and acrosomal status were evaluated at 0, 4, 24, 48, and 72 h of semen storage. A significant increase of the amplitude of lateral sperm head displacement (ALH), and both curvilinear and straight-line velocity (VCL and VSL, respectively) was observed at 72 h of semen storage in samples treated with 0.625 mM AG, probably because of the antioxidant properties of this NOS inhibitor. Contrarily, 0.625 mM L-NAME showed no effect on boar sperm parameters during the entire period of semen storage. Moreover, AG and L-NAME at 10 mM negatively affected sperm kinetics and acrosome integrity, which may provide further support to the notion that low NO levels are necessary for a normal sperm function. The concentrations of SNP used in this study had mostly no or negative effects on boar sperm parameters during semen storage. In conclusion, the results from this study increase the understanding of the role of NO on boar sperm physiology. Copyright© by the Polish Academy of Sciences.

Group X phospholipase A2 is released during sperm acrosome reaction and controls fertility outcome in mice.

PubMed

Escoffier, Jessica; Jemel, Ikram; Tanemoto, Akemi; Taketomi, Yoshitaka; Payre, Christine; Coatrieux, Christelle; Sato, Hiroyasu; Yamamoto, Kei; Masuda, Seiko; Pernet-Gallay, Karin; Pierre, Virginie; Hara, Shuntaro; Murakami, Makoto; De Waard, Michel; Lambeau, Gérard; Arnoult, Christophe

2010-05-01

Ejaculated mammalian sperm must undergo a maturation process called capacitation before they are able to fertilize an egg. Several studies have suggested a role for members of the secreted phospholipase A2 (sPLA2) family in capacitation, acrosome reaction (AR), and fertilization, but the molecular nature of these enzymes and their specific roles have remained elusive. Here, we have demonstrated that mouse group X sPLA2 (mGX) is the major enzyme present in the acrosome of spermatozoa and that it is released in an active form during capacitation through spontaneous AR. mGX-deficient male mice produced smaller litters than wild-type male siblings when crossed with mGX-deficient females. Further analysis revealed that spermatozoa from mGX-deficient mice exhibited lower rates of spontaneous AR and that this was associated with decreased in vitro fertilization (IVF) efficiency due to a drop in the fertilization potential of the sperm and an increased rate of aborted embryos. Treatment of sperm with sPLA2 inhibitors and antibodies specific for mGX blocked spontaneous AR of wild-type sperm and reduced IVF success. Addition of lysophosphatidylcholine, a catalytic product of mGX, overcame these deficiencies. Finally, recombinant mGX triggered AR and improved IVF outcome. Taken together, our results highlight a paracrine role for mGX during capacitation in which the enzyme primes sperm for efficient fertilization and boosts premature AR of a likely phospholipid-damaged sperm subpopulation to eliminate suboptimal sperm from the pool available for fertilization.

Group X phospholipase A2 is released during sperm acrosome reaction and controls fertility outcome in mice

PubMed Central

Escoffier, Jessica; Jemel, Ikram; Tanemoto, Akemi; Taketomi, Yoshitaka; Payre, Christine; Coatrieux, Christelle; Sato, Hiroyasu; Yamamoto, Kei; Masuda, Seiko; Pernet-Gallay, Karin; Pierre, Virginie; Hara, Shuntaro; Murakami, Makoto; De Waard, Michel; Lambeau, Gérard; Arnoult, Christophe

2010-01-01

Ejaculated mammalian sperm must undergo a maturation process called capacitation before they are able to fertilize an egg. Several studies have suggested a role for members of the secreted phospholipase A2 (sPLA2) family in capacitation, acrosome reaction (AR), and fertilization, but the molecular nature of these enzymes and their specific roles have remained elusive. Here, we have demonstrated that mouse group X sPLA2 (mGX) is the major enzyme present in the acrosome of spermatozoa and that it is released in an active form during capacitation through spontaneous AR. mGX-deficient male mice produced smaller litters than wild-type male siblings when crossed with mGX-deficient females. Further analysis revealed that spermatozoa from mGX-deficient mice exhibited lower rates of spontaneous AR and that this was associated with decreased in vitro fertilization (IVF) efficiency due to a drop in the fertilization potential of the sperm and an increased rate of aborted embryos. Treatment of sperm with sPLA2 inhibitors and antibodies specific for mGX blocked spontaneous AR of wild-type sperm and reduced IVF success. Addition of lysophosphatidylcholine, a catalytic product of mGX, overcame these deficiencies. Finally, recombinant mGX triggered AR and improved IVF outcome. Taken together, our results highlight a paracrine role for mGX during capacitation in which the enzyme primes sperm for efficient fertilization and boosts premature AR of a likely phospholipid-damaged sperm subpopulation to eliminate suboptimal sperm from the pool available for fertilization. PMID:20424324

Retained functional integrity of bull spermatozoa after double freezing and thawing using PureSperm density gradient centrifugation.

PubMed

Maxwell, W M C; Parrilla, I; Caballero, I; Garcia, E; Roca, J; Martinez, E A; Vazquez, J M; Rath, D

2007-10-01

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88

The osmotic tolerance of boar spermatozoa and its usefulness as sperm quality parameter.

PubMed

Yeste, Marc; Briz, Mailo; Pinart, Elisabeth; Sancho, Sílvia; Bussalleu, Eva; Bonet, Sergi

2010-06-01

Predicting the fertility outcome of ejaculates is very important in the field of porcine reproduction. The aims of this study were to determine the effects of different osmotic treatments on boar spermatozoa and to correlate them with fertility and prolificacy, assessed as non-return rates within 60 days (NRR(60d)) of the first inseminations, and litter size (LS), respectively. Sperm samples (n=100) from one hundred healthy Piétrain boars were used to assess 48 treatments combining different osmolalities (ranged between 100 and 4000 mOsm kg(-1)), different compounds used to prepare anisotonic solutions, and two different modalities: return and non-return to isotonic conditions. Sperm quality was evaluated before and after applying the treatments on the basis of analyses of sperm viability, motility, morphology and percentages of acrosome-intact spermatozoa. Statistical analyses were performed using a one-way ANOVA and post hoc Tukey's test, linear regression analyses (Pearson correlation and multiple regression) and Jackknife cross-validation. Although three conventional parameters: sperm viability, sperm morphology and the percentages of acrosome-intact spermatozoa were significantly correlated with NRR(60d) and with LS, their respective osmotic tolerance parameters (defined for each parameter and treatment regarding with negative control) presented a higher Pearson coefficient with both fertility and prolificacy in three treatments (150 mOsm kg(-1) with non-return to isotonic conditions, 200 mOsm kg(-1) with return and 500 mOsm kg(-1) using sodium citrate and non-return to isotonic conditions). We conclude that osmotic resistance in sperm viability, sperm morphology and acrosome-intactness in the treatments mentioned above could be assessed along with classical parameters to better predict the fertilising ability of a given ejaculate. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Effect of cholesterol-loaded-cyclodextrin on sperm viability and acrosome reaction in boar semen cryopreservation.

PubMed

Lee, Yong-Seung; Lee, Seunghyung; Lee, Sang-Hee; Yang, Boo-Keun; Park, Choon-Keun

2015-08-01

This study was undertaken to examine the effect of cholesterol-loaded-cyclodextrin (CLC) on boar sperm viability and spermatozoa cryosurvival during boar semen cryopreservation, and methyl-β-cyclodextrin (MBCD) was treated for comparing with CLC. Boar semen treated with CLC and MBCD before freezing process to monitor the effect on survival and capacitation status by flow cytometry with appropriate fluorescent probes. Sperm viability was higher in 1.5mg CLC-treated sperm (76.9±1.01%, P<0.05) than un-treated and MBCD-treated sperm before cryopreservation (58.7±1.31% and 60.3±0.31%, respectively). For CTC patterns, F-pattern was higher in CLC treated sperm than MBCD-treated sperm, for B-pattern was higher in CLC-treated sperm than fresh sperm (P<0.05). For AR pattern (an acrosome-reacted sperm) was lower in CLC-treated sperm than MBCD-treated sperm (P<0.05). Moreover, we examined in vitro development of porcine oocytes after in vitro fertilization using CLC-treated frozen-thawed semen, in which CLC treatment prior to freezing and thawing increased the development of oocytes to blastocyst stage in vitro. In conclusion, CLC could protect the viability of spermatozoa from cryodamage prior to cryopreservation in boar semen. Copyright © 2015 Elsevier B.V. All rights reserved.

Calcium antagonists modulate oxidative stress and acrosomal reaction in rat spermatozoa.

PubMed

Morakinyo, Ayodele; Iranloye, Bolanle; Adegoke, Olufeyisipe

2011-08-01

Calcium ions are vital in many biological processes and qualify as an almost ubiquitous intracellular second messenger. This indicates the multiplicity of the effects associated with drug actions aimed at interfering with calcium ions. To examine the cellular process involved in the induction of infertility in males by calcium antagonist (CA) even in the presence of normal semen parameters, we studied the effects of different CA namely; nifedipine, verapamil and diltiazem on oxidative balance and acrosome reaction in the sperm. For this purpose, lipid peroxidation, antioxidants such as superoxide dismutase, catalase and reduced glutathione, and acrosomal reaction were determined in sperm samples of rats. Calcium antagonist causes significant oxidative stress in the epididymal sperm with increased malondialdehyde level and a concomitant decrease in antioxidant activities of catalase and superoxide dismutase. The percentage value of acrosomal-reacted sperm in the nifedipine, verapamil and diltiazem-treated rats were 41 ±2.45, 39 ±2.92 and 42 ±1.22 respectively, compared with the control group value of 86 ±2.92. It appears CA oxidatively modify the sperm resulting in functional inhibition of acrosomal reaction. Suppression of the sperm acrosomal reaction is known to have serious adverse implications for fertilization.

Sperm Proteasomes Degrade Sperm Receptor on the Egg Zona Pellucida during Mammalian Fertilization

PubMed Central

Zimmerman, Shawn W.; Manandhar, Gaurishankar; Yi, Young-Joo; Gupta, Satish K.; Sutovsky, Miriam; Odhiambo, John F.; Powell, Michael D.; Miller, David J.; Sutovsky, Peter

2011-01-01

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

Improving sperm banking efficiency in endangered species through the use of a sperm selection method in brown bear (Ursus arctos) thawed sperm.

PubMed

Anel-Lopez, L; Ortega-Ferrusola, C; Álvarez, M; Borragán, S; Chamorro, C; Peña, F J; Morrell, J; Anel, L; de Paz, P

2017-06-26

Sperm selection methods such as Single Layer Centrifugation (SLC) have been demonstrated to be a useful tool to improve the quality of sperm samples and therefore to increase the efficiency of other artificial reproductive techniques in several species. This procedure could help to improve the quality of genetic resource banks, which is essential for endangered species. In contrast, these sperm selection methods are optimized and focused on farm animals, where the recovery task is not as important as in endangered species because of their higher sperm availability. The aim of this study was to evaluate two centrifugation methods (300 x g/20 min and 600 x g/10 min) and three concentrations of SLC media (Androcoll-Bear -80, 65 and 50%) to optimise the procedure in order to recover as many sperm with the highest quality as possible. Sperm morphology could be important in the hydrodynamic relationship between the cell and centrifugation medium and thus the effect of sperm head morphometry on sperm yield and its hydrodynamic relationship were studied. The samples selected with Androcoll-Bear 65% showed a very good yield (53.1 ± 2.9) although the yield from Androcoll-Bear 80% was lower (19.3 ± 3.3). The latter showed higher values of motility than the control immediately after post-thawing selection. However, both concentrations of colloid (65 and 80%) showed higher values of viable sperm and viable sperm with intact acrosome than the control. After an incubation of 2 h at 37 °C, the samples from Androcoll-Bear 80% had higher kinematics and proportion of viable sperm with intact acrosome. In the morphometric analysis, the sperm selected by the Androcoll-Bear 80% showed a head with a bigger area which was more elongated than the sperm from other treatments. We conclude that sperm selection with Androcoll-Bear at either 65% or 80% is a suitable technique that allows a sperm population with better quality than the initial sample to be obtained. We recommend the

Evaluation of sperm quality of Erythrolamprus poecilogyrus sublineatus (Cope, 1860) (Serpentes, Dipsadidae).

PubMed

Silva, A C; Varela, A S; Cardoso, T F; Silva, E F; Loebmann, D; Corcini, C D

2017-01-01

Erythrolamprus poecilogyrus sublineatus (Cope, 1860), is a species widely distributed in the Pampa Domain, occurring in Rio Grande do Sul, Argentina and Uruguay, mainlyin the pampa region. In the coastal region of southern Brazil this is serpent is considered one of the most abundant. The purpose of the present study is to describe the techniques of sperm evaluation in vitro for E. poecilogyrus sublineatus in the coastal plain of Rio Grande do Sul, Brazil. After laparatomy the efferent vases were collected and the semen was diluted in 1ml Beltsville Thawing Solution. The characteristics of motility, membrane integrity, mitochondria, acrosome, DNA, cell viability and cellular functionality were evaluated. Fluorescent probes were used for the evaluation of sperm structure in epifluorescence microscope. With the techniques described, it was possible to identify intact and injured cells, enabling the determination of cell characteristics for the spring season (October and November). It was observed in the analyses that 80% of sperm cells were mobile and that 84.1 ± 8.0% of sperm membranes were intact. The standards found were of 48 ± 13.8% of intact acrosome, 73.6 ± 6.0 of perfect DNA and of 91.8 ± 4.0 of functional mitochondria. Thus, these values from the sperm analysis can be used as standards for the species Erythrolamprus poecilogyrus sublineatus.

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. © 2016 American Society of Andrology and European Academy of Andrology.

RIM, Munc13, and Rab3A interplay in acrosomal exocytosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bello, Oscar D.; Zanetti, M. Natalia; Laboratorio de Biologia Reproductiva, Instituto de Histologia y Embriologia, IHEM

2012-03-10

Exocytosis is a highly regulated, multistage process consisting of multiple functionally definable stages, including recruitment, targeting, tethering, priming, and docking of secretory vesicles with the plasma membrane, followed by calcium-triggered membrane fusion. The acrosome reaction of spermatozoa is a complex, calcium-dependent regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. Not much is known about the molecules that mediate membrane docking in this particular fusion model. In neurons, the formation of the ternary RIM/Munc13/Rab3A complex has been suggestedmore » as a critical component of synaptic vesicles docking. Previously, we demonstrated that Rab3A localizes to the acrosomal region in human sperm, stimulates acrosomal exocytosis, and participates in an early stage during membrane fusion. Here, we report that RIM and Munc13 are also present in human sperm and localize to the acrosomal region. Like Rab3A, RIM and Munc13 participate in a prefusion step before the efflux of intra-acrosomal calcium. By means of a functional assay using antibodies and recombinant proteins, we show that RIM, Munc13 and Rab3A interplay during acrosomal exocytosis. Finally, we report by electron transmission microscopy that sequestering RIM and Rab3A alters the docking of the acrosomal membrane to the plasma membrane during calcium-activated acrosomal exocytosis. Our results suggest that the RIM/Munc13/Rab3 A complex participates in acrosomal exocytosis and that RIM and Rab3A have central roles in membrane docking. -- Highlights: Black-Right-Pointing-Pointer RIM and Munc13 are present in human sperm and localize to the acrosomal region. Black-Right-Pointing-Pointer RIM and Munc13 are necessary for acrosomal exocytosis. Black-Right-Pointing-Pointer RIM and Munc13 participate before the acrosomal calcium efflux. Black

Metabolism of exogenous fatty acids, fatty acid-mediated cholesterol efflux, PKA and PKC pathways in boar sperm acrosome reaction.

PubMed

Hossain, Md Sharoare; Afrose, Sadia; Sawada, Tomio; Hamano, Koh-Ichi; Tsujii, Hirotada

2010-03-01

For understanding the roles of fatty acids on the induction of acrosome reaction which occurs under association of cholesterol efflux and PKA or PKC pathways in boar spermatozoa, metabolic fate of alone and combined radiolabeled 14 C-oleic acid and 3 H-linoleic acid incorporated in the sperm was compared, and behavior of cholesterol and effects of PKA and PKC inhibitors upon fatty acid-induced acrosome reaction were examined. Semen was collected from a Duroc boar, and the metabolic activities of fatty acids in the spermatozoa were measured using radioactive compounds and thin layer chromatography. Cholesterol efflux was measured with a cholesterol determination assay kit. Participation of fatty acids on the AR through PKA and PKC pathways was evaluated using a specific inhibitor of these enzymes. Incorporation rate of 14 C-oleic acid into the sperm lipids was significantly higher than that of 3 H-linoleic acid ( P < 0.05). The oxidation of 14 C-oleic acid was higher in combined radiolabeling rather than in one. The highest amounts of 3 H-linoleic acid and 14 C-oleic acid were recovered mainly in the triglycerides and phospholipids fraction, and 14 C-oleic acid distribution was higher than the 3 H-linoleic acid in both labeled ( P < 0.05) sperm lipids. In the 3 H-linoleic and 14 C-oleic acid combined radiolabeling, the incorporation rate of the radioactive fatty acids in all the lipid fractions increased 15 times more than the alone radiolabeling. Boar sperm utilize oleic acid to generate energy for hyperactivation ( P < 0.05). Supplementation of arachidonic acid significantly increased ( P < 0.05) cholesterol efflux in sperm. When spermatozoa were incubated with PKA or PKC inhibitors, there was a significant reduction of arachidonic acid-induced acrosome reaction (AR) ( P < 0.05), and inhibition by PKA inhibitor is stronger than that by PKC inhibitor. Incorporation of unsaturated fatty acids, especially oleic acid, into triglycerides and phospholipids provides

A role for carbohydrate recognition in mammalian sperm-egg binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clark, Gary F., E-mail: clarkgf@health.missouri.edu

Highlights: • Mammalian sperm-egg binding as a carbohydrate dependent species recognition event. • The role of carbohydrate recognition in human, mouse and pig sperm-egg binding. • Historical perspective and future directions for research focused on gamete binding. - Abstract: Mammalian fertilization usually requires three sequential cell–cell interactions: (i) initial binding of sperm to the specialized extracellular matrix coating the egg known as the zona pellucida (ZP); (ii) binding of sperm to the ZP via the inner acrosomal membrane that is exposed following the induction of acrosomal exocytosis; and (iii) adhesion of acrosome-reacted sperm to the plasma membrane of the eggmore » cell, enabling subsequent fusion of these gametes. The focus of this review is on the initial binding of intact sperm to the mammalian ZP. Evidence collected over the past fifty years has confirmed that this interaction relies primarily on the recognition of carbohydrate sequences presented on the ZP by lectin-like egg binding proteins located on the plasma membrane of sperm. There is also evidence that the same carbohydrate sequences that mediate binding also function as ligands for lectins on lymphocytes that can inactivate immune responses, likely protecting the egg and the developing embryo up to the stage of blastocyst hatching. The literature related to initial sperm-ZP binding in the three major mammalian models (human, mouse and pig) is discussed. Historical perspectives and future directions for research related to this aspect of gamete adhesion are also presented.« less

Sperm proteasome and fertilization.

PubMed

Sutovsky, Peter

2011-07-01

The omnipresent ubiquitin-proteasome system (UPS) is an ATP-dependent enzymatic machinery that targets substrate proteins for degradation by the 26S proteasome by tagging them with an isopeptide chain composed of covalently linked molecules of ubiquitin, a small chaperone protein. The current knowledge of UPS involvement in the process of sperm penetration through vitelline coat (VC) during human and animal fertilization is reviewed in this study, with attention also being given to sperm capacitation and acrosome reaction/exocytosis. In ascidians, spermatozoa release ubiquitin-activating and conjugating enzymes, proteasomes, and unconjugated ubiquitin to first ubiquitinate and then degrade the sperm receptor on the VC; in echinoderms and mammals, the VC (zona pellucida/ZP in mammals) is ubiquitinated during oogenesis and the sperm receptor degraded during fertilization. Various proteasomal subunits and associated enzymes have been detected in spermatozoa and localized to sperm acrosome and other sperm structures. By using specific fluorometric substrates, proteasome-specific proteolytic and deubiquitinating activities can be measured in live, intact spermatozoa and in sperm protein extracts. The requirement of proteasomal proteolysis during fertilization has been documented by the application of various proteasome-specific inhibitors and antibodies. A similar effect was achieved by depletion of sperm-surface ATP. Degradation of VC/ZP-associated sperm receptor proteins by sperm-borne proteasomes has been demonstrated in ascidians and sea urchins. On the applied side, polyspermy has been ameliorated by modulating sperm-associated deubiquitinating enzymes. Diagnostic and therapeutic applications could emerge in human reproductive medicine. Altogether, the studies on sperm proteasome indicate that animal fertilization is controlled in part by a unique, gamete associated, extracellular UPS.

Dynamic regulation of sperm interactions with the zona pellucida prior to and after fertilisation.

PubMed

Gadella, B M

2012-01-01

Recent findings have refined our thinking on sperm interactions with the cumulus-oocyte complex (COC) and our understanding of how, at the molecular level, the sperm cell fertilises the oocyte. Proteomic analyses has identified a capacitation-dependent sperm surface reordering that leads to the formation of functional multiprotein complexes involved in zona-cumulus interactions in several mammalian species. During this process, multiple docking of the acrosomal membrane to the plasma membrane takes place. In contrast with the dogma that the acrosome reaction is initiated when spermatozoa bind to the zona pellucida (ZP), it has been established recently that, in mice, the fertilising spermatozoon initiates its acrosome reaction during its voyage through the cumulus before it reaches the ZP. In fact, even acrosome-reacted mouse spermatozoa collected from the perivitelline space can fertilise another ZP-intact oocyte. The oviduct appears to influence the extracellular matrix properties of the spermatozoa as well as the COC. This may influence sperm binding and penetration of the cumulus and ZP, and, in doing so, increase monospermic while decreasing polyspermic fertilisation rates. Structural analysis of the ZP has shed new light on how spermatozoa bind and penetrate this structure and how the cortical reaction blocks sperm-ZP interactions. The current understanding of sperm interactions with the cumulus and ZP layers surrounding the oocyte is reviewed with a special emphasis on the lack of comparative knowledge on this topic in humans, as well as in most farm mammals.

Roles of the zona pellucida and functional exposure of the sperm-egg fusion factor 'IZUMO' during in vitro fertilization in pigs.

PubMed

Tanihara, Fuminori; Nakai, Michiko; Men, Nguyen Thi; Kato, Noriko; Kaneko, Hiroyuki; Noguchi, Junko; Otoi, Takeshige; Kikuchi, Kazuhiro

2014-04-01

The zona pellucida (ZP) is considered to play important roles in the prevention of polyspermy in mammalian oocytes. However, in pigs we have shown that the presence of the ZP accelerates sperm penetration into the ooplasm during in vitro fertilization (IVF). In the present study, we investigated the effects of the ZP on sperm binding, acrosomal status, and functional exposure of IZUMO, a critical factor involved in sperm-egg fusion, during IVF in pigs. We evaluated the numbers and acrosomal statuses of sperm binding to the ZP and oolemma, and being present in the ZP and perivitelline space (PVS) using ZP-intact and ZP-free oocytes. More sperm bound to the ZP than to the oolemma. The average number of sperm present in the PVS was 0.44-0.51 per oocyte, and all sperm had lost their acrosomes. The proportion of sperm that were immunopositive for anti-IZUMO antibody was significantly higher after they were passing or had passed through the ZP. Furthermore, addition of anti-IZUMO antibody to the fertilization medium significantly inhibited the penetration of sperm into ZP-free oocytes. These results suggest that, in pigs, the ZP induces the acrosome reaction, which is associated with the functional exposure of IZUMO, resulting in completion of fertilization. © 2014 Japanese Society of Animal Science.

Maturational changes in motility, acrosomal proteolytic activity, and penetrability of the inner perivitelline layer of fowl sperm, during their passage through the male genital tract.

PubMed

Ahammad, Muslah U; Nishino, C; Tatemoto, H; Okura, N; Kawamoto, Y; Okamoto, S; Nakada, T

2011-10-01

The objective was to examine, in vitro, the motility, acrosomal proteolytic activity (APA), and penetrating ability of fowl sperm recovered from the testis and epididymis, as well as the proximal, middle, and distal vas deferens, to assess the potential fertilizing ability of sperm as a function of maturation. A motile sperm separation technique was used to estimate sperm motility with Accudenz, a gelatin slide technique was used to measure the diameter of the halo around the acrosome of individual sperm as an indication of APA, and a sperm-inner perivitelline layer (IPL) interaction assay was done to estimate the number of hole formations as an indication of sperm penetration into the IPL. Sperm in the testis exhibited the least motility, produced the smallest halos, and created the least number of holes per 0.25 mm(2). Motility, diameter of the halo, and number of holes increased gradually (P < 0.05) from the epididymis to the distal vas deferens and were markedly different (P < 0.05) between testicular and deferent duct sperm. Based on these in vitro experimental findings, we inferred that fowl sperm undergo a gradual process of maturational changes in motility, APA, and penetrability as a means of acquiring potential fertility during their passage throughout the male genital tract. Copyright © 2011 Elsevier Inc. All rights reserved.

Structure, cytoskeleton, and development of the acrosome of Platycleis albopunctata (Orthoptera: Tettigoniidae).

PubMed

Guerra, R; Esponda, P

1999-10-01

The acrosome of Platycleis albopunctata (Orthoptera: Tettigoniidae) is relatively large and complex, consisting of an apical vesicle and two large wing-like extensions that give the spermatozoon the shape of an arrow. The wings have actin microfilaments and microtubules and are covered with a noticeable extracellular material. Actin filaments are present in the acrosome when it first appears in spermatid stages. The acrosome and the acrosomal attachment to the nucleus are more resistant than other structures to the reducing agents DTT and SDS. At the end of spermiogenesis, groups of spermatozoa juxtapose their sperm heads and become joined to form a spermatodesm encircled by an amorphous material. Treatment with the ionophore A23187 rapidly disrupted acrosomes of the free gametes, but acrosomes from spermatozoa contained in the spermatodesm were not disassembled. Packaging of sperm in a spermatodesm appears to protect the acrosome. Copyright 1999 Wiley-Liss, Inc.

Sperm membrane functionality in the dog assessed by flow cytometry.

PubMed

Cheuquemán, C; Bravo, P; Treulén, F; Giojalas, Lc; Villegas, J; Sánchez, R; Risopatrón, J

2012-02-01

The objective assessment of sperm function increases the chances of predicting the fertilizing capacity of a fresh semen sample or diagnosing infertility problems. In this study, the available flow cytometry technique was used to determine the membrane functional capacity of canine spermatozoa. The second fractions of ejaculates from six dogs were pooled, and samples (n = 26) processed to determine the variables: sperm viability and plasma membrane integrity by Sybr-14/Pi staining; phosphatidylserine (PS) translocation by Annexin-V-FITC/PI labelling; acrosome membrane integrity by FITC-conjugated Pisum sativum agglutinin/PI labelling; and mitochondrial membrane potential (ΔΨm) by staining with JC-1. Means for the 26 examined samples indicated that 82.66 ± 2.8% of the viable spermatozoa showed an intact plasma membrane, 8.4 ± 2.6% were moribund, 72.7 ± 16% had an intact acrosome, 80.9 ± 17% had high ΔΨm and 8.1 ± 11% had PS translocation with a PS translocation index of 2.1 ± 3%. Motility was only correlated with PS translocation (R = 0.3901; p = 0.0488), and acrosome membrane integrity was correlated with PS translocation (R = -0.5816; p = 0.0018). This study provides objective physiological data on the functional capacity of canine spermatozoa. © 2011 Blackwell Verlag GmbH.

Trypan blue/giemsa staining to assess sperm membrane integrity in salernitano stallions and its relationship to pregnancy rates.

PubMed

Serafini, R; Longobardi, V; Spadetta, M; Neri, D; Ariota, B; Gasparrini, B; Di Palo, R

2014-02-01

Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty-one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty-five mares were inseminated during the breeding season from April to July. Differences among stallions were found in volume, sperm concentration (p < 0.05) and visual motility (p < 0.01). A decrease in sperm motility, concentration (p < 0.05) and total sperm number was found in June-July (p < 0.01). Live sperm with intact acrosome (LSIA) and proximal droplets (PD) were lower (p < 0.01) in June-July, while acrosome reacted sperm (ARS) percentage increased (p < 0.05). No fertility differences were found among stallions with an average fertility per cycle of 44.6% and a pregnancy rate of 68.6%. Higher percentages of LSIA were found in the ejaculates used to inseminate mares that became pregnant vs those used in mares not pregnant (p < 0.05). The significance of LSIA as test variable to verify the reliability of Trypan blue/Giemsa staining was confirmed by Receiver operating characteristic ROC analysis and the sensitivity of the test was 85% at a cut-off value of 48% LSIA. Trypan blue-Giemsa showed to be an accurate method that can be applied on field to evaluate sperm membrane integrity and to identify poor-quality ejaculates. © 2013 Blackwell Verlag GmbH.

Membrane events in the acrosomal reaction of Limulus sperm. Membrane fusion, filament-membrane particle attachment, and the source and formation of new membrane surface

PubMed Central

1979-01-01

The membranes of Limulus (horseshoe crab) sperm were examined before and during the acrosomal reaction by using the technique of freeze- fracturing and thin sectioning. We focused on three areas. First, we examined stages in the fusion of the acrosomal vacuole with the cell surface. Fusion takes place in a particle-free zone which is surrounded by a circlet of particles on the P face of the plasma membrane and an underlying circlet of particles on the P face of the acrosomal vauole membrane. These circlets of particles are present before induction. Up to nine focal points of fusion occur within the particle-free zone. Second, we describe a system of fine filaments, each 30 A in diameter, which lies between the acrosomal vacuole and the plasma membrane. These filaments change their orientation as the vacuole opens, a process that takes place in less than 50 ms. Membrane particles seen on the P face of the acrosomal vacuole membrane change their orientation at the same time and in the same way as do the filaments, thus indicating that the membrane particles and filaments are probably connected. Third, we examined the source and the point of fusion of new membrane needed to cover the acrosomal process. This new membrane is almost certainly derived from the outer nuclear envelope and appears to insert into the plasma membrane in a particle-free area adjacent to an area rich in particles. The latter is the region where the particles are probably connected to the cytoplasmic filaments. The relevance of these observations in relation to the process of fertilization of this fantastic sperm is discussed. PMID:582596

[Application of spontaneous acrosome reaction of sperm in prediction of outcome of in-vitro fertilization and embryo transfer].

PubMed

Xuan, X J; Xu, C; Zhao, Y R; Wu, K L; Chen, T; Zhang, H B; Li, X; Su, S Z; Ma, G; Tang, R; Sheng, Y; Ma, J L

2016-04-26

To investigate the clinical application of spontaneous acrosome reaction (AR) rate of sperm in predicting the outcome of in-vitro fertilization and embryo transfer (IVF-ET). The spontaneous AR rate of the sperm of patients who underwent IVF-ET treatment in our center during the period from November to December 2014 were studied. The cut-off value from 6% to 12% were set and analyzed its association between the IVF-ET outcomes (including fertility rates, normal fertilization rates and high-quality embryo rates). For those who underwent fresh embryo transplantation, the rates of chemical pregnancy and clinical pregnancy were calculated, and compared the spontaneous AR rates and quantity of acrosomal enzyme according to the pregnancy outcome. There were 202 patients in this study and the mean spontaneous AR rate was 5.99%±5.18%. For patients with the spontaneous AR rate ≥9% versus <9%, the fertility rate, normal fertilization rate and high-quality embryo rate were 81.33% vs 83.85%, 60.53% vs 60.99%, and 51.10% vs 59.67%, respectively, with statistically significant difference in the high-quality embryo rate (P=0.02). For patients who underwent fresh embryo transplantation, when comparison was made between those with spontaneous AR rate ≥8% and those <8%, the rate of chemical pregnancy and clinical pregnancy were 48.57% (17/35) vs 69.64% (78/112) and 37.14% (13/35) vs 63.39% (71/112), respectively, both with statistically significant difference (P=0.02 and P<0.01). The patients with clinical pregnancy had lower spontaneous AR rate than those without clinical pregnancy (5.41%±3.87% vs 7.40%±6.79%, P=0.04), while the quantity of acrosomal enzyme showed no significant difference [(131.79±68.50) vs (153.62±59.59) μU/10(6,) P=0.06]. Logistic regression analysis demonstrated association between spontaneous AR rates and clinical pregnancy (OR=0.93, 95%CI: 0.87-0.99, P=0.03). The spontaneous AR rate of sperm may have clinical significance in predicting the outcome of

Acrosome reaction inducers impose alterations in repulsive strain and hydration barrier in human sperm membranes.

PubMed

Purohit, S B; Laloraya, M; Kumar, G P

1998-06-01

Spin labeling studies of the lipophilic domains of human spermatozoa during capacitation and during acrosome reaction (AR) under the influence of selected AR-inducers were performed. Significantly enhanced rotational function of molecules was obvious during capacitation, with no significant changes in membrane packaging or the lateral diffusion of molecules. The AR inducers appeared to restrict the rotational freedom of molecules, dramatically enhancing the lateral diffusion and ordering coefficients. A significant decrease in superoxide anion generation was observed in the acrosome reacted groups when compared to the non-acrosome reacted groups. A high level of superoxide anion radical (O2.-) level maintained in capacitated spermatozoa would add to the Van der Waal's repulsive forces at the polar head of phospholipids, holding the membrane in strain where the molecular enjoy little freedom for lateral motion. A sudden drop in the levels of O2.- in spermatozoa upon addition of AR inducers could abruptly release the local hydrophobic repulsive strain within the membrane. This loss of hydration barrier explains the observed enhancement in lateral diffusion profiles of lipids and the packaging of molecules. It is reasonable to assume that these phenomena could be amplified further by interplay of Ca2+ by modifying the local charge aggregation. Thus, we would conclude that AR inducers release the oxyradical load in capacitated spermatozoa, which would modify the repulsive strain and hydration barrier forces in the lipophilic domains permitting vesiculation of the membranes. It appears that various acrosome reaction inducers act as effectors of grossly similar physical alterations in sperm membranes and that the resulting signal cascades proceed through intercalating biochemical sequences.

Egg sialoglycans increase intracellular pH and potentiate the acrosome reaction of sea urchin sperm.

PubMed

Hirohashi, Noritaka; Vacquier, Victor D

2002-03-08

Sea urchin egg jelly (EJ) triggers sperm acrosome reaction (AR), an exocytotic event required for membrane fusion of the gametes. Purified fucose sulfate polymer (FSP) in EJ is one inducer of the AR. Binding of FSP to its receptor regulates opening of two distinct calcium channels and also elevates intracellular pH (pH(i)). EJ also contains sialic acid-rich glycans (sialoglycans (SG)) that were isolated by beta-elimination followed by DEAE chromatography. In the presence of limiting amounts of FSP, the SG fraction markedly potentiates the AR; however, by itself SG has no activity. The SG fraction increases the pH(i) of sperm without increasing intracellular Ca(2+). The SG-induced increase in pH(i) is not blocked by nifedipine or high K(+), whereas the FSP-induced pH(i) increase is sensitive to both these agents. Treatment of the SG fraction with neuraminidase or mild metaperiodate that specifically cleaves the glycerol side chain of sialic acid abolishes the AR potentiation and ability of SG to elevate pH(i). These data are the first to show that there are at least two pathways to induce sperm pH(i) increase and that egg surface sialic acid plays a role in triggering the sperm AR.

Cryopreservation of Cynomolgus Macaque (Macaca fascicularis) Sperm by Using a Commercial Egg-YolkFree Freezing Medium.

PubMed

Yan, Yaping; Ao, Lei; Wang, Hong; Duan, Yanchao; Chang, Shaohui; Chen, Bingbing; Zhi, Dalong; Li, Sujuan; Niu, Yuyu; Ji, Weizhi; Si, Wei

2016-11-01

Conventional TRISegg yolk (TEY) freezing medium for the cryopreservation of NHP sperm has the risk of contamination due to widespread zoonotic diseases. This study was aimed at determining the optimal glycerol concentration, freezing rate, and holding time in liquid N2 vapor for the cryopreservation of cynomolgus macaque sperm by using a commercial egg-yolkfree freezing medium (SC medium) designed for human sperm cryopreservation. Sperm motility and acrosomal integrity after freezing were assessed. Sperm in SC medium (dilution ratio, 3:1) frozen at cooling rates of 67 and 183C/min in liquid N2 vapor showed higher post-thaw motility than did samples frozen at 435C/min. At the cooling rate of 183C/min and dilution in SC medium at a 3:1 ratio, post-thaw motility was higher after a holding time of 10 min than after 30 min (recommended by the manufacturer). In addition, post-thaw motility of sperm frozen in SC medium was higher with dilution ratios of 3:1, 4.5:1, and 6:1 compared with 9:1, 10.5:1, and 12:1, and the sample diluted 12:1 showed the lowest percentage of thawed sperm with intact acrosomes. Sperm showed higher post-thaw motility after freezing in TEY than in SC medium; acrosomal integrity did not differ between the 2 media. Our results indicated that cynomolgus macaque sperm can be cryopreserved successfully by using a commercial egg-yolkfree freezing medium, which provides an option for genetic preservation with decreased zoonotic risk in this important NHP species.

A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm1

PubMed Central

Romarowski, Ana; Sánchez-Cárdenas, Claudia; Ramírez-Gómez, Héctor V.; Puga Molina, Lis del C.; Treviño, Claudia L.; Hernández-Cruz, Arturo; Darszon, Alberto; Buffone, Mariano G

2016-01-01

During capacitation, sperm acquire the ability to undergo the acrosome reaction (AR), an essential step in fertilization. Progesterone produced by cumulus cells has been associated with various physiological processes in sperm, including stimulation of AR. An increase in intracellular Ca2+ ([Ca2+]i) is necessary for AR to occur. In this study, we investigated the spatiotemporal correlation between the changes in [Ca2+]i and AR in single mouse spermatozoa in response to progesterone. We found that progesterone stimulates an [Ca2+]i increase in five different patterns: gradual increase, oscillatory, late transitory, immediate transitory, and sustained. We also observed that the [Ca2+]i increase promoted by progesterone starts at either the flagellum or the head. We validated the use of FM4-64 as an indicator for the occurrence of the AR by simultaneously detecting its fluorescence increase and the loss of EGFP in transgenic EGFPAcr sperm. For the first time, we have simultaneously visualized the rise in [Ca2+]i and the process of exocytosis in response to progesterone and found that only a specific transitory increase in [Ca2+]i originating in the sperm head promotes the initiation of AR. PMID:26819478

Zona pellucida from fertilised human oocytes induces a voltage-dependent calcium influx and the acrosome reaction in spermatozoa, but cannot be penetrated by sperm

PubMed Central

Patrat, Catherine; Auer, Jana; Fauque, Patricia; Leandri, Roger L; Jouannet, Pierre; Serres, Catherine

2006-01-01

Background The functions of three zona glycoproteins, ZP1, ZP2 and ZP3 during the sperm-zona pellucida (ZP) interaction are now well established in mice. The expression of an additional zona glycoprotein, ZPB/4, in humans, led us to reconsider the classical mouse model of gamete interaction. We investigated the various functions of human ZP (hZP) during the interaction of spermatozoa with fertilised and unfertilised oocytes. Results The hZP of fertilised oocytes retained their ability to bind sperm (albeit less strongly than that from unfertilised oocytes), to induce an intraspermatic calcium influx through voltage-dependent channels similar to that observed with hZP from unfertilised oocytes and to promote the acrosome reaction at a rate similar to that induced by the ZP of unfertilised oocytes (61.6 ± 6.2% vs60.7 ± 9.1% respectively). Conversely, the rate of hZP penetrated by sperm was much lower for fertilised than for unfertilised oocytes (19% vs 57% respectively, p < 0.01). We investigated the status of ZP2 in the oocytes used in the functional tests, and demonstrated that sperm binding and acrosome reaction induction, but not ZP penetration, occurred whether or not ZP2 was cleaved. Conclusion The change in ZP function induced by fertilisation could be different in human and mouse species. Our results suggest a zona blocking to polyspermy based at the sperm penetration level in humans. PMID:17147816

Addition of cholesterol-loaded cyclodextrins to the thawing extender: effects on boar sperm quality.

PubMed

Tomás, C; Gómez-Fernández, J; Gómez-Izquierdo, E; Mocé, E; de Mercado, E

2014-06-01

The aim of the present study was to evaluate the effect that the addition of cholesterol-loaded cyclodextrins (CLC) to the thawing extender has on the quality of frozen-thawed boar sperm. Pooled semen (n = 5) from three boars was used for the experiments. The semen was cryopreserved with an egg-yolk-based extender, it was diluted after thawing in Beltsville thawing solution (BTS) supplemented with different concentrations of CLC (0, 12.5, 25, 50 or 100 mg/500 × 10(6) sperm), and these samples were incubated at 37°C for 150 min. The following parameters of sperm quality were evaluated 30 and 150 min after incubation: sperm with intact plasma membrane (SIPM; %), sperm with normal acrosomal ridge (NAR; %), total motile sperm (TMS; %), progressively motile sperm (PMS; %) and kinetic parameters. Both SIPM and NAR increased (p < 0.05) when the thawing extender was supplemented with 12.5, 25 and 50 mg CLC/500 × 10(6) sperm. Nevertheless, motility decreased (p < 0.05) when the concentration of CLC exceeded 12.5 mg CLC/500 × 10(6) sperm. In conclusion, our results suggest that the supplementation of thawing extenders with CLC improves sperm viability and reduces acrosome damage after freezing/thawing. © 2014 Blackwell Verlag GmbH.

Regulation of acrosomal exocytosis. II. The zona pellucida-induced acrosome reaction of bovine spermatozoa is controlled by extrinsic positive regulatory elements.

PubMed

Florman, H M; First, N L

1988-08-01

The effects of accessory sex gland secretions on the zona pellucida-induced acrosome reaction of bovine spermatozoa were investigated. Soluble extracts of zonae pellucidae initiated exocytosis in ejaculated spermatozoa. This process had an ED50 of 20 ng/microliter zona pellucida protein and saturated at 50 ng/microliter (Florman and First, 1988. Dev. Biol. 128, 453-463). In epididymal sperm this dose-response relationship was shifted toward greater agonist concentrations by at least a factor of 10(3). Reconstitution of high potency agonist response was achieved in vitro by incubation of epididymal sperm with bovine seminal plasma. Reconstitution was dependent on the seminal plasma protein concentration. The ED50 of this process was 62 micrograms protein/10(8) sperm and saturation was observed with 124 micrograms protein/10(8) sperm. Agonist responses in reconstituted epididymal sperm and in ejaculated sperm were indistinguishable with regard to dependence on the zona pellucida protein concentration and the kinetics of induced acrosome reactions. Kinetic studies suggest that reconstitution is due to adsorption of regulatory factors from seminal plasma. In addition to the positive regulatory elements responsible for reconstituting activity, seminal plasma also contains negative regulatory elements which inhibit agonist response. These negative factors are inactivated during sperm capacitation, permitting the expression of positive regulators. Acting together, these regulatory elements could coordinate high affinity agonist response with the availability of eggs in vivo.

Cryopreservation of Cynomolgus Macaque (Macaca fascicularis) Sperm by Using a Commercial Egg-Yolk–Free Freezing Medium

PubMed Central

Yan, Yaping; Ao, Lei; Wang, Hong; Duan, Yanchao; Chang, Shaohui; Chen, Bingbing; Zhi, Dalong; Li, Sujuan; Niu, Yuyu; Ji, Weizhi; Si, Wei

2016-01-01

Conventional TRIS–egg yolk (TEY) freezing medium for the cryopreservation of NHP sperm has the risk of contamination due to widespread zoonotic diseases. This study was aimed at determining the optimal glycerol concentration, freezing rate, and holding time in liquid N2 vapor for the cryopreservation of cynomolgus macaque sperm by using a commercial egg-yolk–free freezing medium (SC medium) designed for human sperm cryopreservation. Sperm motility and acrosomal integrity after freezing were assessed. Sperm in SC medium (dilution ratio, 3:1) frozen at cooling rates of –67° and –183°C/min in liquid N2 vapor showed higher post-thaw motility than did samples frozen at –435 °C/min. At the cooling rate of –183 °C/min and dilution in SC medium at a 3:1 ratio, post-thaw motility was higher after a holding time of 10 min than after 30 min (recommended by the manufacturer). In addition, post-thaw motility of sperm frozen in SC medium was higher with dilution ratios of 3:1, 4.5:1, and 6:1 compared with 9:1, 10.5:1, and 12:1, and the sample diluted 12:1 showed the lowest percentage of thawed sperm with intact acrosomes. Sperm showed higher post-thaw motility after freezing in TEY than in SC medium; acrosomal integrity did not differ between the 2 media. Our results indicated that cynomolgus macaque sperm can be cryopreserved successfully by using a commercial egg-yolk–free freezing medium, which provides an option for genetic preservation with decreased zoonotic risk in this important NHP species. PMID:27931311

Assessment of sperm hyperactivated motility and acrosome reaction can discriminate the use of spermatozoa for conventional in vitro fertilisation or intracytoplasmic sperm injection: preliminary results.

PubMed

Wiser, A; Sachar, S; Ghetler, Y; Shulman, A; Breitbart, H

2014-04-01

Basic semen analysis is insufficient for determining the fertility potential. The aim of this study was to determine if hyperactivated motility (HAM) and acrosome reaction (AR) can be useful tests for evaluating semen quality during male infertility evaluations and to help the clinician decide whether regular insemination or intracytoplasmic sperm injection (ICSI) is preferable during in vitro fertilisation. A prospective study was conducted. Patients with normal sperm according to World Health Organization guidelines who underwent IVF treatment and planned regular insemination were asked to participate. A portion of sperm sample was evaluated for HAM and AR on day of ovum pick up. In HAM assessment, 93.3% of patients with increased HAM had a high fertilisation rate compared with 64% in the group without increased HAM (P = 0.059). For the AR evaluation, 91.7% of samples with a low rate of spontaneous AR had a high fertilisation rate compared with 39.3% in the group with a high rate of spontaneous AR (P = 0.004). © 2013 Blackwell Verlag GmbH.

Infertility in a Ram Associated with a Knobbed Acrosome Abnormality of the Spermatozoa

PubMed Central

Savage, N. C.

1984-01-01

A yearling Rambouillet ram with an asymmetrical scrotum was examined for potential breeding soundness prior to use in a synchronized mating program in a purebred flock of 20 ewes. Initial sperm cell evaluation revealed 78% knobbed acrosomes associated with few other abnormalities of the head and midpiece. Use of the ram resulted in no conception in one group of ten synchronized ewes. One month later, the proportion of sperm cells with knobbed acrosomes was 80%. ImagesFigure 1.Figure 2. PMID:17422370

Lactose-egg yolk diluent supplemented with N-acetyl-D-glucosamine affect acrosome morphology and motility of frozen-thawed boar sperm.

PubMed

Yi, Y J; Im, G S; Park, C S

2002-12-16

These experiments were carried out to investigate the effect of N-acetyl-D-glucosamine, and to obtain additional information about the effect of orvus es paste (OEP) and egg yolk concentration in the freezing of boar sperm in the maxi-straw. The highest post-thaw acrosomes of normal apical ridge (NAR) and motility were obtained with 0.025 or 0.05% N-acetyl-D-glucosamine concentration in the first diluent. However, there were no effects of N-acetyl-D-glucosamine among the diluents with or without N-acetyl-D-glucosamine at the second dilution. The N-acetyl-D-glucosamine in the first and second diluents was added at room temperatures (20-23 degrees C) and 5 degrees C, respectively. It is suggested that the temperature of N-acetyl-D-glucosamine addition is important for the effect of boar sperm protection during freezing and thawing. When the 0.05% N-acetyl-D-glucosamine was supplemented in the first diluent, the optimum final OEP content was 0.5%. The optimum content of egg yolk in the diluent with 0.05% N-acetyl-D-glucosamine concentration was 20% and egg yolk was one of the main cryoprotective agents. In conclusion, we found out that the diluent with 0.025 or 0.05% soluble N-acetyl-D-glucosamine in the first diluent, 0.5% final orvus es paste concentration and 20% egg yolk concentration significantly enhanced NAR acrosomes and motility of boar sperm after freezing and thawing. Copyright 2002 Elsevier Science B.V.

MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization.

PubMed

Ferrer, Marvin; Rodriguez, Hilma; Zara, Lindsay; Yu, Yang; Xu, Wei; Oko, Richard

2012-09-01

Sperm-zona pellucida (ZP) penetration during fertilization is a process that most likely involves enzymatic digestion of this extracellular coat by spermatozoa. Since the inner acrosomal membrane (IAM) is the leading edge of spermatozoa during penetration and proteins required for secondary binding of sperm to the zona are present on it, the IAM is the likely location of these enzymes. The objectives of this study were to identify and characterize proteinases present on the IAM, confirm their localization and provide evidence for their role in fertilization. Gelatin zymography of detergent extracts of the IAM revealed bands of enzymatic activity identified as serine and matrix metallo-proteinases (MMPs). Specific inhibitors to MMPs revealed that MMP activity was due to MMP2. Immunoblotting determined that the serine protease activity on the zymogram was due to acrosin and also confirmed the MMP2 activity. Immunogold labeling of spermatozoa at the electron microscope level showed that acrosin and MMP2 were confined to the apical and principal segments of the acrosome in association with the IAM, confirming our IAM isolation technique. Immunohistochemical examination of acrosin and MMP2 during spermiogenesis showed that both proteins originate in the acrosomic granule during the Golgi phase and later redistribute to the acrosomal membrane. Anti-MMP2 antibodies and inhibitors incorporated into in vitro fertilization media significantly decreased fertilization rates. This is the first study to demonstrate that MMP2 and acrosin are associated with the IAM and introduces the possibility of their cooperation in enzymatic digestion of the ZP during penetration.

Computer-assisted sperm analysis of fresh epididymal cat spermatozoa and the impact of cool storage (4 degrees C) on sperm quality.

PubMed

Filliers, M; Rijsselaere, T; Bossaert, P; De Causmaecker, V; Dewulf, J; Pope, C E; Van Soom, A

2008-12-01

Epididymal cat sperm is commonly used for in vitro fertilization. Because of the high variability in preparation protocols and methods of evaluation, sperm quality may vary considerably between experiments and laboratories. The aims of the present study were (1) to describe an epididymal sperm preparation protocol to produce clean, highly motile samples using density gradient centrifugation, (2) to provide reference values of computer-assisted semen analysis (CASA) parameters of fresh epididymal cat sperm after density gradient centrifugation and (3) to investigate the effect of cool storage on various spermatozoa characteristics. After slicing the epididymides, viable and motile sperm cells were isolated using Percoll centrifugation. Sperm motility parameters were subsequently assessed using CASA in experiment 1. In experiment 2, fresh (day 0) sperm samples were evaluated for motility parameters (HTR) and stained for assessment of acrosomal status (FITC-PSA), morphology (eosin/nigrosin (E/N)), membrane integrity (E/N and SYBR((R))14-PI) and DNA fragmentation (TUNEL). After addition of a Tris-glucose-citrate diluent containing 20% egg yolk, samples were cooled to 4 degrees C and reassessed on d1, d3, d5, d7 and d10. Cool storage impaired most motility and velocity parameters: MOT, PMOT, VAP, VSL, VCL, BCF, RAPID and the percentage of normal spermatozoa showed a decrease over time (P<0.05) as compared to fresh samples. In contrast, STR, ALH, membrane integrity, DNA fragmentation and the percentage of acrosome intact spermatozoa were not affected by cool storage. However, the influence of cool storage of cat spermatozoa on subsequent in vitro embryo development and quality after IVF requires further investigation.

Formation of the acrosome complex in the bush cricket Gampsocleis gratiosa (Orthoptera: Tettigoniidae).

PubMed

Su, Cai Xia; Chen, Jie; Shi, Fu Ming; Guo, Ming Shen; Chang, Yan Lin

2017-07-01

The acrosome complex plays an indispensable role in the normal function of mature spermatozoa. However, the dynamic process of acrosome complex formation in insect remains poorly understood. Gampsocleis gratiosa Brunner von Wattenwyl possesses the typical characteristic of insect sperms, which is tractable in terms of size, and therefore was selected for the acrosome formation study in this report. The results show that acrosome formation can be divided into six phases: round, rotating, rhombic, cylindrical, transforming and mature phase, based on the morphological dynamics of acrosome complex and nucleus. In addition, the cytoskeleton plays a critical role in the process of acrosome formation. The results from this study indicate that: (1) glycoprotein is the major component of the acrosome proper; (2) the microfilament is one element of the acrosome complex, and may mediate the morphologic change of the acrosome complex; (3) the microtubules might also shape the nucleus and acrosome complex during the acrosome formation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Use of two conventional staining methods to assess the acrosomal status of stallion spermatozoa.

PubMed

Runcan, E E; Pozor, M A; Zambrano, G L; Benson, S; Macpherson, M L

2014-07-01

The acrosome is a highly specialised region of the spermatozoon that is essential for fertilisation. Defects or dysfunction of this structure have been associated with fertility problems in man and various domestic species including stallions. Current methods of evaluating the acrosome of stallion spermatozoa are time consuming and require specialised equipment, which is cost prohibitive to the average practitioner. To evaluate 2 conventional stains (Dip Quick and Spermac) and determine their usefulness in assessing acrosome integrity in stallions as compared with specific acrosomal labelling with a fluorescein-conjugated lectin - a method that has been validated for acrosome status evaluation in stallions. In vivo experimental design. Semen from 6 mature Miniature horse stallions of known fertility was collected on 5 separate occasions. To increase the number of reacted acrosomes, portions of each ejaculate were incubated with the calcium ionophore, A23187. Ejaculates were divided and semen samples were processed according to recommendations for fluorescein-conjugated peanut lectin, Pisum sativum agglutin, Dip Quick, and Spermac staining methods. Slides were evaluated independently by 2 separate investigators. Spermatozoa were classified as having intact, reacting, reacted or defective acrosomes. All parameters obtained by both investigators, using all 3 staining methods were highly correlated (P<0.001). There was no statistical difference (P>0.05) between investigators or staining method for the percentages of intact or reacted acrosomes. However, there was a significant difference between investigators and staining methods for determining reacting acrosome percentages (P<0.05). Dip Quick and Spermac stains are useful for determining intact vs. reacted acrosomes for stallion spermatozoa. © 2013 EVJ Ltd.

The effect of glycosaminoglycan enzymes and proteases on the viscosity of alpaca seminal plasma and sperm function.

PubMed

Kershaw-Young, C M; Stuart, C; Evans, G; Maxwell, W M C

2013-05-01

In order to advance the development of cryopreservation and other assisted reproductive technologies in camelids it is necessary to eliminate the viscous component of the seminal plasma without impairing sperm function. It has been postulated that glycosaminoglycans (GAGs) or proteoglycans are responsible for this viscosity. This study investigated the effect of the GAG enzymes hyaluronidase, chondroitinase ABC and keratanase and the proteases papain and proteinase K on seminal plasma viscosity and sperm function in order to aid identification of the cause of seminal plasma viscosity and propose methods for the reduction of viscosity. Sperm motility, DNA integrity, acrosome integrity and viability were assessed during 2h incubation. All enzymes reduced seminal plasma viscosity compared to control (P<0.001) although papain was most effective, completely eliminating viscosity within 30 min of treatment. Sperm motility and DNA integrity was not affected by enzyme treatment. The proportion of viable, acrosome intact sperm was reduced in all enzyme treated samples except those treated with papain (P<0.001). These findings suggest that proteins, not GAGs are the main cause of alpaca seminal plasma viscosity. Papain treatment of alpaca semen may be a suitable technique for reduction of seminal plasma viscosity prior to sperm cryopreservation. Copyright © 2013 Elsevier B.V. All rights reserved.

Effect of post-thaw incubation on sperm kinematics and acrosomal integrity of ram spermatozoa cryopreserved in medium-sized French straws.

PubMed

Bag, Sadhan; Joshi, Anil; Naqvi, S M K; Mittal, J P

2004-08-01

The objectives were to assess the effect of post-thaw in vitro incubation on motion characteristics and acrosomal integrity of ram spermatozoa of native Malpura and Bharat Merino breeds maintained under a semi-arid tropical environment. Good quality semen samples of both breeds were diluted, packaged in medium-sized straws, and frozen under controlled conditions. Straws were thawed at 60 degrees C for 10s and thawed samples were incubated at 37 degrees C for 4h. Post-thaw motion characteristics and acrosomal integrity of incubated spermatozoa were assessed (by computer-aided semen analysis and Giemsa staining, respectively) just prior to incubation and at hourly intervals thereafter. There was a significant effect of incubation time on motility characteristics and the proportion of spermatozoa with normal acrosomes; 81.4% (arcsin transformed value, 65.2) of spermatozoa were motile at the start of incubation, with 47.9% (arcsin transformed value, 44.4) motile after 4h. At the corresponding times, there were normal acrosomes in 65.8 (arcsin transformed value, 54.8) and 55.7% (arcsin transformed value, 48.9) of spermatozoa, respectively. The percentage straightness of spermatozoa varied during incubation (P < 0.01). However, there was no significant change in percentage linearity, curvilinear velocity, average path velocity, straight line velocity, lateral head displacement, and beat cross frequency of spermatozoa during incubation. There were no breed variations in any motility parameters during incubation, except percentage straightness (P < 0.05), lateral head displacement (P < 0.05) and beat cross frequency (P < 0.01). That sperm motility and acrosomal morphology were very acceptable immediately post-thaw and after 4h of incubation indicated the efficacy of cryopreserving ram spermatozoa under controlled conditions in medium-sized straws.

Short-Term Storage of Rat Sperm in the Presence of Various Extenders

PubMed Central

Varisli, Omer; Agca, Cansu; Agca, Yuksel

2013-01-01

Sperm preservation protocols differ among animal species because of different sperm characteristics among species. Rat sperm have extreme sensitivity to suboptimal conditions in centrifugation, pipetting and chilling due to their longer tail, the shape and size of the sperm head, and membrane composition. The aim of this study was to determine optimal conditions for short-term storage of rat sperm by evaluating their motility and membrane and acrosomal integrity in response to various extender solutions, temperatures, and durations. Motility of rat sperm was highest when stored at 22 °C; motility was 28% and 14% at 72 h in TL-HEPES and PBS extenders, respectively. The motility and membrane integrity of rat sperm fell significantly within 24 h at 4 and 37 °C. Although cold storage did not have a detrimental effect on acrosomal integrity of sperm, room temperature storage reduced acrosomal integrity after 24 h. LEY extender caused the highest loss in acrosomal integrity at 48 and 72 h. In conclusion, storage at 4 or 37 °C reduced the motility and membrane integrity of rat sperm even with short incubation periods. Rat sperm stored in TL-HEPES or PBS remained motile for at least 3 d when held at 22 °C. PMID:24351761

Bovine sperm separation by Swim-up and density gradients (Percoll and BoviPure): Effect on sperm quality, function and gene expression.

PubMed

Arias, María Elena; Andara, Katherine; Briones, Evelyn; Felmer, Ricardo

2017-06-01

This study assesses the effect of bovine sperm (obtained from three bulls) separation using density gradients (Percoll and BoviPure) and Swim-up on sperm function and gene expression. Sperm evaluations included the plasma membrane integrity (SYBR14/PI), acrosomal integrity (PNA-FITC/PI), oxidative stress (ROS; CH2FDDA), DNA fragmentation (TUNEL assay) and mitochondrial membrane potential (ΔYm; TMRM) using flow cytometry. Sperm motility was evaluated by computer-assisted sperm analysis (CASA) and gene expression using RT-qPCR. The results showed that separation by Percoll achieves a higher proportion of sperm with intact plasma and acrosomal membranes (89.8 and 87.5%, respectively) than the unseparated control (70.3 and 62.4%, respectively), as well as by Swim-up (74.9 and 63.3%, respectively) and BoviPure (83.3 and 80.4%, respectively). No differences were observed in the proportion of spermatozoa with high ΔΨm between Percoll and BoviPure (84.3% and 83.5%, respectively), which were higher than Swim-up and the unseparated control (72.8% and 43.8%, respectively). The ROS levels were higher in the spermatozoa separated by Percoll and no differences were observed in the sperm DNA integrity between all groups. The motility analysis showed that the separation methods improve (p<0.05) total and progressive motility compared to the control, with Percoll proving the most efficient in this regard. Finally, the gene expression analysis of leptin (LEP), aromatase cytochrome P450 (CYP19) and protamine I (PRM1), after validation of 6 reference genes, showed no differences between groups. In conclusion, bovine sperm separation using density gradient improves the parameters of motility and sperm function without affecting the gene expression. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Novel Flow Cytometry Analyses of Boar Sperm Viability: Can the Addition of Whole Sperm-Rich Fraction Seminal Plasma to Frozen-Thawed Boar Sperm Affect It?

PubMed

Torres, Mariana Andrade; Díaz, Rommy; Boguen, Rodrigo; Martins, Simone Maria Massami Kitamura; Ravagnani, Gisele Mouro; Leal, Diego Feitosa; Oliveira, Melissa de Lima; Muro, Bruno Bracco Donatelli; Parra, Beatriz Martins; Meirelles, Flávio Vieira; Papa, Frederico Ozanan; Dell'Aqua, José Antônio; Alvarenga, Marco Antônio; Moretti, Aníbal de Sant'Anna; Sepúlveda, Néstor; de Andrade, André Furugen Cesar

2016-01-01

Boar semen cryopreservation remains a challenge due to the extension of cold shock damage. Thus, many alternatives have emerged to improve the quality of frozen-thawed boar sperm. Although the use of seminal plasma arising from boar sperm-rich fraction (SP-SRF) has shown good efficacy; however, the majority of actual sperm evaluation techniques include a single or dual sperm parameter analysis, which overrates the real sperm viability. Within this context, this work was performed to introduce a sperm flow cytometry fourfold stain technique for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential. We then used the sperm flow cytometry fourfold stain technique to study the effect of SP-SRF on frozen-thawed boar sperm and further evaluated the effect of this treatment on sperm movement, tyrosine phosphorylation and fertility rate (FR). The sperm fourfold stain technique is accurate (R2 = 0.9356, p > 0.01) for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential (IPIAH cells). Centrifugation pre-cryopreservation was not deleterious (p > 0.05) for any analyzed variables. Addition of SP-SRF after cryopreservation was able to improve total and progressive motility (p < 0.05) when boar semen was cryopreserved without SP-SRF; however, it was not able to decrease tyrosine phosphorylation (p > 0.05) or improve IPIAH cells (p > 0.05). FR was not (p > 0.05) statistically increased by the addition of seminal plasma, though females inseminated with frozen-thawed boar semen plus SP-SRF did perform better than those inseminated with sperm lacking seminal plasma. Thus, we conclude that sperm fourfold stain can be used to simultaneously evaluate plasma and acrosomal membrane integrity and mitochondrial membrane potential, and the addition of SP-SRF at thawed boar semen cryopreserved in absence of SP-SRF improve its total and progressive motility.

Novel Flow Cytometry Analyses of Boar Sperm Viability: Can the Addition of Whole Sperm-Rich Fraction Seminal Plasma to Frozen-Thawed Boar Sperm Affect It?

PubMed Central

Díaz, Rommy; Boguen, Rodrigo; Martins, Simone Maria Massami Kitamura; Ravagnani, Gisele Mouro; Leal, Diego Feitosa; Oliveira, Melissa de Lima; Muro, Bruno Bracco Donatelli; Parra, Beatriz Martins; Meirelles, Flávio Vieira; Papa, Frederico Ozanan; Dell’Aqua, José Antônio; Alvarenga, Marco Antônio; Moretti, Aníbal de Sant’Anna; Sepúlveda, Néstor

2016-01-01

Boar semen cryopreservation remains a challenge due to the extension of cold shock damage. Thus, many alternatives have emerged to improve the quality of frozen-thawed boar sperm. Although the use of seminal plasma arising from boar sperm-rich fraction (SP-SRF) has shown good efficacy; however, the majority of actual sperm evaluation techniques include a single or dual sperm parameter analysis, which overrates the real sperm viability. Within this context, this work was performed to introduce a sperm flow cytometry fourfold stain technique for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential. We then used the sperm flow cytometry fourfold stain technique to study the effect of SP-SRF on frozen-thawed boar sperm and further evaluated the effect of this treatment on sperm movement, tyrosine phosphorylation and fertility rate (FR). The sperm fourfold stain technique is accurate (R2 = 0.9356, p > 0.01) for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential (IPIAH cells). Centrifugation pre-cryopreservation was not deleterious (p > 0.05) for any analyzed variables. Addition of SP-SRF after cryopreservation was able to improve total and progressive motility (p < 0.05) when boar semen was cryopreserved without SP-SRF; however, it was not able to decrease tyrosine phosphorylation (p > 0.05) or improve IPIAH cells (p > 0.05). FR was not (p > 0.05) statistically increased by the addition of seminal plasma, though females inseminated with frozen-thawed boar semen plus SP-SRF did perform better than those inseminated with sperm lacking seminal plasma. Thus, we conclude that sperm fourfold stain can be used to simultaneously evaluate plasma and acrosomal membrane integrity and mitochondrial membrane potential, and the addition of SP-SRF at thawed boar semen cryopreserved in absence of SP-SRF improve its total and progressive motility. PMID:27529819

Influence of different anaesthetic protocols over the sperm quality on the fresh, chilled (4°C) and frozen-thawed epididymal sperm samples in domestic dogs.

PubMed

Batista, M; Vilar, J; Rosario, I; Terradas, E

2016-10-01

This study assessed the influence of three different anaesthetic protocols on semen quality obtained from the epididymis. Sixty male dogs undergoing to routine sterilization were assigned to three anaesthetic protocols: thiopental group (TG, n = 20), propofol group (PG, n = 20) and ketamine-dexmedetomidine group (KDG, n = 20). Immediately after orchidectomy, the cauda epididymides and vas deferent ducts were isolated and then a retrograde flushing was performed to collect spermatozoa. In experiment 1, after the initial evaluation of the semen (sperm concentration, sperm motility and the percentages of live spermatozoa, abnormal spermatozoa and acrosome membrane integrity), semen samples were diluted in Tris-glucose-egg yolk extender and chilled for 48 hr, and the sperm motility was assessed at 6, 24 and 48 hr. In experiment 2, semen samples were diluted in Tris-glucose-egg yolk extender and chilled for 24 hr, and then samples were frozen in two extenders with different glycerol concentrations, to reach a final concentration of 50-100 × 10(6) spermatozoa ml(-1) , 20% egg yolk, 0.5% Equex and 4% and 5% glycerol, respectively. Mean values of total sperm concentration, sperm viability and the percentages of intact acrosome and abnormal spermatozoa were not significantly different between experimental groups, and therefore, the anaesthetic protocols assessed did not affect sperm parameters mentioned above. However, our study confirmed a detrimental effect of the use of thiopental (TG) over the total sperm motility (p < 0.05) and progressive sperm motility (p < 0.05) of the fresh and chilled epididymal sperm samples. The anaesthetic protocols including the application of propofol or ketamine-dexmedetomidine can be used to recover sperm in domestic canids without significant changes in sperm quality compared when semen is collected routinely and these techniques could be applicable to endangered wild canids. © 2016 Blackwell Verlag GmbH.

In vitro sperm characterization and development of a sperm cryopreservation method using directional solidification in the killer whale (Orcinus orca).

PubMed

Robeck, T R; Gearhart, S A; Steinman, K J; Katsumata, E; Loureiro, J D; O'Brien, J K

2011-07-15

Research was conducted to characterize seminal traits and to develop a sperm cryopreservation method using directional freezing (DF) for the killer whale (Orcinus orca). Experiments evaluated effects of: (i) freezing rate (SLOW, MED, FAST) by diluent (BF5F, Biladyl®, EYC) in 0.5 mL straws; and (ii) freezing method (straw or DF) by glycerol (3, 6, or 9% final concentration, v:v) on in vitro sperm quality. Fresh ejaculates (n = 161) were (mean ± SD) 7.8 ± 7.4 mL at 740 × 10(6) sperm/mL with 92.2 ± 6.3% total motility (TM), 85.4 ± 6.9% progressive motility (PM), 89.6 ± 9.0% viability and 89.8 ± 9.2% acrosome integrity. Samples frozen using straws by the MED or SLOW method were improved (P < 0.05) over FAST across all diluents. At 3 h post thaw (PT), TM, PM, Rapid motility (RM), VAP, VCL, ALH and viability for 3% and 6% glycerol were improved (P < 0.05) over 9% glycerol. Directional freezing samples at 0 h and 3 h PT, at all glycerol concentrations, displayed higher (P < 0.001) TM, PM, RM, VAP, VSL, VCL and viability /intact acrosomes (PI/FITC-PNA) than straw. These data provided the first information on ejaculate characteristics and the development of a semen cryopreservation method using DF in the killer whale. Copyright © 2011 Elsevier Inc. All rights reserved.

Sperm quality variables as indicators of bull fertility may be breed dependent.

PubMed

Morrell, Jane M; Nongbua, Thanapol; Valeanu, Sabina; Lima Verde, Isabel; Lundstedt-Enkel, Katrin; Edman, Anders; Johannisson, Anders

2017-10-01

A means of discriminating among bulls of high fertility based on sperm quality is needed by breeding centers. The objective of the study was to examine parameters of sperm quality in bulls of known fertility to identify useful indicators of fertility. Frozen semen was available from bulls of known fertility (Viking Genetics, Skara, Sweden): Swedish Red (n=31), Holstein (n=25) and Others (one each of Charolais, Limousin, Blonde, SKB). After thawing, the sperm samples were analyzed for motility (computer assisted sperm analysis), plasma membrane integrity, chromatin integrity, acrosome status, mitochondrial activity and reactive oxygen species. A fertility index score based on the adjusted 56-day non-return rate for >1000 inseminations was available for each bull. Multivariate data analysis (Partial Least Squares Regression and Orthogonal Partial Least Squares Regression) was performed to identify variables related to fertility; Pearson univariate correlations were made on the parameters of interest. Breed of bull affected the relationship of sperm quality variables and fertility index score, as follows: Swedish Red: %DNA Fragmentation Index, r=-0.56, P<0.01; intact plasma membrane, r=0.40, P<0.05; membrane damaged, not acrosome reacted, r=-0.6, P<0.01; Linearity, r=0.37, P<0.05; there was a trend towards significance for Wobble, r=0.34, P=0.08. Holstein: Linearity was significant r=0.46, P<0.05; there was a trend towards significance for Wobble, r=0.45, P=0.08. In conclusion, breed has a greater effect on sperm quality than previously realized; different parameters of sperm quality are needed to indicate potential fertility in different breeds. Copyright © 2017 Elsevier B.V. All rights reserved.

Versatile Action of Picomolar Gradients of Progesterone on Different Sperm Subpopulations

PubMed Central

Uñates, Diego Rafael; Guidobaldi, Héctor Alejandro; Gatica, Laura Virginia; Cubilla, Marisa Angélica; Teves, María Eugenia; Moreno, Ayelén; Giojalas, Laura Cecilia

2014-01-01

High step concentrations of progesterone may stimulate various sperm physiological processes, such as priming and the acrosome reaction. However, approaching the egg, spermatozoa face increasing concentrations of the hormone, as it is secreted by the cumulus cells and then passively diffuses along the cumulus matrix and beyond. In this context, several questions arise: are spermatozoa sensitive to the steroid gradients as they undergo priming and the acrosome reaction? If so, what are the functional gradual concentrations of progesterone? Do spermatozoa in different physiological states respond differentially to steroid gradients? To answer these questions, spermatozoa were confronted with progesterone gradients generated by different hormone concentrations (1 pM to 100 µM). Brief exposure to a 10 pM progesterone gradient stimulated priming for the acrosome reaction in one sperm subpopulation, and simultaneously induced the acrosome reaction in a different sperm subpopulation. This effect was not observed in non-capacitated cells or when progesterone was homogeneously distributed. The results suggest a versatile role of the gradual distribution of very low doses of progesterone, which selectively stimulate the priming and the acrosome reaction in different sperm subpopulations. PMID:24614230

Puma (Puma concolor) epididymal sperm morphometry.

PubMed

Cucho, Hernán; Alarcón, Virgilio; Ordóñez, César; Ampuero, Enrique; Meza, Aydee; Soler, Carles

2016-01-01

The Andean puma (Puma concolor) has not been widely studied, particularly in reference to its semen characteristics. The aim of the present study was to define the morphometry of puma sperm heads and classify their subpopulations by cluster analysis. Samples were recovered postmortem from two epididymides from one animal and prepared for morphological observation after staining with the Hemacolor kit. Morphometric data were obtained from 581 spermatozoa using a CASA-Morph system, rendering 13 morphometric parameters. The principal component (PC) analysis was performed followed by cluster analysis for the establishment of subpopulations. Two PC components were obtained, the first related to size and the second to shape. Three subpopulations were observed, corresponding to elongated and intermediate-size sperm heads and acrosomes, to large heads with large acrosomes, and to small heads with short acrosomes. In conclusion, puma spermatozoa showed no uniform sperm morphology but three clear subpopulations. These results should be used for future work in the establishment of an adequate germplasm bank of this species.

Loss of the Na+/H+ exchanger NHE8 causes male infertility in mice by disrupting acrosome formation.

PubMed

Oberheide, Karina; Puchkov, Dmytro; Jentsch, Thomas J

2017-06-30

Mammalian sperm feature a specialized secretory organelle on the anterior part of the sperm nucleus, the acrosome, which is essential for male fertility. It is formed by a fusion of Golgi-derived vesicles. We show here that the predominantly Golgi-resident Na + /H + exchanger NHE8 localizes to the developing acrosome of spermatids. Similar to wild-type mice, Nhe8 -/- mice generated Golgi-derived vesicles positive for acrosomal markers and attached to nuclei, but these vesicles failed to form large acrosomal granules and the acrosomal cap. Spermatozoa from Nhe8 -/- mice completely lacked acrosomes, were round-headed, exhibited abnormal mitochondrial distribution, and displayed decreased motility, resulting in selective male infertility. Of note, similar features are also found in globozoospermia, one of the causes of male infertility in humans. Germ cell-specific, but not Sertoli cell-specific Nhe8 disruption recapitulated the globozoospermia phenotype, demonstrating that NHE8's role in spermiogenesis is germ cell-intrinsic. Our work has uncovered a crucial role of NHE8 in acrosome biogenesis and suggests that some forms of human globozoospermia might be caused by a loss of function of this Na + /H + exchanger. It points to NHE8 as a candidate gene for human globozoospermia and a possible drug target for male contraception. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Genome-Wide Association Study Implicates Testis-Sperm Specific FKBP6 as a Susceptibility Locus for Impaired Acrosome Reaction in Stallions

PubMed Central

Raudsepp, Terje; Dobson, Lauren; Vishnoi, Monika; Fritz, Krista L.; Schaefer, Robert; Rendahl, Aaron K.; Derr, James N.; Love, Charles C.; Varner, Dickson D.; Chowdhary, Bhanu P.

2012-01-01

Impaired acrosomal reaction (IAR) of sperm causes male subfertility in humans and animals. Despite compelling evidence about the genetic control over acrosome biogenesis and function, the genomics of IAR is as yet poorly understood, providing no molecular tools for diagnostics. Here we conducted Equine SNP50 Beadchip genotyping and GWAS using 7 IAR–affected and 37 control Thoroughbred stallions. A significant (P<6.75E-08) genotype–phenotype association was found in horse chromosome 13 in FK506 binding protein 6 (FKBP6). The gene belongs to the immunophilins FKBP family known to be involved in meiosis, calcium homeostasis, clathrin-coated vesicles, and membrane fusions. Direct sequencing of FKBP6 exons in cases and controls identified SNPs g.11040315G>A and g.11040379C>A (p.166H>N) in exon 4 that were significantly associated with the IAR phenotype both in the GWAS cohort (n = 44) and in a large multi-breed cohort of 265 horses. All IAR stallions were homozygous for the A-alleles, while this genotype was found only in 2% of controls. The equine FKBP6 was exclusively expressed in testis and sperm and had 5 different transcripts, of which 4 were novel. The expression of this gene in AC/AG heterozygous controls was monoallelic, and we observed a tendency for FKBP6 up-regulation in IAR stallions compared to controls. Because exon 4 SNPs had no effect on the protein structure, it is likely that FKBP6 relates to the IAR phenotype via regulatory or modifying functions. In conclusion, FKBP6 was considered a susceptibility gene of incomplete penetrance for IAR in stallions and a candidate gene for male subfertility in mammals. FKBP6 genotyping is recommended for the detection of IAR–susceptible individuals among potential breeding stallions. Successful use of sperm as a source of DNA and RNA propagates non-invasive sample procurement for fertility genomics in animals and humans. PMID:23284302

Assessment of basic seminal characteristics, sperm cryopreservation and heterologous in vitro fertilisation in the fishing cat (Prionailurus viverrinus).

PubMed

Thiangtum, Khongsak; Swanson, William F; Howard, JoGayle; Tunwattana, Wanchai; Tongthainan, Dakara; Wichasilpa, Wisid; Patumrattanathan, Pornchai; Pinyopoommintr, Tanu

2006-01-01

Conservation of the fishing cat, a threatened south-east Asian felid, could benefit from effective ex situ genetic management and breeding programmes, including the use of assisted reproduction. The aims of the present study were to: (1) characterise basal seminal traits of fishing cats in Thailand zoos; and (2) investigate the effect of cryopreservation on sperm motility, acrosomal integrity and in vitro function. Seminal traits were evaluated in electroejaculates collected from eight males. Spermatozoa were diluted in n-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid Tris (TEST)-yolk buffer (TYB) without glycerol, then diluted further with TYB with glycerol (4% final concentration) at either 25 degrees C or after slow cooling to 5 degrees C and frozen in straws over liquid nitrogen vapour. After thawing, sperm function was assessed by insemination of viable domestic cat oocytes. Fishing cat ejaculates averaged (+/- s.e.m.) 43.6 +/- 14.2 x 10(6) motile spermatozoa with 33.5 +/- 6.8% normal sperm morphology. Semen processing had a negligible effect (P > 0.05) on sperm motility and acrosomal integrity, but values were reduced (P < 0.05) after thawing. All thawed samples fertilised domestic cat oocytes, with 62.1% (36/58) of mature oocytes cleaving. Glycerol addition at 5 degrees C resulted in higher (P < 0.05) post-thaw motility and intact acrosomes than glycerol addition at 25 degrees C. In conclusion, good-quality ejaculates can be obtained from Thai fishing cats and their spermatozoa exhibit adequate function after cryopreservation for in vitro fertilisation procedures.

Pig Spermatozoa Defect in Acrosome Formation Caused Poor Motion Parameters and Fertilization Failure through Artificial Insemination and In vitro Fertilization.

PubMed

Lee, Won Young; Lee, Ran; Kim, Hee Chan; Lee, Kyung Hoon; Cui, Xiang Shun; Kim, Nam Hyung; Kim, Sang Hyun; Lee, Il Joo; Uhm, Sang Jun; Yoon, Min Jung; Song, Hyuk

2014-10-01

The selection of morphologically normal spermatozoa is critical to obtain high breeding performances in boar breeding farms and artificial insemination (AI) centers. Parameters for the selection of semen mainly include total sperm motility, concentration, and morphology. However, these primary parameters are often not reliable for discriminating between normal and abnormal, non-fertilizable spermatozoa. The present study was designed to compare the motion characteristics, fertilization ability using in vitro fertilization (IVF), and acrosome formation of the semen from boars having low (boar number 2012) and normal (boar number 2004 and 2023) breeding performances. The ultimate goal was to identify additional simple and easy criteria for the selection of normal sperm. There was no significant difference between boar 2004 and boar 2023 sperm total motility in computer assisted sperm analysis. However, boar number 2012 semen presented a significantly reduced population of rapid moving spermatozoa and an increased population of slow moving spermatozoa compared to boar numbers 2004 and 2023. Analysis of detailed motion characteristics revealed that sperm from boar number 2012 had significantly reduced motility in progressiveness, average path velocity, straight-line velocity (VSL), curvilinear velocity (VCL), straightness, and linearity. The assessment of the fertilizing ability by IVF also showed that sperm from boar number 2012 showed a fertility rate of 3.4%, whereas sperm from boar number 2023 had a fertility rate of 75.45%. Interestingly, most of the sperm nuclei were found on the peripheral area of the oocytes, suggesting that the sperm from boar number 2012 lacked penetration ability into the oocyte zonapellucida. The acrosome formation analysis using Pisum sativum agglutinin staining demonstrated that the sperm from boar number 2012 had a defect in acrosome formation. Consequently, primary parameters for selecting semen before AI such as motility are not

Pig Spermatozoa Defect in Acrosome Formation Caused Poor Motion Parameters and Fertilization Failure through Artificial Insemination and In vitro Fertilization

PubMed Central

Lee, Won Young; Lee, Ran; Kim, Hee Chan; Lee, Kyung Hoon; Cui, Xiang Shun; Kim, Nam Hyung; Kim, Sang Hyun; Lee, Il Joo; Uhm, Sang Jun; Yoon, Min Jung; Song, Hyuk

2014-01-01

The selection of morphologically normal spermatozoa is critical to obtain high breeding performances in boar breeding farms and artificial insemination (AI) centers. Parameters for the selection of semen mainly include total sperm motility, concentration, and morphology. However, these primary parameters are often not reliable for discriminating between normal and abnormal, non-fertilizable spermatozoa. The present study was designed to compare the motion characteristics, fertilization ability using in vitro fertilization (IVF), and acrosome formation of the semen from boars having low (boar number 2012) and normal (boar number 2004 and 2023) breeding performances. The ultimate goal was to identify additional simple and easy criteria for the selection of normal sperm. There was no significant difference between boar 2004 and boar 2023 sperm total motility in computer assisted sperm analysis. However, boar number 2012 semen presented a significantly reduced population of rapid moving spermatozoa and an increased population of slow moving spermatozoa compared to boar numbers 2004 and 2023. Analysis of detailed motion characteristics revealed that sperm from boar number 2012 had significantly reduced motility in progressiveness, average path velocity, straight-line velocity (VSL), curvilinear velocity (VCL), straightness, and linearity. The assessment of the fertilizing ability by IVF also showed that sperm from boar number 2012 showed a fertility rate of 3.4%, whereas sperm from boar number 2023 had a fertility rate of 75.45%. Interestingly, most of the sperm nuclei were found on the peripheral area of the oocytes, suggesting that the sperm from boar number 2012 lacked penetration ability into the oocyte zonapellucida. The acrosome formation analysis using Pisum sativum agglutinin staining demonstrated that the sperm from boar number 2012 had a defect in acrosome formation. Consequently, primary parameters for selecting semen before AI such as motility are not

EDC IMPACT: Chemical UV filters can affect human sperm function in a progesterone-like manner

PubMed Central

Rehfeld, A; Egeberg, D L; Almstrup, K; Petersen, J H; Dissing, S

2018-01-01

Human sperm cell function must be precisely regulated to achieve natural fertilization. Progesterone released by the cumulus cells surrounding the egg induces a Ca2+ influx into human sperm cells via the CatSper Ca2+-channel and thereby controls sperm function. Multiple chemical UV filters have been shown to induce a Ca2+ influx through CatSper, thus mimicking the effect of progesterone on Ca2+ signaling. We hypothesized that these UV filters could also mimic the effect of progesterone on sperm function. We examined 29 UV filters allowed in sunscreens in the US and/or EU for their ability to affect acrosome reaction, penetration, hyperactivation and viability in human sperm cells. We found that, similar to progesterone, the UV filters 4-MBC, 3-BC, Meradimate, Octisalate, BCSA, HMS and OD-PABA induced acrosome reaction and 3-BC increased sperm penetration into a viscous medium. The capacity of the UV filters to induce acrosome reaction and increase sperm penetration was positively associated with the ability of the UV filters to induce a Ca2+ influx. None of the UV filters induced significant changes in the proportion of hyperactivated cells. In conclusion, chemical UV filters that mimic the effect of progesterone on Ca2+ signaling in human sperm cells can similarly mimic the effect of progesterone on acrosome reaction and sperm penetration. Human exposure to these chemical UV filters may impair fertility by interfering with sperm function, e.g. through induction of premature acrosome reaction. Further studies are needed to confirm the results in vivo. PMID:28874401

EDC IMPACT: Chemical UV filters can affect human sperm function in a progesterone-like manner.

PubMed

Rehfeld, A; Egeberg, D L; Almstrup, K; Petersen, J H; Dissing, S; Skakkebæk, N E

2018-01-01

Human sperm cell function must be precisely regulated to achieve natural fertilization. Progesterone released by the cumulus cells surrounding the egg induces a Ca 2+ influx into human sperm cells via the CatSper Ca 2+ -channel and thereby controls sperm function. Multiple chemical UV filters have been shown to induce a Ca 2+ influx through CatSper, thus mimicking the effect of progesterone on Ca 2+ signaling. We hypothesized that these UV filters could also mimic the effect of progesterone on sperm function. We examined 29 UV filters allowed in sunscreens in the US and/or EU for their ability to affect acrosome reaction, penetration, hyperactivation and viability in human sperm cells. We found that, similar to progesterone, the UV filters 4-MBC, 3-BC, Meradimate, Octisalate, BCSA, HMS and OD-PABA induced acrosome reaction and 3-BC increased sperm penetration into a viscous medium. The capacity of the UV filters to induce acrosome reaction and increase sperm penetration was positively associated with the ability of the UV filters to induce a Ca 2+ influx. None of the UV filters induced significant changes in the proportion of hyperactivated cells. In conclusion, chemical UV filters that mimic the effect of progesterone on Ca 2+ signaling in human sperm cells can similarly mimic the effect of progesterone on acrosome reaction and sperm penetration. Human exposure to these chemical UV filters may impair fertility by interfering with sperm function, e.g. through induction of premature acrosome reaction. Further studies are needed to confirm the results in vivo . © 2018 The authors.

Influence of Boar and Semen Parameters on Motility and Acrosome Integrity in Liquid Boar Semen Stored for Five Days

PubMed Central

2002-01-01

Ninety ejaculates from a total of 76 AI boars were extended in Beltsville Thawing Solution (BTS). Boar identity, breed, weight of the ejaculate and sperm concentration were registered. Motility and acrosome integrity were assessed after storage at 16–18°C for 6, 30, 54, 78, and 102 h. Storage time had a significant influence on both motility (p < 0.01) and acrosome integrity (p < 0.001). The Least Square Means for percentage of motility showed a small decline from 79.8% after 6 h of storage to 78.4% at 102 h. Motility at 78 and 102 h was significantly different from motility at 6 h (p < 0.05). The percentage of sperm cells with normal acrosomes declined throughout the experiment. The Least Square Means for 6, 30, 54, 78, and 102 h of storage were 93.9%, 90.6%, 88.0%, 84.8%, and 78.2%, respectively. The decrease in acrosome integrity from one storage time to the next was highly significant throughout the trial (p < 0.001). There was a significant influence of boar (p < 0.001) and sperm concentration (p < 0.01) on motility, while acrosome integrity was affected only by boar (p < 0.001). Breed of the boars and weight of the ejaculate did not influence the dependent variables. PMID:12071116

Collection and evaluation of epididymal sperm in captive agoutis (Dasyprocta aguti).

PubMed

Ferraz, M S; de Menezes, D J A; Pessoa, G T; Cabral, R M; Illera, M J; Silva, A R; Carvalho, M A M

2011-02-01

The objective was to establish a protocol for the collection and evaluation of epididymal sperm in agoutis. Eight males (1-2 y old) underwent left orchidectomy and epididymal sperma were collected by retrograde flush. Average values were flush volume 32 μL, pH 6.9, sperm concentration 748 x 10(6) sperm/mL, with motility 86.5% and vigor 4.6. Viable sperm were present in all flush samples; 66% of sperm were alive, and 41.9% of sperm responded positively to the hypoosmotic test (using distilled water). There were 21.1% morphologically abnormal sperm, of which 2.0 and 19.1% were primary and secondary defects, respectively. The acrosome was intact in 99.5% of sperm. The sperm head was 4.89 ± 0.41 μm long and 3.13 ± 0.35 μm wide, with an area of 13.01 ± 2.01 μm(2). Midpieces were 5.33 ± 0.44 μm long and 0.98 ± 0.13 wide, sperm tails were 29.91 ± 2.29 μm, and overall sperm length was 40.12 ± 2.44 μm. In conclusion, epididymal sperm collection from agoutis was satisfactory; the collected sperm has the potential to be stored, facilitating development of other reproductive biotechnologies for this species. Copyright © 2011 Elsevier Inc. All rights reserved.

Ca2+-stores in sperm: their identities and functions

PubMed Central

Costello, Sarah; Michelangeli, Francesco; Nash, Kate; Lefievre, Linda; Morris, Jennifer; Machado-Oliveira, Gisela; Barratt, Christopher; Kirkman-Brown, Jackson; Publicover, Stephen

2013-01-01

Intracellular Ca2+ stores play a central role in the regulation of cellular [Ca2+]i and the generation of complex [Ca2+] signals such as oscillations and waves. Ca2+ signalling is of particular significance in sperm cells, where it is a central regulator in many key activities (including capacitation, hyperactivation, chemotaxis and acrosome reaction) yet mature sperm lack endoplasmic reticulum and several other organelles which serve as Ca2+ stores in somatic cells. Here we review (i) the evidence for the expression in sperm of the molecular components (pumps and channels) which are functionally significant in the activity of Ca2+ stores of somatic cells and (ii) the evidence for the existence of functional Ca2+ stores in sperm. This evidence supports the existence of at least two storage organelles in mammalian sperm, one in the acrosomal region and another in the region of the sperm neck and midpiece. We then go on to discuss the likely identity of these organelles and their discrete functions: regulation by the acrosome of its own secretion and regulation by membranous organelles at the sperm neck (and possibly by the mitochondria) of flagellar activity and hyperactivation. Finally we consider the ability of the sperm discretely to control mobilisation of these stores and the functional interaction of stored Ca2+ at the sperm neck/midpiece with CatSper channels in the principal piece in regulation of the activities of mammalian sperm. PMID:19542252

Puma (Puma concolor) epididymal sperm morphometry

PubMed Central

Cucho, Hernán; Alarcón, Virgilio; Ordóñez, César; Ampuero, Enrique; Meza, Aydee; Soler, Carles

2016-01-01

The Andean puma (Puma concolor) has not been widely studied, particularly in reference to its semen characteristics. The aim of the present study was to define the morphometry of puma sperm heads and classify their subpopulations by cluster analysis. Samples were recovered postmortem from two epididymides from one animal and prepared for morphological observation after staining with the Hemacolor kit. Morphometric data were obtained from 581 spermatozoa using a CASA-Morph system, rendering 13 morphometric parameters. The principal component (PC) analysis was performed followed by cluster analysis for the establishment of subpopulations. Two PC components were obtained, the first related to size and the second to shape. Three subpopulations were observed, corresponding to elongated and intermediate-size sperm heads and acrosomes, to large heads with large acrosomes, and to small heads with short acrosomes. In conclusion, puma spermatozoa showed no uniform sperm morphology but three clear subpopulations. These results should be used for future work in the establishment of an adequate germplasm bank of this species. PMID:27678466

[Expression and localization of transmembrane protein CMTM2 in human testis and sperm].

PubMed

Zhang, X W; Lan, K; Yang, W B; Li, Q; Zhao, Y P; Yin, H Q; Kite, B; Bai, W J; Xu, T

2017-08-18

To study the expression of transmembrane protein CMTM2 in the testis and sperm of adult males and to approach the potential function of the protein in the male reproductive system. The expression of CMTM2 in human testis and sperm was confirmed by Western blot. Immunohistochemical staining was used for detecting CMTM2 localization in the testis tissue, TRITC-CMTM2 and FITC-Hoechst double immunofluorescence staining was performed to examine the subcellular localization of CMTM2 in the human sperm before and after acrosome reaction, that is, immunofluorescent staining was used for detecting CMTM2 localization in both the testis and sperm before and after the acrosome reaction. CMTM2 was presented in both human testis and sperm. In the testis, CMTM2 immunoreactive particles were observed mainly in the membrane of the different stages of spermatogenic cells. In the human sperm, its immunoreactivity was restrictively localized to the posterior head where sperm-egg fusion occurred, and the CMTM2 localization was not affected by sperm acrosome reaction. CMTM2 was widely expressed in seminiferous tubules of the human testis, mainly in the cell membranes of spermatogenic cells, which was consistent with the previous reports. The immunofluorescence performed on frozen human testis slides showed similar findings with immunohistochemistry, which gave weight to the localization of CMTM2 in the cell membranes of spermatogenic cells at different stages. TRITC-CMTM2 and FITC-Hoechst double immunofluorescence staining was performed to examine the subcellular localization of CMTM2 in the human sperm before and after acrosome reaction. CMTM2 was localized at the posterior head of sperm before and after acrosome reaction. The localization and expression of CMTM2 were not affected by sperm acrosome reaction. Expression of CMTM2 in the male reproductive system of the adult human exhibits cell- and region-specific patterns, which suggests that they may play an important role in

Delta opioid receptor on equine sperm cells: subcellular localization and involvement in sperm motility analyzed by computer assisted sperm analyzer (CASA)

PubMed Central

2010-01-01

Background Opioid receptors and endogenous opioid peptides act not only in the control of nociceptive pathways, indeed several reports demonstrate the effects of opiates on sperm cell motility and morphology suggesting the importance of these receptors in the modulation of reproduction in mammals. In this study we investigated the expression of delta opioid receptors on equine spermatozoa by western blot/indirect immunofluorescence and its relationship with sperm cell physiology. Methods We analyzed viability, motility, capacitation, acrosome reaction and mitochondrial activity in the presence of naltrindole and DPDPE by means of a computer assisted sperm analyzer and a fluorescent confocal microscope. The evaluation of viability, capacitation and acrosome reaction was carried out by the double CTC/Hoechst staining, whereas mitochondrial activity was assessed by means of MitoTracker Orange dye. Results We showed that in equine sperm cells, delta opioid receptor is expressed as a doublet of 65 and 50 kDa molecular mass and is localized in the mid piece of tail; we also demonstrated that naltrindole, a delta opioid receptor antagonist, could be utilized in modulating several physiological parameters of the equine spermatozoon in a dose-dependent way. We also found that low concentrations of the antagonist increase sperm motility whereas high concentrations show the opposite effect. Moreover low concentrations hamper capacitation, acrosome reaction and viability even if the percentage of cells with active mitochondria seems to be increased; the opposite effect is exerted at high concentrations. We have also observed that the delta opioid receptor agonist DPDPE is scarcely involved in affecting the same parameters at the employed concentrations. Conclusions The results described in this paper add new important details in the comprehension of the mammalian sperm physiology and suggest new insights for improving reproduction and for optimizing equine breeding. PMID

Correlation analysis of the progesterone-induced sperm acrosome reaction rate and the fertilisation rate in vitro.

PubMed

Jiang, T; Qin, Y; Ye, T; Wang, Y; Pan, J; Zhu, Y; Duan, L; Li, K; Teng, X

2015-10-01

In this study, we aimed to investigate whether progesterone-induced acrosome reaction (AR) rate could be an indicator for fertilisation rate in vitro. Twenty-six couples with unexplained infertility and undergoing in vitro fertilisation (IVF) treatment were involved. On the oocytes retrieval day after routine IVF, residual sperm samples were collected to receive progesterone induction (progesterone group) or not (control group). AR rate was calculated and fertilisation rate was recorded. The correlation between progesterone-induced AR and fertilisation rate and between sperm normal morphology and 3PN (tripronuclear) were analysed using the Spearman correlation analysis. The AR rate of progesterone group was statistically higher than that of the control group (15.6 ± 5.88% versus 9.66 ± 5.771%, P < 0.05), but not significantly correlated with fertilisation rate (r = -0.053, P > 0.01) or rate of high-quality embryo development (r = -0.055, P > 0.01). Normal sperm morphology also showed no significant correlation with the amount of 3PN zygotes (r = 0.029, P > 0.01), rate of 3PN zygotes production (r = 0.20, P > 0.01), rate of 3PN embryo development (r = -0.406, P > 0.01), fertilisation rate (r = -0.148, P > 0.01) or progesterone-induced AR rate (r = 0.214, P > 0.01). Progesterone can induce AR in vitro significantly; however, the progesterone-induced AR may not be used to indicate fertilisation rate. © 2014 Blackwell Verlag GmbH.

Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein

PubMed Central

Jagadish, Nirmala; Rana, Ritu; Selvi, Ramasamy; Mishra, Deepshikha; Garg, Manoj; Yadav, Shikha; Herr, John C.; Okumura, Katsuzumi; Hasegawa, Akiko; Koyama, Koji; Suri, Anil

2005-01-01

We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561–565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an α-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38α or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa–egg interaction. PMID:15693750

Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein.

PubMed

Jagadish, Nirmala; Rana, Ritu; Selvi, Ramasamy; Mishra, Deepshikha; Garg, Manoj; Yadav, Shikha; Herr, John C; Okumura, Katsuzumi; Hasegawa, Akiko; Koyama, Koji; Suri, Anil

2005-07-01

We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561-565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an alpha-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38alpha or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa-egg interaction.

TRPM8, a Versatile Channel in Human Sperm

PubMed Central

Ocampo, Ana Y.; Serrano, Carmen J.; Castellano, Laura E.; Hernández-González, Enrique O.; Chirinos, Mayel; Larrea, Fernando; Beltrán, Carmen; Treviño, Claudia L.

2009-01-01

Background The transient receptor potential channel (TRP) family includes more than 30 proteins; they participate in various Ca2+ dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca2+ ([Ca2+]i). Ca2+ triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored. Principal Findings Here we document with RT-PCR, western blot and immunocitochemistry analysis the presence of TRPM8 in human sperm. We also examined the participation of this channel in sperm function using specific agonists (menthol and temperature) and antagonists (BCTC and capsazepine). Computer-aided sperm analysis revealed that menthol did not significantly alter human sperm motility. In contrast, menthol induced the acrosome reaction in human sperm. This induction was inhibited about 70% by capsazepine (20 µM) and 80% by BCTC (1.6 µM). Activation of TRPM8 either by temperature or menthol induced [Ca2+]i increases in human sperm measured by fluorescence in populations or individual sperm cells, effect that was also inhibited by capsazepine (20 µM) and BCTC (1.6 µM). However, the progesterone and ZP3-induced acrosome reaction was not inhibited by capsazepine or BCTC, suggesting that TRPM8 activation triggers this process by a different signaling pathway. Conclusions This is the first report dealing with the presence of a thermo sensitive channel (TRPM8) in human sperm. This channel could be involved in cell signaling events such as thermotaxis or chemotaxis. PMID:19582168

Effects of hypothermic liquid storage and cryopreservation on basal and induced plasma membrane phospholipid disorder and acrosome exocytosis in boar spermatozoa.

PubMed

Guthrie, H D; Welch, G R

2005-01-01

Flow cytometry was utilised to determine whether short-term (Day 1) or long-term hypothermic liquid storage (Day 5), or cryopreservation of boar spermatozoa (1) caused changes in plasma membrane phospholipid disorder (MPLD) and acrosome exocytosis (AE), indicative of an advanced stage of capacitation or acrosome status, and (2) facilitated or inhibited the induction of capacitation and the acrosome reaction. Merocyanine with Yo-Pro-1 and peanut agglutinin-fluorescein isothiocyanate with propidium iodide were used to identify MPLD and AE, respectively, in viable spermatozoa. The incidence of basal sperm MPLD and AE in fresh semen was very low (1.1 and 2.2%, respectively) and was increased (P < 0.05) only a small amount in Day 5 and cryopreserved semen (3-8%). Compared to no bicarbonate, incubation with bicarbonate increased MPLD, but the response was greatest (P < 0.05) in fresh sperm (52.3%) compared with Day 1 (36.6%), Day 5 (13.9%) and cryopreserved sperm (13.6%). Incubation with calcium ionophore A23187 increased AE in spermatozoa, but the response was less (P < 0.05) for fresh (34%) and cryopreserved (27%) semen than for Day 1 (45%) and Day 5 (57%) semen. In summary, hypothermic liquid storage and cryopreservation of boar spermatozoa did not advance capacitation or acrosome status in viable spermatozoa, but did alter their responses to induction of capacitation and the acrosome reaction.

Ejaculate quality and constraints in relation to sperm competition levels among eutherian mammals.

PubMed

Lüpold, Stefan

2013-10-01

The outcome of sperm competition is influenced by the relative quantity and quality of sperm among competing ejaculates. Whereas it is well established that individual ejaculate traits evolve rapidly under postcopulatory sexual selection, little is known about other factors that might influence the evolution of ejaculates. For example, the metabolic rate is likely to affect the sperm production rate and the cellular activity or metabolism of sperm, and it has recently been suggested to constrain the evolution of sperm length in large but not small mammals. I thus examined in eutherian mammals how ejaculate quality traits vary with one another and with testis mass, body size, and metabolism. I found all ejaculate traits to covary positively with one another and to increase with relative testis mass. When controlling for testis mass, small-bodied species showed superior sperm quality (but not sperm number). Furthermore, sperm motility and viability were positively associated with the mass-corrected metabolic rate, but the percentage of morphologically normal and acrosome-intact sperm were not. These results indicate that body size and the energy budget may also influence the evolution of ejaculate quality, although these influences appear to vary among traits. © 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.

Comparison of the frequency of defective sperm-zona pellucida (ZP) binding and the ZP-induced acrosome reaction between subfertile men with normal and abnormal semen.

PubMed

Liu, De Yi; Liu, Ming Li; Garrett, Claire; Baker, H W Gordon

2007-07-01

The aim of this study was to compare the frequency of defective sperm-zona pellucida (ZP) binding (DSZPB) and defective ZP-induced acrosome reaction (DZPIAR) in subfertile men (i.e. male partners of infertile couples) with normal and abnormal semen analyses. A total of 1030 subfertile men with normal semen analysis (n=255), oligozoospermia (count<20x10(6)/ml, n=136), severe teratozoospermia (strict normal morphologysperm injection were used for sperm-ZP interaction tests. After 2 h incubation of motile sperm with four oocytes, sperm tightly bound to the ZP, and the AR of ZP-bound sperm (ZPIAR) were assessed. An average of <40 sperm bound/ZP and <16% ZPIAR were used for diagnosis of DSZPB or DZPIAR. For the groups of men with normal semen or mild-moderate teratozoospermia, severe teratozoospermia and oligozoospermia, the frequencies of DSZPB were: 13, 21, 29 and 28%, respectively, and in those normal SZPB, DZPIAR were 27, 36, 56 and 68%, respectively. Overall DSZPB and ZPIAR were 36, 49, 68 and 77% for the four groups, respectively. The highest frequencies of defective sperm-ZP interaction were in the oligozoospermia and severe teratozoospermia groups. In the normal and teratozoospermia groups, subjects with a relatively low sperm concentration (20-60x10(6)/ml) had a significantly higher frequency of DZPIAR. Defective sperm-ZP interaction is a major mechanism of male infertility. DZPIAR is more frequent than DSZPB in subfertile men with either normal or abnormal semen, suggesting that sequential sperm-ZP interaction tests are essential to detect these sperm defects.

Validation of simple and cost-effective stains to assess acrosomal status, DNA damage and mitochondrial activity in rooster spermatozoa.

PubMed

Rui, Bruno R; Angrimani, Daniel S R; Losano, João Diego A; Bicudo, Luana de Cássia; Nichi, Marcílio; Pereira, Ricardo J G

2017-12-01

Several methods have been developed to evaluate spermatozoa function in birds but many of these are sometimes complicated, costly and not applicable to field studies (i.e., performed within poultry breeding facilities). The objective was, therefore, to validate efficient, practical and inexpensive procedures to determine DNA fragmentation, acrosomal integrity, and mitochondrial activity in poultry spermatozoa. Initially, ejaculates were individually diluted and divided into control (4°C, 4h) and UV-irradiated aliquots (room temperature, 4h), and then samples containing different percentages of DNA-damaged spermatozoa (0%, 25%, 50%, 75% and 100%) were subjected to Toluidine Blue (TB) and Sperm Chromatin Dispersion assessments (SCD). Fast Green-Rose Bengal (FG-RB) and FITC-PSA staining protocols were subsequently used to assess acrosome status in aliquots comprising assorted amounts of acrosome-reacted spermatozoa. Furthermore, to validate 3,3'-diaminobenzidine (DAB) assay, ejaculates containing different gradients of spermatozoa with great amounts of mitochondrial activity were concurrently evaluated using DAB and JC-1 stains. The proportion of spermatozoa with abnormal DNA integrity when evaluated using the TB assessment correlated significantly with the expected percentages of UV-irradiated spermatozoa and with SCD results. A significant linear regression coefficient was also observed between expected amounts of acrosome-intact spermatozoa and FG-RB readings, and there was a significant correlation of the data when FG-RB and FITC-PSA were used. Likewise, the use of the DAB assay enabled for accurately ascertaining percentages of rooster spermatozoa with greater and lesser mitochondrial function, and results were highly correlated to results with staining with JC-1. Altogether, findings of the present study indicate acrosomal status, DNA integrity and mitochondrial activity in rooster spermatozoa can be easily and reliably determined using FG-RB, TB and DAB stains

Sperm DNA fragmentation in boars is delayed or abolished by using sperm extenders.

PubMed

Pérez-Llano, Begoña; Enciso, María; García-Casado, Pedro; Sala, Rubén; Gosálvez, Jaime

2006-12-01

The semen quality of seven young adult boars was assessed for percentages of sperm motility, normal acrosomes, abnormal sperm, cells positive to sHOST (short Hipoosmotic Swelling Test), HPNA cells (sHOST Positive with Normal Acrosome cells) and the percentage of sperm heads, which exhibited DNA fragmentation using the Sperm Chromatin Dispersion test (SCD). These parameters were analysed in sperm samples both undiluted and diluted using a commercial extender and stored at 15 degrees C for 21 days. Results showed that semen quality decreases faster in the undiluted semen samples from day 0 to day 7 compared to diluted semen samples that remained with a high quality up to day 11. The undiluted semen exhibited a low DNA fragmentation index (DFI) during the first days and then a significant increase from day 7 up to day 21. This increase in the DFI coincided with the lowest levels of the other semen quality parameters. On the contrary, the samples diluted in the commercial extender showed very low levels of DNA fragmentation in all boars during the preservation period. When the evolution of DNA fragmentation was analysed in the undiluted samples, differences were found among boars. These differences were not shown in the samples diluted in the extender where the basal DFI remained stable during the 21 days. The main conclusion of this study was that some sperm extenders delay or partially prevent sperm DNA fragmentation.

Partial deletion of chromosome 8 β-defensin cluster confers sperm dysfunction and infertility in male mice.

PubMed

Zhou, Yu S; Webb, Sheila; Lettice, Laura; Tardif, Steve; Kilanowski, Fiona; Tyrrell, Christine; Macpherson, Heather; Semple, Fiona; Tennant, Peter; Baker, Tina; Hart, Alan; Devenney, Paul; Perry, Paul; Davey, Tracey; Barran, Perdita; Barratt, Chris L; Dorin, Julia R

2013-10-01

β-defensin peptides are a family of antimicrobial peptides present at mucosal surfaces, with the main site of expression under normal conditions in the male reproductive tract. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. We show here that homozygous deletion of a cluster of nine β-defensin genes (DefbΔ9) in the mouse results in male sterility. The sperm derived from the mutants have reduced motility and increased fragility. Epididymal sperm isolated from the cauda should require capacitation to induce the acrosome reaction but sperm from the mutants demonstrate precocious capacitation and increased spontaneous acrosome reaction compared to wild-types but have reduced ability to bind the zona pellucida of oocytes. Ultrastructural examination reveals a defect in microtubule structure of the axoneme with increased disintegration in mutant derived sperm present in the epididymis cauda region, but not in caput region or testes. Consistent with premature acrosome reaction, sperm from mutant animals have significantly increased intracellular calcium content. Thus we demonstrate in vivo that β-defensins are essential for successful sperm maturation, and their disruption leads to alteration in intracellular calcium, inappropriate spontaneous acrosome reaction and profound male infertility.

Partial Deletion of Chromosome 8 β-defensin Cluster Confers Sperm Dysfunction and Infertility in Male Mice

PubMed Central

Zhou, Yu S.; Webb, Sheila; Lettice, Laura; Tardif, Steve; Kilanowski, Fiona; Tyrrell, Christine; MacPherson, Heather; Semple, Fiona; Tennant, Peter; Baker, Tina; Hart, Alan; Devenney, Paul; Perry, Paul; Davey, Tracey; Barran, Perdita; Barratt, Chris L.; Dorin, Julia R.

2013-01-01

β-defensin peptides are a family of antimicrobial peptides present at mucosal surfaces, with the main site of expression under normal conditions in the male reproductive tract. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. We show here that homozygous deletion of a cluster of nine β-defensin genes (DefbΔ9) in the mouse results in male sterility. The sperm derived from the mutants have reduced motility and increased fragility. Epididymal sperm isolated from the cauda should require capacitation to induce the acrosome reaction but sperm from the mutants demonstrate precocious capacitation and increased spontaneous acrosome reaction compared to wild-types but have reduced ability to bind the zona pellucida of oocytes. Ultrastructural examination reveals a defect in microtubule structure of the axoneme with increased disintegration in mutant derived sperm present in the epididymis cauda region, but not in caput region or testes. Consistent with premature acrosome reaction, sperm from mutant animals have significantly increased intracellular calcium content. Thus we demonstrate in vivo that β-defensins are essential for successful sperm maturation, and their disruption leads to alteration in intracellular calcium, inappropriate spontaneous acrosome reaction and profound male infertility. PMID:24204287

An alkaline follicular fluid fraction induces capacitation and limited release of oviduct epithelium-bound stallion sperm.

PubMed

Leemans, Bart; Gadella, Bart M; Stout, Tom A E; Nelis, Hilde; Hoogewijs, Maarten; Van Soom, Ann

2015-09-01

Induction of hyperactivated motility is considered essential for triggering the release of oviduct-bound mammalian spermatozoa in preparation for fertilization. In this study, oviduct-bound stallion spermatozoa were exposed for 2 h to: i) pre-ovulatory and ii) post-ovulatory oviductal fluid; iii) 100% and iv) 10% follicular fluid (FF); v) cumulus cells, vi) mature equine oocytes, vii) capacitating and viii) non-capacitating medium. None of these triggered sperm release or hyperactivated motility. Interestingly, native FF was detrimental to sperm viability, an effect that was negated by heat inactivation, charcoal treatment and 30 kDa filtration alone or in combination. Moreover, sperm suspensions exposed to treated FF at pH 7.9 but not pH 7.4 showed Ca(2+)-dependent hypermotility. Fluo-4 AM staining of sperm showed elevated cytoplasmic Ca(2+) in hyperactivated stallion spermatozoa exposed to treated FF at pH 7.9 compared to a modest response in defined capacitating conditions at pH 7.9 and no response in treated FF at pH 7.4. Moreover, 1 h incubation in alkaline, treated FF induced protein tyrosine phosphorylation in 20% of spermatozoa. None of the conditions tested induced widespread release of sperm pre-bound to oviduct epithelium. However, the hyperactivating conditions did induce release of 70-120 spermatozoa per oviduct explant, of which 48% showed protein tyrosine phosphorylation and all were acrosome-intact, but capable of acrosomal exocytosis in response to calcium ionophore. We conclude that, in the presence of elevated pH and extracellular Ca(2+), a heat-resistant, hydrophilic, <30 kDa component of FF can trigger protein tyrosine phosphorylation, elevated cytoplasmic Ca(2+) and hyperactivated motility in stallion sperm, but infrequent release of sperm pre-bound to oviduct epithelium. © 2015 Society for Reproduction and Fertility.

Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form

PubMed Central

Zheng, Heping; Mandal, Arabinda; Shumilin, Igor A.; Chordia, Mahendra D.; Panneerdoss, Subbarayalu; Herr, John C.; Minor, Wladek

2016-01-01

Sperm Lysozyme-Like Protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75Å in diameter with a 25Å central pore comprised of six monomers per helix turn repeating every 33Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally-observed SLLP1/SAS1B interaction involved in fertilization. PMID:26198801

Seasonal functional relevance of sperm characteristics in equine spermatozoa.

PubMed

Gamboa, S; Rodrigues, A S; Henriques, L; Batista, C; Ramalho-Santos, J

2010-04-15

A group of stallions with different reproductive indexes were used to study seasonal variations in sperm quality (Equus caballus). Semen samples were collected from late September to July and analyzed according to four seasonal periods: late September-December, January-March, late March-May, and June-July. Parameters monitored included sperm concentration, sperm motility, sperm morphology, sperm viability, acrosomal status, plasma membrane stability, and sperm mitochondrial membrane potential. Overall, seminal parameters monitored are affected mostly by time period, followed by animal and lastly by fertility, stressing the importance of individual variations in out-bred animal models. The analysis of multiple ejaculates from the same animals showed clear seasonal-based differences (P<0.05) with poor semen quality in winter and a noticeable improvement in sperm quality with increasing photoperiod. Better semen quality was observed between late March and May. Interactions between month period, animal, and fertility were evident (P<0.05) for sperm concentration, head and tail sperm anomalies, and acrosomal integrity. Thus, it may be advisable to adjust the use of stallion semen according to seasonal variations. Copyright 2010 Elsevier Inc. All rights reserved.

Spermine reduces reactive oxygen species levels and decreases cryocapacitation in canine sperm cryopreservation.

PubMed

Setyawan, Erif Maha Nugraha; Kim, Min Jung; Oh, Hyun Ju; Kim, Geon A; Jo, Young Kwang; Lee, Seok Hee; Choi, Yoo Bin; Lee, Byeong Chun

2016-10-28

The objective of this study was to determine the ability of spermine to act as an antioxidant in scavenging reactive oxygen species (ROS), maintaining sperm function and decreasing cryocapacitation after cryopreservation. Although motility did not increase with spermine treatment, values for membrane integrity were significantly increased (P < 0.05). Higher percentages of linearity and straightness with a lower amplitude of lateral head displacement (ALH) indicated that spermine inhibits hyperactivation. Concentrations of intracellular and extracellular ROS were decreased in the treatment group (P < 0.05). Higher expression of an anti-apoptotic gene (Bcl-2) and lower expression of a pro-apoptotic gene (Bax), together with decreased expression of the mitochondrial ROS modulator ROMO1, DNA repair due to oxidative damage (OGG1), spermine synthase (SMS), NADPH oxidase associated with motility (NOX5) and spermine amino oxidase (SMOX), all showed that 5.0 mM spermine treatment was beneficial to spermatozoa. Furthermore, the proportion of live spermatozoa with intact acrosomes after thawing in the treatment group was higher than in the control. After incubation in canine capacitating medium, numbers of live capacitated spermatozoa with reacted acrosomes were higher than in the control. Our results indicate that 5.0 mM spermine is an optimal concentration for maintaining sperm function, reducing ROS production, preventing apoptosis and adverse effects of cryocapacitation during canine sperm cryopreservation. Copyright © 2016 Elsevier Inc. All rights reserved.

Analysis of the functional aspects and seminal plasma proteomic profile of sperm from smokers.

PubMed

Antoniassi, Mariana Pereira; Intasqui, Paula; Camargo, Mariana; Zylbersztejn, Daniel Suslik; Carvalho, Valdemir Melechco; Cardozo, Karina H M; Bertolla, Ricardo Pimenta

2016-11-01

To evaluate the effect of smoking on sperm functional quality and seminal plasma proteomic profile. Sperm functional tests were performed in 20 non-smoking men with normal semen quality, according to the World Health Organization (2010) and in 20 smoking patients. These included: evaluation of DNA fragmentation by alkaline Comet assay; analysis of mitochondrial activity using DAB staining; and acrosomal integrity evaluation by PNA binding. The remaining semen was centrifuged and seminal plasma was used for proteomic analysis (liquid chromatography-tandem mass spectrometry). The quantified proteins were used for Venn diagram construction in Cytoscape 3.2.1 software, using the PINA4MS plug-in. Then, differentially expressed proteins were used for functional enrichment analysis of Gene Ontology categories, Kyoto Encyclopedia of Genes and Genomes and Reactome, using Cytoscape software and the ClueGO 2.2.0 plug-in. Smokers had a higher percentage of sperm DNA damage (Comet classes III and IV; P < 0.01), partially and fully inactive mitochondria (DAB classes III and IV; P = 0.001 and P = 0.006, respectively) and non-intact acrosomes (P < 0.01) when compared with the control group. With respect to proteomic analysis, 422 proteins were identified and quantified, of which one protein was absent, 27 proteins were under-represented and six proteins were over-represented in smokers. Functional enrichment analysis showed the enrichment of antigen processing and presentation, positive regulation of prostaglandin secretion involved in immune response, protein kinase A signalling and arachidonic acid secretion, complement activation, regulation of the cytokine-mediated signalling pathway and regulation of acute inflammatory response in the study group (smokers). In conclusion, cigarette smoking was associated with an inflammatory state in the accessory glands and in the testis, as shown by enriched proteomic pathways. This state causes an alteration in sperm functional quality

Ultrastructural Characteristics of the Testis, Spermatogenesis and Taxonomic Values of Sperm Morphology in Male Ruditapes philippinarum in Western Korea.

PubMed

Kim, Jin Hee; Chung, Jae Seung; Lee, Ki-Young

2013-06-01

Ultrastructural characteristics of the germ cells and accessory cells in testis during spermatogenesis and taxonomic values of mature sperm morphology of Ruditapes philippinarum were investigated by the transmission electron microscope and scanning electron microscope observations. The testis is the diffuse organ that consists of branching acini containing developing germ cells and accessory cells associated with spermatogenesis. The morphology of the spermatozoon is of the primitive type and is somewhat different to those of other bivalves. The morphologies of the sperm nucleus type and the acrosome shape of this species have a cylinderical type and a modified cone shape, respectively. As some ultrastructural characteristics of the acrosomal vesicle, the peripheral parts of two basal rings show electron opaque part, while the apex part of the acrosome shows electron lucent part. These characteristics of sperm belong to the family Veneridae in the subclass Heterodonta, unlike a characteristic of the subclass Pteriomorphia showing all part of the acrosome being composed of electron opaque part. In particular, a cylinder-like nucleus of the sperm is curved. The spermatozoon is approximately 48-51 μm in length, including a long acrosome (about 2.40 μm in length), a curved sperm nucleus (about 3.40 μm in length), and a tail flagellum. The axoneme of the sperm tail shows a 9+2 structure.

Studies on the membrane integrity of human sperm treated with a new injectable male contraceptive.

PubMed

Chaudhury, K; Bhattacharyya, A K; Guha, S K

2004-08-01

The aim of this study was to evaluate the integrity of sperm surface characteristics in the presence of a new male contraceptive, RISUG [1 mg styrene maleic anhydride (SMA)/100 microl dimethylsulphoxide (DMSO) in 1 ml sperm solution]. Progressively motile human sperm were treated in vitro with RISUG. The cells were analysed for the release of 5'-nucleotidase (5'-NT) (a plasma membrane marker) using 3 mmol/l 5'-AMP and 3 mmol/l beta-glycerophosphate as substrates. Hyaluronidase (an acrosomal membrane marker) was analysed using hyaluronic acid as a substrate. The contents of free and total acrosin, and % proacrosin (all acrosome markers) were assayed using 0.5 mmol/l alpha-N-benzoyl-L-arginine ethylester (BAEE). RISUG caused almost complete disintegration of the plasma membrane leading to significant (P < 0.0001) release of 5'-NT into the surrounding media. Complete dissolution of the acrosome with concomitant vesiculation of the membrane system, as judged from the loss of hyaluronidase, was observed. Total acrosin content in the sperm was also reduced to almost 10%, and proacrosin dropped to 13.2% in the presence of RISUG in comparison to 90.2% in control (P < 0.0001), indicating dispersion of acrosomal contents. Under in vitro conditions, RISUG, at a concentration of 1 mg SMA dissolved in 100 microl of DMSO, caused significant damage to the acrosome and its contents, indicating loss of functional ability of sperm. Copyright 2004 European Society of Human Reproduction and Embryology

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

Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation.

PubMed

Chuaychu-Noo, Napapach; Thananurak, Pachara; Chankitisakul, Vibantita; Vongpralub, Thevin

2017-02-01

Little is known about the effects of Cholesterol-Loaded Cyclodextrin (CLC) on post-thaw semen quality in chicken. The aim of the present study is to investigate the efficacy of CLC levels (0, 1, 2 and 3 mg/mL Schramm diluent) on post-thawed semen quality and fertility in two breeds of chicken Pradu Hang Dum (native chicken) and Rhode Island Red. Semen samples of each breed were pooled, divided into 4 aliquots and diluted with Schramm diluents, cooled to 5 °C when DMF was added (6% of final volume). Semen straws were subjected to cryopreservation using the liquid nitrogen vapor method. Post-thawed sperm motility, viability, acrosome integrity, mitochondrial function, and the Malondialdehyde (MDA) level were determined. The fertility of frozen semen was tested by inseminating laying hens. Post-thaw motility between Pradu Hang Dum and Rhode Island Red was no different; but Rhode Island Red had a higher semen viability and live cell intact acrosomes than Pradu Hang Dum (P < 0.05). The percentage of high functioning mitochondria in the Pradu Hang Dum was higher than the Rhode Island Red. CLC at 2 and 3 mg/mL supplementation was associated with improved viability of frozen semen; that is, acrosome integrity and mitochondrial function (P < 0.01), albeit having no effect on MDA levels. The sperm with 1 mg/mL CLC yielded a significantly better fertility (P < 0.01). CLC (1 mg/mL) improved the quality of frozen rooster semen. There was no interaction among breeds and CLC on post-thaw semen quality and fertility. Copyright © 2017 Elsevier Inc. All rights reserved.

Cryopreservation of canine sperm using egg yolk and soy bean based extenders.

PubMed

Sánchez-Calabuig, María Jesús; Maillo, Verónica; Beltrán-Breña, Paula; de la Fuente Martínez, Julio; Galera-Carrillo, Silvestre; Pérez-Gutiérrez, José Félix; Pérez-Cerezales, Serafín

2017-09-01

Animal protein-based extenders are widely used despite being a potential source of bacterial or mycoplasma contamination. Its replacement with vegetal protein-based extenders could represent an interesting alternative for dog sperm cryopreservation. This technique could be further improved by the addition of Tris-Glucose-Citric acid (TGC) that could physically protect the spermatozoa and improve its homeostasis. The aim of this study was to evaluate a cryopreservation protocol for dog spermatozoa using a soybean-based extender (LP1 ℗ ) as well as the effects of the addition of (TGC) immediately after the semen collection. Eleven ejaculates from purebred adult dogs were collected, centrifuged in the absence or presence of TGC and processed as fresh or cryopreserved spermatozoa with: egg yolk-based extender (CaniPRO) or LP1 ℗ . Freezing the spermatozoa in LP1 ℗ reduced the amplitude of the lateral head displacement, the percentage of spermatozoa that showed the intact acrosome and the mitochondrial function (P<0.05). These samples also showed a trend towards increased percentage of apoptotic spermatozoa (P<0.05). The addition of TGC before centrifugation did not improve the seminal parameters and adversely affected motility (P<0.05) in the spermatozoa cryopreserved in CaniPRO. However, TGC did not affect motility and increased (P<0.05) the percentage of intact acrosomes in the spermatozoa cryopreserved in LP1 ℗ , reaching similar values than those cryopreserved in CaniPRO. In conclusion, LP1 ® plus TGC provide the same level of protection to dog spermatozoa cryopreservation than the egg yolk based extender CaniPRO when comparing standard post-thaw sperm quality parameters. Copyright © 2017. Published by Elsevier Urban & Partner Sp. z o.o.

Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form.

PubMed

Zheng, H; Mandal, A; Shumilin, I A; Chordia, M D; Panneerdoss, S; Herr, J C; Minor, W

2015-07-01

Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization. © 2015 American Society of Andrology and European Academy of Andrology.

Rapid freezing without cooling equilibration in canine sperm.

PubMed

Kim, Suhee; Lee, Yongcheol; Yang, Honghyun; Kim, Yong-Jun

2012-01-01

The aim of this study was to develop a rapid method of canine semen freezing without cooling equilibration using treatment with different cryoprotectant agents (CPAs) and freezing in liquid nitrogen (LN(2)) vapor in a 0.5-mL straw via modifying vitrification. Ejaculates from eight beagle dogs were frozen with different CPAs (CPA-free, 5% glycerol, 5% ethylene glycol, and 10% ethylene glycol) and freezing times (direct plunging into LN(2) or freezing for 1, 2, 3, or 10 min in LN(2) vapor before plunging into LN(2)). Frozen-thawed sperm were evaluated for motility, viability, normal morphology, and plasma- and acrosome-membrane integrities. The 5% glycerol treatment resulted in improved sperm motility, plasma-membrane integrity and acrosome-membrane integrity (P<0.05). Freezing in LN(2) vapor showed improved sperm motility, viability, and plasma membrane integrity (P<0.05), and freezing for more than 2 min in LN(2) vapor increased acrosome-membrane integrity compared with direct plunging into LN(2) (P<0.05). The direct plunging into LN(2) showed no motile sperm. However, freezing for more than 2 min in LN(2) vapor increased the total abnormalities compared to direct plunging into LN(2) (P<0.05). In conclusion, use of 5% glycerol and freezing in LN(2) vapor were essential for the rapid freezing of canine sperm without cooling equilibration. In particular, holding for 2 min in LN(2) vapor was sufficient to yield successful rapid freezing. This rapid freezing method is simple and effective in canine sperm and would be helpful to offer information for trial of vitrification in large volumes of canine sperm. Copyright © 2012 Elsevier B.V. All rights reserved.

Cryopreservation of spermatozoa from wild-born Namibian cheetahs (Acinonyx jubatus) and influence of glycerol on cryosurvival.

PubMed

Crosier, Adrienne E; Pukazhenthi, Budhan S; Henghali, Josephine N; Howard, Jogayle; Dickman, Amy J; Marker, Laurie; Wildt, David E

2006-04-01

Sperm cryopreservation is a valuable tool for the genetic management of ex situ populations. This study was conducted to assess: (1) semen characteristics of wild-born cheetahs; and (2) the impact of three types of glycerol influence (duration of exposure, temperature, and method of addition) on sperm cryosensitivity. To evaluate the impact of duration of glycerol exposure, spermatozoa were incubated in Test Yolk Buffer (TYB) with 4% glycerol at ambient temperature (approximately 22 degrees C) for 15 vs. 60 min before cryopreservation. To evaluate the influence of temperature and method of glycerol addition, spermatozoa were resuspended at ambient temperature either in TYB with 0% glycerol followed by addition of 8% glycerol (1:1 v/v; at ambient temperature vs. 5 degrees C) or directly in TYB with 4% glycerol. All samples were cryopreserved in straws over liquid nitrogen vapor and evaluated for sperm motility and acrosomal integrity after thawing. Semen samples (n = 23; n = 13 males) contained a high proportion (78%) of pleiomorphic spermatozoa. Ejaculates also contained a high proportion of acrosome-intact (86%) and motile spermatozoa (78%). Immediately after thawing, a significant proportion of spermatozoa retained intact acrosomes (range, 48-67%) and motility (range, 40-49%). After thawing, incubation in glycerol for 60 min at ambient temperature before freezing decreased (p < 0.05) sperm motility and acrosomal integrity at one time-point each (pre-centrifugation and post-centrifugation, respectively). However, method or temperature of glycerol addition had no (p > 0.05) impact on sperm cryosurvival. In summary, (1) wild-born cheetahs produce high proportions of pleiomorphic spermatozoa but with a high proportion of intact acrosomes; and (2) resuspension in 4% glycerol, followed by exposure for up to 60 min at ambient temperature, had minimal effect on sperm motility and acrosomal integrity after cryopreservation. Results indicate the feasibility of

Ejaculate traits and sperm cryopreservation in the endangered Baird's tapir (Tapirus bairdii).

PubMed

Pukazhenthi, Budhan S; Togna, Gina Della; Padilla, Luis; Smith, Diorene; Sanchez, Carlos; Pelican, Katey; Sanjur, Oris I

2011-01-01

There is little information on the reproductive biology of the male Baird's tapir (Tapirus bairdii). In this study, we characterized the ejaculate traits and evaluated the efficacy of 2 cryodiluents on sperm cryosurvival. Ejaculates were assessed for volume, pH, sperm motility, forward progression, osmolality, sperm concentration, sperm morphology, and acrosomal integrity. For cryopreservation, ejaculates with >50% total sperm motility were washed, and sperm pellets were resuspended in either Botu-Crio (CryoVital, Grandau, Germany) or INRA 96 containing 2% egg yolk and 2.5% each of methyl- and dimethylformamide (INRA 96), and they were cryopreserved over liquid nitrogen vapor. Thawed samples were incubated in vitro (25 °C) and evaluated for percent total sperm motility, forward progression, and acrosomal integrity at hourly intervals for 4 hours. Spermic ejaculates were obtained from all males, and the mean seminal volume, sperm concentration per milliliter, percent sperm motility, progressive status, and percent morphologically normal cells were 20.4 ± 4.3 mL, 101.2 ± 24.0 × 10(6)/mL, 46.1% ± 5.0%, 2.9 ± 0.1, and 6.9% ± 1.4%, respectively. There was a positive significant correlation between percent normal sperm and animal age (r = 0.66; P < .004). Cryopreservation in either Botu-Crio or INRA 96 resulted in a decline (P < .05) in percent sperm motility and acrosomal integrity. Sperm forward progression remained unaffected immediately after thawing in INRA 96 but continued to decline over time. These results characterize, for the first time, the ejaculate traits of the tapir; demonstrate that tapir spermatozoa can be cryopreserved in diluents containing amides alone or in combination with glycerol; and provide fundamental information critical for development of assisted reproductive technologies for the Baird's tapir.

Effect of colloid (Androcoll-Bear, Percoll, and PureSperm) selection on the freezability of brown bear (Ursus arctos) sperm.

PubMed

Álvarez-Rodríguez, M; Álvarez, M; Anel-López, L; López-Urueña, E; Manrique, P; Borragán, S; Morrell, J M; de Paz, P; Anel, L

2016-04-01

The development of a species-specific conservation protocol that involves artificial insemination with frozen semen needs to validate an effective methodology for freezing semen. Colloid centrifugation has been suggested and widely applied as an effective tool for selecting animal spermatozoa for artificial breeding. The objective of the present study was to compare different methods of centrifugation, single layer using Androcoll-Bear and Percoll and double layer using PureSperm 100 (in two different discontinuous gradients 40%-80% and 45%-90%), for the selection of fresh brown bear sperm samples. In the before freezing group, all selected samples showed a higher progressive motility and viability (except Percoll for motility 43.0 ± 5.3 [P < 0.05]); all colloids except PureSperm 45/90% rendered samples with fewer damaged acrosomes. In the after thawing group, all tested centrifugation colloids showed a good capacity to decrease the number of damaged acrosomes. Furthermore, PureSperm treatment (45/90%) resulted in an increase in apoptotic-like changes not only immediately after thawing but also after the incubation test, leading us to suggest that this gradient could induce some kind of deleterious effects on the sperm samples. On the other hand, PureSperm treatment (40/80%) yielded a quality preservation capacity similar to Androcoll-Bear in number of damaged acrosomes, different relative to the control (control, 5.3 ± 0.6; PureSperm 80, 2.0 ± 0.3; Androcoll, 2.1 ± 0.9 [P < 0.05]) but a decrease in the number of viable spermatozoa recovered after thawing relative to the control (control, 21.2 ± 3.1; PureSperm 80, 13.7 ± 2.7 [P < 0.05]). In conclusion, Androcoll-Bear constitutes a useful tool for handling of brown bear ejaculates owing to its simple handling and procedure with a reliable sperm selection and freezability. This colloid yielded an improvement in several sperm parameters in brown bear frozen-thawed semen; the selected spermatozoa of fresh samples

The ultrastructure of the spermatozoon of Paradynomene tuberculata Sakai, 1963 (Crustacea, Brachyura, Dynomenidae): Synapomorphies with dromiid sperm

NASA Astrophysics Data System (ADS)

Jamieson, B. G. M.; Guinot, D.; de Forges, B. Richer

1993-10-01

The dynomenid spermatozoon, exemplified here by Paradynomene tuberculata, resembles the spermatozoa of the Dromiidae, Homolidae and lyreidine raninoids and differs markedly from those of other crabs (the heterotreme, thoracotremes, raninines and raninoidines) in the depressed, discoidal form of the acrosome and the capitate form of the perforatorium. Four or five apparent dynomenid—dromiid sperm synapomorphies are recognizable. (1) Dynomenids ( P. tuberculata) and dromiids differ from homolids and lyreidines in the greater depression of the acrosome (ratio of length to width=0.3); (2) the capitate head of the perforatorium is bilaterally prolonged in P. tuberculata as in dromiids though symmetrical in homolids; (3) dynomenid and dromiid sperm lack the—albeit variably developed—posterior median process of the nucleus seen in homolids, anomurans, raninoids and lower heterotremes; (4) P. tuberculata, like dromiids and less distinctly homolids, has an apical protuberance of subopercular material through the opercular perforation, unknown in other crabs, being distinct from the apical button of thoracotreme sperm; (5) a less certain synapomorphy is the anterolateral electron-pale peripheral zone of the acrosome. These synapomorphies endorse a sister-group relationship of dynomenids and dromiids, P. tuberculata sperm differs notably from the sperm of dromiids in the more complex zonation of the acrosome. The perforatorium lacks the radial rays (“spiked wheel”) of homolid sperm and does not show the “amoeboid” form seen in lyreidines. Absence of internal corrugations of the perforatorial chamber is a major difference from all examined raninids. Centrioles are only very tentatively identifiable. Nuclear arms are absent in glutaraldehyde fixed spermatozoa of P. tuberculata and have not been observed in the dromiid Petalomera lateralis but are present as three small radial vertices in the dromiid Dromidiopsis edwardsi and in homolids. P. tuberculata resembles

Manchette-acrosome disorders during spermiogenesis and low efficiency of seminiferous tubules in hypercholesterolemic rabbit model

PubMed Central

Simón, Layla; Funes, Abi K.; Yapur, Martín A.; Cabrillana, María E.; Monclus, María A.; Boarelli, Paola V.; Vincenti, Amanda E.

2017-01-01

Hypercholesterolemia is a marker for several adult chronic diseases. Recently we demonstrated that sub/infertility is also associated to Hypercholesterolemia in rabbits. Seminal alterations included: abnormal sperm morphology, decreased sperm number and declined percentage of motile sperm, among others. In this work, our objective was to evaluate the effects of hypercholesterolemia on testicular efficiency and spermiogenesis, as the latter are directly related to sperm number and morphology respectively. Tubular efficiency was determined by comparing total number of spermatogenic cells with each cell type within the proliferation/differentiation compartments. We found lower testicular efficiency related to both a decrease in spermatogonial cells and an increase in germ cell apoptosis in hypercholesterolemic rabbits. On the other hand, spermiogenesis–the last step of spermatogenesis involved in sperm shaping–was detaily analyzed, particularly the acrosome-nucleus-manchette complex. The manchette is a microtubular-based temporary structure responsible in sperm cell elongation. We analyzed the contribution of actin filaments and raft microdomains in the arrangement of the manchette. Under fluorescence microscopy, spermatocyte to sperm cell development was followed in cells isolated from V to VIII tubular stages. In cells from hypercholesterolemic rabbits, abnormal development of acrosome, nucleus and inaccurate tail implantation were associated with actin–alpha-tubulin–GM1 sphingolipid altered distribution. Morphological alterations were also observed at electron microscopy. We demonstrated for the first time that GM1-enriched microdomains together with actin filaments and microtubules are involved in allowing the correct anchoring of the manchette complex. In conclusion, cholesterol enriched diets promote male fertility alterations by affecting critical steps in sperm development: spermatogenesis and spermiogenesis. It was also demonstrated that

Performance of Rodent Spermatozoa Over Time Is Enhanced by Increased ATP Concentrations: The Role of Sperm Competition.

PubMed

Tourmente, Maximiliano; Villar-Moya, Pilar; Varea-Sánchez, María; Luque-Larena, Juan J; Rial, Eduardo; Roldan, Eduardo R S

2015-09-01

Sperm viability, acrosome integrity, motility, and swimming velocity are determinants of male fertility and exhibit an extreme degree of variation among closely related species. Many of these sperm parameters are associated with sperm ATP content, which has led to predictions of trade-offs between ATP content and sperm motility and velocity. Selective pressures imposed by sperm competition have been proposed as evolutionary causes of this pattern of diversity in sperm traits. Here, we examine variation in sperm viability, acrosome integrity, motility, swimming velocity, and ATP content over time, among 18 species of closely related muroid rodents, to address the following questions: (a) Do sperm from closely related species vary in ATP content after a period of incubation? (b) Are these differences in ATP levels related to differences in other sperm traits? (c) Are differences in ATP content and sperm performance over time explained by the levels of sperm competition in these species? Our results revealed a high degree of interspecific variability in changes in sperm ATP content, acrosome integrity, sperm motility and swimming velocity over time. Additionally, species with high sperm competition levels were able to maintain higher levels of sperm motility and faster sperm swimming velocity when they were incubated under conditions that support sperm survival. Furthermore, we show that the maintenance of such levels of sperm performance is correlated with the ability of sperm to sustain high concentrations of intracellular ATP over time. Thus, sperm competition may have an important role maximizing sperm metabolism and performance and, ultimately, the fertilizing capacity of spermatozoa. © 2015 by the Society for the Study of Reproduction, Inc.

Quantitative light and scanning electron microscopy of ferret sperm.

PubMed

Van der Horst, G; Curry, P T; Kitchin, R M; Burgess, W; Thorne, E T; Kwiatkowski, D; Parker, M; Atherton, R W

1991-11-01

Sperm were obtained via electroejaculation from Domestic ferret, (Mustela putorius furo), Siberian ferret (M. eversmanni), Black-footed ferret (M. nigripes), and a hybrid between Siberian and Domestic, called the Fitch ferret (M. sp.). Comparisons of sperm were made by four different microscopy techniques to determine whether differences exist among species. First, Nomarski differential interference microscopy could be used to distinguish domestic ferret sperm from the others on the basis of the structure of the posterior part of the acrosome. Second, both silver staining, which demonstrates argentophilic protein distribution, and scanning electron microscopy (SEM), revealed differences among the morphology of sperm for each species; variation in the unique appearance of the acrosome in ferret sperm was detected especially well by SEM. To quantify differences in morphology, five sperm head parameters were measured using image analysis; light microscopy produced significantly larger values than did SEM (all parameters and all species but Fitch), and there were significant differences owing to species for all parameters but one. Generally, our data demonstrate the value of complementary techniques to distinguish among sperm of closely related species and more specifically may help establish evolutionary relationships among the ferret species studied. In addition, they provide baseline data important for the captive breeding of the endangered Black-footed ferret.

Incubation of spermatozoa with Anandamide prior to cryopreservation reduces cryocapacitation and improves post-thaw sperm quality in the water buffalo (Bubalus bubalis).

PubMed

Kumar, Puneeth; Mohanty, Tushar Kumar; Kumaresan, Arumugam; Nag, Pradeep; Saraf, Kaustubh Kishor; Kumar, Vimlesh; Lathika, Sreela; Nayak, Samiksha; Bhakat, Mukesh

2018-02-01

Anandamide (AEA), an endocannabinoid, has been shown to reduce capacitation and acrosomal exocytosis in human spermatozoa. Because buffalo spermatozoa are highly susceptible to cryopreservation induced damage, AEA was assessed as to whether it could protect spermatozoa from cryo-damage. Six ejaculates from six Murrah buffalo bulls (total 36 ejaculates) were utilized for the study. Each ejaculate was divided into four aliquots; spermatozoa in Aliquot 1 were extended in Tris-Citrate-Egg Yolk and frozen as per the standard protocol. Spermatozoa in Aliquots 2, 3 and 4 were incubated with AEA at 1 nM, 1 μM and 10 μM, respectively in Tris-Citrate extender for 15 min at 37 °C before cryopreservation. Cryopreserved spermatozoa were thawed at 37 °C for 30 s before assessment of sperm motility, membrane integrity, capacitation, acrosome reaction, mitochondrial membrane potential (MMP) and lipid peroxidation status. The proportion of motile and membrane intact spermatozoa were greater (P < 0.05) with use of 1 μM AEA incorporated group compared with other groups. The proportion of un-capacitated and acrosome intact spermatozoa was greater (P < 0.05) with use of 1 or 10 μM of AEA compared with the other groups. When compared to the control group, use of 1 μM AEA resulted in a greater proportion of spermatozoa with high MMP (P < 0.05). There was no significant difference in the lipid peroxidation status of spermatozoa among any of the four groups. It was inferred that the protective role of AEA during cryopreservation of buffalo spermatozoa was dose dependent and incubation of spermatozoa with AEA at 1 μM concentration prior to cryopreservation reduced cryo-capacitation and improved post-thaw sperm quality in buffalo. Copyright © 2017 Elsevier B.V. All rights reserved.

Magnetic-activated cell sorting for sperm preparation reduces spermatozoa with apoptotic markers and improves the acrosome reaction in couples with unexplained infertility.

PubMed

Lee, Tsung-Hsien; Liu, Chung-Hsien; Shih, Yang-Tse; Tsao, Hui-Mei; Huang, Chun-Chia; Chen, Hsiu-Hui; Lee, Maw-Sheng

2010-04-01

Couples with unexplained infertility (UI) tend to have low fertilization rates with current IVF procedures. Here, we attempted to identify spermatozoa with apoptotic markers in couples with UI and unsuccessful intrauterine insemination (IUI) and we investigated the efficiency and benefit of magnetic-activated cell sorting (MACS) for sperm preparation in such patients. Sixty couples with UI and two IUI failures were recruited. The sperm were prepared by conventional density gradient centrifugation (DGC) and divided into two aliquots. One aliquot was used as a control and the other was further processed by MACS (D + M). Apoptotic markers were identified using fluorescence-labeled dye and flow cytometry, including externalization of phosphatidylserine (EPS), disrupted mitochondrial membrane potential (MMP) and DNA fragmentation. The fertilization potential of prepared spermatozoa was analyzed by basic semen analysis, computer-aided sperm analysis and the induced acrosome reaction test (IART). After DGC, spermatozoa showed 18.6% EPS, 28.3% disrupted MMP and 13.5% DNA fragmentation. Numbers of spermatozoa with apoptotic markers were significantly reduced by D + M, versus DGC alone (P < 0.001). Although the motility of spermatozoa was slightly decreased after MACS, most sperm motion characteristics were not impaired. Interestingly, the IART significantly improved after D + M, versus DGC alone, especially for the couples with a normal hemizona assay (P < 0.001). The spermatozoa prepared by D + M showed a reduced level of apoptotic markers. Improvement in the IART suggests a high fertilization potential of the processed spermatozoa. The identification of apoptotic markers and use of MACS may be helpful in directing the management plan for patients with UI and multiple IUI failures.

Sperm quality improvement after date seed oil in vitro supplementation in spontaneous and induced oxidative stress.

PubMed

Fatma, Ben A; Nozha, Chakroun F; Ines, Dammak; Hamadi, Attia; Basma, Hentati; Leila, Ammar K

2009-05-01

In vitro supplementation with date seed oil (DSO) can protect spermatozoa against hydrogen peroxide (H2O2)-mediated damage and can improve sperm function, possibly owing to antioxidant properties. We tested the antioxidant effects of DSO on human sperm motility, sperm viability, reacted acrosome and lipid peroxidation assessed in vitro after H2O2-mediated oxidative damage in spermatozoa. Sixteen patients (mean age: 35 years; range: 25-45 years) referred to the Histology-Embryology Laboratory of the Medicine Faculty of Sfax for semen analysis after 12-24 months of sexual intercourse without conception were selected. After spermiogram, sperm selection by two-interface discontinuous Sill Select gradient was performed, and selected spermatozoa were used in four experimental assays: control; incubation with 100 microm H2O2; incubation with 0.1% DSO; and co-incubation with 0.1% DSO and 100 microm H2O2. Motility and viability were determined using World Health Organization criteria. Acrosome reaction and lipid peroxidation were assessed by staining with fluorescein isothiocyanate-Pisum sativum and spectrophotometric measurement of malondialdehyde, respectively. Results showed that incubation with H2O2 alone led to a significant increase in lipid peroxidation (57.83%, P<0.05) associated with a significant decrease in sperm motility, sperm viability (after 30 min and 24 h) and percentage of reacted acrosome (P<0.05). Date seed oil improved sperm motility after 24 h of incubation (P<0.05) and protected spermatozoa against the deleterious effects of H2O2 on motility, viability, acrosome reaction and lipid peroxidation. We conclude that supplementation with DSO may have a function in antioxidant protection against male infertility.

Studies on trypsin-like enzymes in sperm and early embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Penn, A.

1975-12-09

Results are reported from a study of acrosomal proteinase, a trypsin-like enzyme (TLE), found in the acrosome of all eutherian mammals studied to date. It has been implicated in the dissolution of a passage for the sperm through the zona pellucida of the egg, a step necessary for in vivo fertilization. A cytochemical procedure employing autoradiographic film as a gelatin substrate is described for in situ detection and localization of acrosomal proteolytic activity. A role for TLE in the early development of embryos is suggested. (CH)

Aflatoxin B1 impairs sperm quality and fertilization competence.

PubMed

Komsky-Elbaz, A; Saktsier, M; Roth, Z

2018-01-15

Aflatoxins are poisonous byproducts of the soilborne fungus Aspergillus, involved in the decomposition of plant materials. Aflatoxins can be found in various food products, such as maize, sorghum, millet, rice and wheat. AFB1 is the most toxic of these, classified as a carcinogen and mutagen for both humans and animals. AFB1 has been detected in human cord blood and placenta; however, its toxic effect on sperm is less known. The current study examines sperm responses associated with AFB1 exposure. These included acrosome integrity and function, mitochondrial polarity, DNA fragmentation, fertilization competence and early embryonic development. Spermatozoa were obtained from bull ejaculate and epididymis and capacitated in vitro for 4h with 0, 0.1, 1, 10 and 100μM AFB1. Following capacitation, acrosome reaction (AR) was induced by Ca 2+ ionophore. The integrity and functionality of sperm were examined simultaneously by florescent staining. A Halosperm DNA fragmentation kit was used to evaluate DNA integrity. An in-vitro culture system was used to evaluate fertilization competence and blastocyst formation rate, using bovine oocytes. Findings indicate dose-responsive variation among compartments to AFB1 exposure. Sperm viability, expressed by integrity of the plasma membrane, was lower in sperm isolated from ejaculate or epididymis after culturing with AFB1. Exposure to AFB1 reduced the proportion of sperm from the epididymis tail undergoing acrosome reaction induced by Ca 2+ ionophore. AFB1 impaired mitochondrial membrane potential (ΔYm) in sperm isolated from ejaculate and the epididymis tail. Exposing ejaculated sperm to AFB1 increased the proportion of sperm with fragmented DNA and reduced the proportion of embryos that cleaved to the 2- to 4-cell stage, 42h postfertilization, however, the proportion of embryos that developed to blastocysts, 7days postfertilization, did not differ among groups. The findings explore the harmful effects of AFB1 on sperm viability

TMEM16A inhibition impedes capacitation and acquisition of hyperactivated motility in guinea pig sperm.

PubMed

Cordero-Martínez, Joaquín; Reyes-Miguel, Tania; Rodríguez-Páez, Lorena; Garduño-Siciliano, Leticia; Maldonado-García, Deneb; Roa-Espitia, Ana L; Hernández-González, Enrique O

2018-07-01

Ca 2+ -activated Cl - channels (CaCCs) are anionic channels that regulate many important physiological functions associated with chloride and calcium flux in some somatic cells. The molecular identity of CaCCs was revealed to be TMEM16A and TMEM16B (also known as Anoctamin or ANO1 and ANO2, respectively) in all eukaryotes. A recent study suggests the presence of TMEM16A in human sperm and a relationship with the rhZP-induced acrosome reaction. However, to the best of our knowledge, little is known about the role of TMEM16A in other spermatic processes such as capacitation or motility. In this study, we evaluated the effects of two TMEM16A antagonists on capacitation, acrosome reaction, and motility in guinea pig sperm; these antagonists were T16Ainh-A01, belonging to a second generation of potent antagonists of TMEM16A, and niflumic acid (NFA), a well-known antagonist of TMEM16A (CaCCs). First of all, we confirmed that the absence of Cl - in the capacitation medium changes motility parameters, capacitation, and the progesterone-induced acrosome reaction. Using a specific antibody, TMEM16A was found as a protein band of ∼120 kDa, which localization was in the apical crest of the acrosome and the middle piece of the flagellum. Inhibition of TMEM16A by T16Ainh-A01 affected sperm physiology by reducing capacitation, blocking the progesterone-induced acrosome reaction under optimal capacitation conditions, inhibiting progressive motility, and the acquisition of hyperactivated motility, diminishing [Ca 2+ ]i, and increasing [Cl - ]i. These changes in sperm kinematic parameters provide new evidence of the important role played by TMEM16A in the production of sperm capable of fertilizing oocytes. © 2018 Wiley Periodicals, Inc.

The influence of sperm concentration, length of the gamete co-culture and the evolution of different sperm parameters on the in vitro fertilization of prepubertal goat oocytes.

PubMed

Palomo, M J; Mogas, T; Izquierdo, D; Paramio, M T

2010-11-01

The aims of the present study were: (1) to evaluate the influence of sperm concentration (ranging from 0.5 × 10(6) to 4 × 10(6) spermatozoa/ml) and length of the gamete co-incubation time (2, 4, 6, 8, 10, 12, 16, 20, 24 or 28 h) on in vitro fertilization (IVF), assessing the sperm penetration rate; (2) to investigate the kinetics of different semen parameters as motility, viability and acrosome status during the co-culture period; and (3) to analyse the effect of the presence of cumulus-oocytes complexes (COCs) on these parameters. To achieve these objectives, several experiments were carried out using in vitro matured oocytes from prepubertal goats. The main findings of this work are that: (1) in our conditions, the optimum sperm concentration is 4 × 10(6) sperm/ml, as this sperm:oocyte ratio (approximately 28,000) allowed us to obtain the highest penetration rate, without increasing polyspermy incidence; (2) the highest percentage of viable acrosome-reacted spermatozoa is observed between 8-12 h of gamete co-culture, while the penetration rate is maximum at 12 h of co-incubation; and (3) the presence of COCs seems to favour the acrosome reaction of free spermatozoa on IVF medium, but not significantly. In conclusion, we suggest that a gamete co-incubation for 12-14 h, with a concentration of 4 × 10(6) sperm/ml, would be sufficient to obtain the highest rate of penetration, reducing the exposure of oocytes to high levels of reactive oxygen species produced by spermatozoa, especially when a high sperm concentration is used to increase the caprine IVF outcome.

Effect of various commercial buffers on sperm viability and capacitation.

PubMed

Andrisani, Alessandra; Donà, Gabriella; Ambrosini, Guido; Bonanni, Guglielmo; Bragadin, Marcantonio; Cosmi, Erich; Clari, Giulio; Armanini, Decio; Bordin, Luciana

2014-08-01

A wide variety of sperm preparation protocols are currently available for assisted conception. They include density gradient separation and washing methods. Both aim at isolating and capacitating as much motile sperm as possible for subsequent oocyte fertilization. The aim of this study was to examine the effects of four commercial sperm washing buffers on sperm viability and capacitation. Semen samples from 48 healthy donors (normal values of sperm count, motility, morphology, and volume) were analyzed. After separation (density gradient 40/80%), sperm were incubated in various buffers then analysed for reactive oxygen species (ROS) production, viability, tyrosine phosphorylation (Tyr-P), cholera toxin B subunit (CTB) labeling, and the acrosome reaction (AR). The buffers affected ROS generation in various ways resulting either in rapid cell degeneration (when the amount of ROS was too high for cell survival) or the inability of the cells to maintain correct functioning (when ROS were too few). Only when the correct ROS generation curve was maintained, suitable membrane reorganization, evidenced by CTB labeling was achieved, leading to the highest percentages of both Tyr-P- and acrosome-reacted-cells. Distinguishing each particular pathological state of the sperm sample would be helpful to select the preferred buffer treatment since both ROS production and membrane reorganization can be significantly altered by commercial buffers.

The Rab3A-22A Chimera Prevents Sperm Exocytosis by Stabilizing Open Fusion Pores*

PubMed Central

Quevedo, María F.; Lucchesi, Ornella; Bustos, Matías A.; Pocognoni, Cristian A.; De la Iglesia, Paola X.

2016-01-01

At the final stage of exocytotis, a fusion pore opens between the plasma and a secretory vesicle membranes; typically, when the pore dilates the vesicle releases its cargo. Sperm contain a large dense-core secretory granule (the acrosome) whose contents are secreted by regulated exocytosis at fertilization. Minutes after the arrival of the triggering signal, the acrosomal and plasma membranes dock at multiple sites and fusion pores open at the contact points. It is believed that immediately afterward, fusion pores dilate spontaneously. Rab3A is an essential component of human sperm exocytotic machinery. Yet, recombinant, persistently active Rab3A halts calcium-triggered secretion when introduced after docking into streptolysin O-permeabilized cells; so does a Rab3A-22A chimera. Here, we applied functional assays, electron and confocal microscopy to show that the secretion blockage is due to the stabilization of open fusion pores. Other novel findings are that sperm SNAREs engage in α-SNAP/NSF-sensitive complexes at a post-fusion stage. Complexes are disentangled by these chaperons to achieve vesiculation and acrosomal contents release. Thus, post-fusion regulation of the pores determines their expansion and the success of the acrosome reaction. PMID:27613869

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

Organic zinc and copper supplementation on antioxidant protective mechanism and their correlation with sperm functional characteristics in goats.

PubMed

Narasimhaiah, M; Arunachalam, A; Sellappan, S; Mayasula, V K; Guvvala, P R; Ghosh, S K; Chandra, V; Ghosh, J; Kumar, H

2018-06-01

Trace minerals feeding had significant effects on sperm production and fertility with better absorption and proper utilization within the body for optimum reproductive function. Several studies have shown that more influenced trace elements in the diets of animals are copper (Cu) and zinc (Zn). Bucks showing deficiency of this mineral might affect the quality of semen production which in turn would affect the fertility. This experiment was thus designed to test the effects of organic Cu and Zn supplementation on antioxidants enzyme activities and sperm functional attributes in fresh semen of bucks. Forty bucks (n = 40, Aged 5 months) were assigned to ten groups of four animals in each group, supplemented (for a period of 8 months) with different levels of organic Zn: 20 mg (T2), 40 mg (T3) and 60 mg (T4), organic Cu: 12.5 mg (T5), 25 mg (T6), 37.5 mg (T7) and combined organic Zn and Cu: 20 + 12.5 mg (T8), 40 + 25 mg (T9), 60 + 37.5 mg (T10), respectively, per kg dry matter and no additional mineral diet (control; T1). One hundred and sixty semen samples were collected through electro-ejaculator and analysed for sperm quantity, quality, acrosome intactness and plasma membrane integrity and correlated with the catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase enzyme activities in seminal plasma. The results indicated organic Cu and zinc supplemented bucks produced more sperm cells, had higher sperm concentrations, maintained higher (p < .01) sperm livability, plasma membrane and acrosome integrities, more motility and velocity. The increased antioxidant enzyme activities, reduced oxidative stress and lowered lipid peroxidation were positively correlated (p < .05) with the sperm functional attributes. In conclusion, organic Cu and Zn supplement to male goats showed protective roles against oxidative damage and maintained better fresh semen characteristics. © 2018 Blackwell Verlag GmbH.

Cryopreservation of Peruvian Paso horse spermatozoa: dimethylacetamide preserved an optimal sperm function compared to dimethyl sulfoxide, ethylene glycol and glycerol.

PubMed

Santiani, A; Evangelista-Vargas, S; Vargas, S; Gallo, S; Ruiz, L; Orozco, V; Rosemberg, M

2017-08-01

The objective was to evaluate the effect of different cryoprotectant agents in the cryopreservation of Peruvian Paso horse semen. Twenty semen samples were collected from five Peruvian Paso horse stallions. Each sample was divided into 12 parts to form the groups: dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol (GLY), at 3%, 4% and 5%. Samples were frozen using a rate-controlled freezer. Sperm parameters evaluated were motility and viability/acrosomal status. After thawing, progressive motility in DMA group was higher (p < .05) than in DMSO, EG and GLY groups. Similarly, viable acrosome-intact spermatozoa were higher (p < .05) using DMA in comparison with DMSO. No differences were found when comparing concentrations for any of the cryoprotectant agents. In conclusion, DMA seems to be a good cryoprotectant agent for the cryopreservation of Peruvian Paso horse stallion semen. © 2016 Blackwell Verlag GmbH.

Characterization and possible function of glyceraldehyde-3-phosphate dehydrogenase-spermatogenic protein GAPDHS in mammalian sperm.

PubMed

Margaryan, Hasmik; Dorosh, Andriy; Capkova, Jana; Manaskova-Postlerova, Pavla; Philimonenko, Anatoly; Hozak, Pavel; Peknicova, Jana

2015-03-08

Sperm proteins are important for the sperm cell function in fertilization. Some of them are involved in the binding of sperm to the egg. We characterized the acrosomal sperm protein detected by a monoclonal antibody (MoAb) (Hs-8) that was prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperms and we tested the possible role of this protein in the binding assay. Indirect immunofluorescence and immunogold labelling, gel electrophoresis, Western blotting and protein sequencing were used for Hs-8 antigen characterization. Functional analysis of GAPDHS from the sperm acrosome was performed in the boar model using sperm/zona pellucida binding assay. Monoclonal antibody Hs-8 is an anti-human sperm antibody that cross-reacts with the Hs-8-related protein in spermatozoa of other mammalian species (boar, mouse). In the immunofluorescence test, Hs-8 antibody recognized the protein localized in the acrosomal part of the sperm head and in the principal piece of the sperm flagellum. In immunoblotting test, MoAb Hs-8 labelled a protein of 45 kDa in the extract of human sperm. Sequence analysis identified protein Hs-8 as GAPDHS (glyceraldehyde 3-phosphate dehydrohenase-spermatogenic). For this reason, commercial mouse anti-GAPDHS MoAb was applied in control tests. Both antibodies showed similar staining patterns in immunofluorescence tests, in electron microscopy and in immunoblot analysis. Moreover, both Hs-8 and anti-GAPDHS antibodies blocked sperm/zona pellucida binding. GAPDHS is a sperm-specific glycolytic enzyme involved in energy production during spermatogenesis and sperm motility; its role in the sperm head is unknown. In this study, we identified the antigen with Hs8 antibody and confirmed its localization in the apical part of the sperm head in addition to the principal piece of the flagellum. In an indirect binding assay, we confirmed the potential role of GAPDHS as a binding protein that is involved in the secondary sperm

The Behavior and Acrosomal Status of Mouse Spermatozoa In Vitro, and Within the Oviduct During Fertilization after Natural Mating.

PubMed

Hino, Toshiaki; Muro, Yuko; Tamura-Nakano, Miwa; Okabe, Masaru; Tateno, Hiroyuki; Yanagimachi, Ryuzo

2016-09-01

Although 90%-100% of mouse oocytes can be fertilized in vitro with capacitated spermatozoa within 1 h after insemination, oocytes within the oviduct are fertilized one by one over a period of several hours. In vitro experiments showed that both acrosome-intact and acrosome-reacted spermatozoa entered the cumulus oophorus, but that acrosome-reacted spermatozoa reached the surface of oocytes more readily than acrosome-intact spermatozoa. During the period of fertilization within the oviduct, acrosome-reacted spermatozoa were seen throughout the isthmus, but with higher incidence in the upper than in the mid- and lower segments of the isthmus. Very few spermatozoa were present in the ampulla, and almost all were acrosome reacted. Although the cumulus oophorus and zona pellucida are known to be able to induce or facilitate the acrosome reaction of spermatozoa, this picture makes it likely that almost all fertilizing mouse spermatozoa within the oviduct begin to react before ascending from the isthmus to the ampulla. We witnessed a reacted spermatozoon that stayed on the zona pellucida of a fertilized oocyte for a while; it then moved out of the cumulus before reaching the zona pellucida of the nearby unfertilized oocyte. We noted that only a few spermatozoa migrate from the isthmus to the ampulla during the progression of fertilization, and this must be one of the reasons why we do not see many spermatozoa swarming around a single oocyte during in vivo fertilization. © 2016 by the Society for the Study of Reproduction, Inc.

Cryopreservation of boar semen by egg yolk-based extenders containing lactose or fructose is better than sorbitol.

PubMed

Chanapiwat, Panida; Kaeoket, Kampon; Tummaruk, Padet

2012-03-01

The present study determined the effect of different types of sugars (lactose, fructose, glucose and sorbitol) used in egg yolk-based extender on the post-thawed boar semen quality. Twenty-two ejaculates from 6 fertility-proven Yorkshire boars were cryopreserved by liquid nitrogen vapor method. Sperm motility, viability, acrosome integrity and intact functional plasma membrane were determined at 0, 2 and 4 hr after thawing. It was found that the lactose-based extender resulted in a higher percentage of post-thawed sperm motility, viability, intact acrosome and functional plasma membrane than sorbitol-based extender (P<0.05) and fructose-based extender yielded a higher post-thawed sperm motility and viability than sorbitol-based extender (P<0.05). It could be concluded that sorbitol was not an effective sugar for the cryopreservation in boar semen.

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

Specific strychnine binding sites on acrosome-associated membranes of golden hamster spermatozoa.

PubMed

Llanos, Miguel N; Ronco, Ana M; Aguirre, María C

2003-06-27

This study demonstrates for the first time, that membrane vesicles originated from the hamster sperm head after the occurrence of the acrosome reaction, possess specific strychnine binding sites. [3H]Strychnine binding was saturable and reversible, being displaced by unlabeled strychnine (IC(50)=26.7+/-2.3 microM). Kinetic analysis revealed one binding site with K(d)=120nM and B(max)=142fmol/10(6) spermatozoa. Glycine receptor agonists beta-alanine and taurine inhibited strychnine binding by 20-30%. Surprisingly, glycine stimulated binding by about 40-50%. Results obtained in this study strongly suggest the presence of glycine receptors-with distinctive kinetic properties on the periacrosomal plasma membrane of hamster spermatozoa. Localization of this receptor fits well with its previously proposed role in acrosomal exocytosis during mammalian fertilization.

Epigallocatechin-3-Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm-Zona Pellucida Heterologous Binding.

PubMed

Plaza Dávila, M; Bucci, D; Galeati, G; Peña, F J; Mari, G; Giaretta, E; Tamanini, C; Spinaci, M

2015-12-01

Stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production to achieve normal sperm function and to fuel the motility. The aim of this study was to evaluate the response of equine sperm under capacitating conditions to the inhibition of mitochondrial complex I by rotenone and to test whether epigallocatechin-3-gallate (EGCG), a natural polyphenol component of green tea, could counteract this effect. After 2-h incubation of stallion spermatozoa in modified Tyrode's medium, rotenone (100 nm, 500 nm and 5 μm) and EGCG (10, 20 and 60 μm), alone or in combination, did not induce any significant difference on the percentage of viable cells, live sperm with active mitochondria and spermatozoa with intact acrosome. The inhibition of complex I of mitochondrial respiratory chain of stallion sperm with rotenone exerted a negative effect on heterologous ZP binding ability. EGCG at the concentrations of 10 and 20 μm (but not of 60 μm) induced a significant increase in the number of sperm bound to the ZP compared with that for control. Moreover, when stallion sperm were treated with rotenone 100 nm, the presence of EGCG at all the concentrations tested (10, 20 and 60 μm) significantly increased the number of sperm bound to the ZP up to control levels, suggesting that this green tea polyphenol is able to reduce the toxicity of rotenone. © 2015 Blackwell Verlag GmbH.

Labelling of living mammalian spermatozoa with the fluorescent thiol alkylating agent, monobromobimane (MB): immobilization upon exposure to ultraviolet light and analysis of acrosomal status

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cummins, J.M.; Fleming, A.D.; Crozet, N.

1986-03-01

Living spermatozoa of seven mammalian species were treated with the thiol-alkylating fluorescent labelling compound, monobromobimane (MBBR). MB-labelling alone had no effect on sperm motility, nor on the time course or ability of golden hamster spermatozoa to undergo the acrosome reaction when capacitated in vitro. Exposure of MB-labelled spermatozoa to ultraviolet (UV) light and excitation of the MB fluorochrome resulted in virtually immediate immobilization of the spermatozoa without affecting acrosomal status. UV exposure of unlabelled spermatozoa for up to 30 sec had no effect upon motility. Immobilization of MB-labelled spermatozoa depended on the midpiece being irradiated, as irradiation of the headmore » alone, or of the more distal parts of the principal piece, had little or no effect upon motility. Labelling with MB followed by immobilization of individually selected spermatozoa was most useful for detailing the course and site of occurrence of the acrosome reaction during penetration of the cumulus oophorus by golden hamster spermatozoa in vitro. In these often hyperactivated spermatozoa, precise determination of the acrosomal status could not often otherwise be made due to the difficulty in visualizing the acrosomal region of a vigorously thrashing, hyperactivated spermatozoon. This technique should prove valuable in a variety of studies on sperm motility, capacitation and fertilization, and could also be extended to other cell systems.« less

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. Copyright © 2014 Elsevier Inc. All rights reserved.

Brown bear sperm double freezing: Effect of elapsed time and use of PureSperm(®) gradient between freeze-thaw cycles.

PubMed

Alvarez-Rodríguez, Manuel; Alvarez, Mercedes; López-Urueña, Elena; Martínez-Rodriguez, Carmen; Borragan, Santiago; Anel-López, Luis; de Paz, Paulino; Anel, Luis

2013-12-01

The use of sexed spermatozoa has great potential to captive population management in endangered wildlife. The problem is that the sex-sorting facility is a long distance from the semen collection place and to overcome this difficulty two freeze-thaw cycles may be necessary. In this study, effects of refreezing on brown bear electroejaculated spermatozoa were analyzed. We carried out two experiments: (1) to assess the effects of the two freezing-thawing cycles on sperm quality and to analyze three different elapsed times between freezing-thawing cycles (30, 90 and 180 min), and (2) to analyze the use of PureSperm between freezing-thawing cycles to select a more motile and viable sperm subpopulation which better survived first freezing. The motility, viability and undamaged acrosomes were significantly reduced after the second thawing respect to first thawing into each elapsed time group, but the elapsed times did not significantly affect the viability and acrosome status although motility was damaged. Our results with the PureSperm gradient showed higher values of viability in freezability of select sample (pellet) respect to the rest of the groups and it also showed a significant decrease in the number of acrosome damaged. In summary, the double freezing of bear semen selected by gradient centrifugation is qualitatively efficient, and thus could be useful to carry out a sex-sorting of frozen-thawed bear spermatozoa before to send the cryopreserved sample to a biobank. Given the low recovery of spermatozoa after applying a selection gradient, further studies will be needed to increase the recovery rate without damaging of the cell quality. Copyright © 2013 Elsevier Inc. All rights reserved.

Does natural honey act as an alternative to antibiotics in the semen extender for cryopreservation of crossbred ram semen?

PubMed Central

Banday, M. N.; Lone, F. A.; Rasool, F.; Rather, H. A.; Rather, M. A.

2017-01-01

Antibiotics are added to semen extenders to take care of heavy microbial load, however, their continuous use poses a constant threat of developing antibiotic resistance by the common microbes present in the semen. Our hypothesis was that natural honey, having antibacterial activity and rich in fructose could replace the use of antibiotics and fructose in the semen extender. Twenty-four ejaculates from six crossbred rams were obtained and extended with tris-based extender without (control) and with honey at 2.5% (T1), 5% (T2) and 7% (T3). Sperm quality was measured in terms of percentage sperm motility, live sperm count, intact acrosome and hypo-osmotic swelling test (HOST) reacted spermatozoa. The semen samples at post-thaw were also evaluated for total viable count (colony forming units/ml). At post-thaw, control exhibited significantly (P<0.05) higher sperm motility in comparison to T2 and T3. The percent of live sperm count, intact acrosome and HOST reacted spermatozoa were significantly higher (P<0.05) for control than all other treatment groups at post-thaw. Among treatment groups, T1 maintained significantly higher (P<0.05) percentage of live sperm count, intact acrosome and HOST reacted spermatozoa than T2 and T3. The total viable count at post-thaw was significantly lower (P<0.05) for control than all the treatment groups. In conclusion, honey cannot be used as an alternative to antibiotics to take care of heavy microbial load in semen, however, levels up to 2.5% may be supplemented to semen as an energy source. PMID:29387098

Metformin blocks mitochondrial membrane potential and inhibits sperm motility in fresh and refrigerated boar spermatozoa.

PubMed

Hurtado de Llera, A; Martin-Hidalgo, D; Garcia-Marin, L J; Bragado, M J

2018-06-01

Metformin is clinically used to treat diabetes. Given its role-impacting metabolism, metformin has been also added to semen cryopreservation media showing specie-dependent effects. We aimed to investigate metformin effects in both fresh (38.5°C for 2, 24 hr) and refrigerated (17°C for 10 days) boar spermatozoa. Metformin (2 hr) does not affect fresh sperm viability, membrane lipid organization nor acrosome integrity. However, metformin (24 hr) blocks sperm ΔΨm and significantly reduces % motile spermatozoa (65%), % progressive spermatozoa (50%), % rapid (100%), velocities VCL (69%), VSL (86%), VAP (78%) and motility coefficients. Metformin-including extender does not modify sperm viability, membrane lipid organization or acrosome integrity. Furthermore, it significantly reduces high ΔΨ-population spermatozoa at refrigeration day 4. Metformin also significantly reduces sperm motility during refrigeration. Summarizing, metformin inhibits both boar sperm ΔΨ and motility in any sperm condition studied: fresh and refrigerated. These findings dissuade metformin as an additive to improve boar sperm quality. © 2018 Blackwell Verlag GmbH.

Induction of ultra-morphological features of apoptosis in mature and immature sperm.

PubMed

Grunewald, Sonja; Fitzl, Guenther; Springsguth, Christopher

2017-01-01

There is a fundamental body of evidence suggesting that activated apoptosis signaling in ejaculated human sperm negatively influences their fertilization potential. However, it is still controversial whether this apoptotic signaling is a relic of an abortive apoptosis related to spermatogenesis or if it should be regarded as a functional preformed pathway in mature sperm leading to stereotypical morphological changes reflecting nuclear disassembly. To address this question, apoptosis was induced using betulinic acid in mature and immature ejaculated human sperm enriched by density gradient centrifugation. Execution of apoptosis was monitored by observing ultra-morphological changes via transmission electron microscopy. Typical morphological signs of apoptosis in somatic cells include plasma membrane blebbing with the formation of apoptotic bodies, impaired mitochondrial integrity, defects of the nuclear envelope, and nuclear fragmentation; these morphologies have also been observed in human sperm. In addition, these apoptotic characteristics were more frequent in immature sperm compared to mature sperm. Following betulinic acid treatment, apoptosis-related morphological changes were induced in mature sperm from healthy donors. This effect was much less pronounced in immature sperm. Moreover, in both fractions, the betulinic acid treatment increased the percentage of acrosome-reacted sperm. The results of our ultra-morphological study prove the functional competence of apoptosis in mature ejaculated human sperm. The theory of a sole abortive process may be valid only for immature sperm. The induction of the acrosome reaction by stimulating apoptosis might shed light on the biological relevance of sperm apoptosis.

A patch clamp study on reconstituted calcium permeable channels of human sperm plasma membranes.

PubMed

Ma, X H; Shi, Y L

1999-10-01

Ionic flux is thought to be important in the initiating process of gamete interaction such as acrosome reaction. However, modern electrophysiological methods, intracellular recording and patch-clamping, are difficult to approach the ion channels in mammal sperm membrane of an intact sperm due to its small size. In this work, by reconstituting the channel protein into lipid bilayer, Ca2+ channels in human spermatozoa were investigated with voltage clamp technique. Membrane proteins isolated from human sperm of 12 healthy donors were incorporated into lipid bilayer via fusion. In a cis 50//trans 10 mmol/L CaCl2 solution system, two types of channel events with similar reversal potential near the value of a perfect Ca2+ electrode, and sensitive to nifedipine and verapamil, were observed. Their unit conductance was 40 and 25 pS respectively. Percentage of channel open time was not dependent to holding potential for the former. However, for the channels of 25 pS, the percentage increased when the holding potential was changed from -20 to 100 mV. Ca(2+)-permeable channels were also detected from the spermatozoon samples of two infertile donors. Abnormal open time of these channels indicates that there are some defects in the conformation of the channel protein of infertile sperm membrane.

The Rab3A-22A Chimera Prevents Sperm Exocytosis by Stabilizing Open Fusion Pores.

PubMed

Quevedo, María F; Lucchesi, Ornella; Bustos, Matías A; Pocognoni, Cristian A; De la Iglesia, Paola X; Tomes, Claudia N

2016-10-28

At the final stage of exocytotis, a fusion pore opens between the plasma and a secretory vesicle membranes; typically, when the pore dilates the vesicle releases its cargo. Sperm contain a large dense-core secretory granule (the acrosome) whose contents are secreted by regulated exocytosis at fertilization. Minutes after the arrival of the triggering signal, the acrosomal and plasma membranes dock at multiple sites and fusion pores open at the contact points. It is believed that immediately afterward, fusion pores dilate spontaneously. Rab3A is an essential component of human sperm exocytotic machinery. Yet, recombinant, persistently active Rab3A halts calcium-triggered secretion when introduced after docking into streptolysin O-permeabilized cells; so does a Rab3A-22A chimera. Here, we applied functional assays, electron and confocal microscopy to show that the secretion blockage is due to the stabilization of open fusion pores. Other novel findings are that sperm SNAREs engage in α-SNAP/NSF-sensitive complexes at a post-fusion stage. Complexes are disentangled by these chaperons to achieve vesiculation and acrosomal contents release. Thus, post-fusion regulation of the pores determines their expansion and the success of the acrosome reaction. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Slow and ultra-rapid freezing protocols for cryopreserving mouflon (Ovis musimon) and fallow deer (Dama dama) epididymal sperm.

PubMed

Bóveda, P; Esteso, M C; Castaño, C; Toledano-Díaz, A; López-Sebastián, A; Muñiz, A; Prieto, P; Mejía, O; Ungerfeld, R; Santiago-Moreno, J

2018-05-01

This study examines the effectiveness of two methods for cryopreserving post-mortem epididymal sperm - conventional slow freezing employing a short equilibration time with glycerol, and ultra-rapid freezing - from the wild ruminant species Ovis musimon (mouflon) and Dama dama (fallow deer). A Tris-citric acid-glucose (TCG) + 12% egg yolk-based medium was used for the conventional slow freezing of the fallow deer sperm, whereas a Tes-Tris-glucose (TEST) + 6% egg yolk-based medium was used for the mouflon sperm. Glycerol was added to a final concentration of 5% to both media. The same diluents were used for ultra-rapid freezing but replacing the glycerol with 100 mM of sucrose. Sperm variables (motility, viability, acrosome integrity, membrane integrity, and morphological abnormalities) were analyzed before and after cryopreservation. Although values were generally better after the thawing of the conventionally cryopreserved sperm, total sperm motility (38.40 ± 4.44% in mouflon and 31.25 ± 3.37% in fallow deer) and total live sperm (47.19 ± 5.18% in mouflon and 43.13 ± 2.43% in fallow deer) were acceptable for the ultra-rapidly cooled sperm. Independent of the cryopreservation method, membrane integrity, acrosome integrity and the percentages of dead sperm and sperms with a damaged acrosome were better for the cryopreserved mouflon sperm than the fallow deer sperm (P < 0.05). Despite exerting a more harmful effect on sperm variables than conventional freezing, ultra-rapid freezing may be a useful alternative for the cryopreservation of these species' epididymal sperm in the field, as this simple technique does not require sophisticated equipment and expertise. Copyright © 2018 Elsevier B.V. All rights reserved.

Lactoferrin increases sperm membrane functionality of frozen equine semen.

PubMed

Martins, H S; da Silva, G C; Cortes, S F; Paes, F O; Martins Filho, O A; Araujo, Mss; Stahlberg, R; Lagares, M A

2018-06-01

During cryopreservation, sperm was submitted to an increase in reactive oxygen species generation. This work aimed to improve the quality of frozen equine sperm after the addition of antioxidants lactoferrin (Lf) and catalase (Cat) to a freezing extender. Semen from six stallions was frozen with the extenders: F1) control, INRA 82 freezing extender, F2) F1 + 500 μg/ml Lf and F3) F1 + 200 IU/ml Cat. After thawing, sperm motility parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome-reacted sperm were evaluated with a computer-assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite, hydroperoxide and iron concentrations of frozen semen were measured with spectrophotometry. The percentage of functional membrane sperm treated with Lf was higher (50.7% ± 11.6%) compared to that of the control (37.6% ± 15.6%), while the iron (61.4 ± 11.6 vs 73.3 ± 13.8 mg/dl) and nitrite concentrations (16.3 ± 7.1 vs 25.9 ± 4.2 μM/μg protein) were lower, respectively (p < .05). Thus, it can be suggested that Lf protect stallion spermatozoon during freezing as it has increased the percentage of sperm with functional membrane and decreased the lipid oxidant agents. © 2018 Blackwell Verlag GmbH.

Metabolites involved in cellular communication among human cumulus-oocyte-complex and sperm during in vitro fertilization.

PubMed

Gómez-Torres, María José; García, Eva María; Guerrero, Jaime; Medina, Sonia; Izquierdo-Rico, María José; Gil-Izquierdo, Ángel; Orduna, Jesús; Savirón, María; González-Brusi, Leopoldo; Ten, Jorge; Bernabeu, Rafael; Avilés, Manuel

2015-11-09

Fertilization is a key physiological process for the preservation of the species. Consequently, different mechanisms affecting the sperm and the oocyte have been developed to ensure a successful fertilization. Thus, sperm acrosome reaction is necessary for the egg coat penetration and sperm-oolema fusion. Several molecules are able to induce the sperm acrosome reaction; however, this process should be produced coordinately in time and in the space to allow the success of fertilization between gametes. The goal of this study was to analyze the metabolites secreted by cumulus-oocyte-complex (COC) to find out new components that could contribute to the induction of the human sperm acrosome reaction and other physiological processes at the time of gamete interaction and fertilization. For the metabolomic analysis, eighteen aliquots of medium were used in each group, containing: a) only COC before insemination and after 3 h of incubation; b) COC and capacitated spermatozoa after insemination and incubated for 16-20 hours; c) only capacitated sperm after 16-20 h in culture and d) only fertilization medium as control. Six patients undergoing assisted reproduction whose male partners provided normozoospermic samples were included in the study. Seventy-two COC were inseminated. The metabolites identified were monoacylglycerol (MAG), lysophosphatidylcholine (LPC) and phytosphingosine (PHS). Analysis by PCR and in silico of the gene expression strongly suggests that the cumulus cells contribute to the formation of the PHS and LPC. LPC and PHS are secreted by cumulus cells during in vitro fertilization and they could be involved in the induction of human acrosome reaction (AR). The identification of new molecules with a paracrine effect on oocytes, cumulus cells and spermatozoa will provide a better understanding of gamete interaction.

Treatment of rats during pubertal development with 2,3,7,8-tetrachlorodibenzo-p-dioxin alters both signaling kinase activities and epidermal growth factor receptor binding in the testis and the motility and acrosomal reaction of sperm.

PubMed

el-Sabeawy, F; Wang, S; Overstreet, J; Miller, M; Lasley, B; Enan, E

1998-06-01

Different doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (0.1, 1, 5, and 10 micrograms/kg body wt) were administered i.p. to 21-day-old male Sprague-Dawley rats. Control animals received the same volume of the vehicle (acetone:corn oil, 1:19). Body weight and daily food intake were recorded during the 90-day time course of the study. Random samples of five rats were sacrificed at 34, 49, 62, and 90 days of age. Epidermal growth factor receptor (EGFR) in whole testis was measured, as were the activities of c-Src kinase, protein tyrosine kinase (PTK), mitogen-activated protein 2 kinase (MAP2K also termed as Erk2), protein kinase A (PKA), and protein kinase C (PKC). Testicular tissue from 90-day-old rats was evaluated for histopathology, and sperm numbers in whole testis were counted to estimate daily sperm production. The motility of sperm in the vas deferens and caudal segments of the epididymis of 90-day-old rats was measured by computer assisted sperm analysis (CASA) and the function of the sperm was tested by assessment of acrosome reactions. A dose of 10 micrograms/kg resulted in testicular atrophy and histopathologic examination revealed a decrease in the diameter of the seminiferous tubules. Sertoli cell nuclei were clearly seen, but the spermatogonial population was totally absent. Lower doses of TCDD did not affect testicular histology, but doses as low as 1 microgram/kg significantly decreased testicular sperm numbers and affected some sperm functions (motility parameters and acrosome reactions) in 90-day-old rats. Significant decreases in EGFR were found in 34-day-old rats and this effect on EGFR was sustained until the end of the experiment (90 days). Although TCDD significantly increased c-Src kinase activity in immature and mature rats, opposite effects of TCDD on activities of PTK, PKA, and PKC were found in 34-day-old rats vs 49-, 62-, and 90-day-old rats. When 10 micrograms TCDD/kg was administered to 21-day-old rat, 24-h after c-Src kinase

Direct action of endocrine disrupting chemicals on human sperm

PubMed Central

Schiffer, Christian; Müller, Astrid; Egeberg, Dorte L; Alvarez, Luis; Brenker, Christoph; Rehfeld, Anders; Frederiksen, Hanne; Wäschle, Benjamin; Kaupp, U Benjamin; Balbach, Melanie; Wachten, Dagmar; Skakkebaek, Niels E; Almstrup, Kristian; Strünker, Timo

2014-01-01

Synthetic endocrine disrupting chemicals (EDCs), omnipresent in food, household, and personal care products, have been implicated in adverse trends in human reproduction, including infertility and increasing demand for assisted reproduction. Here, we study the action of 96 ubiquitous EDCs on human sperm. We show that structurally diverse EDCs activate the sperm-specific CatSper channel and, thereby, evoke an intracellular Ca2+ increase, a motility response, and acrosomal exocytosis. Moreover, EDCs desensitize sperm for physiological CatSper ligands and cooperate in low-dose mixtures to elevate Ca2+ levels in sperm. We conclude that EDCs interfere with various sperm functions and, thereby, might impair human fertilization. PMID:24820036

Normal sperm morphology and changes of semen characteristics and abnormal morphological spermatozoa among peri-mating seasons in captive japanese black bears (Ursus thibetanus japonicus).

PubMed

Okano, Tsukasa; Murase, Tetsuma; Nakamura, Sachiko; Komatsu, Takeshi; Tsubota, Toshio; Asano, Makoto

2009-04-01

The objectives of this study were to obtain morphological data for normal spermatozoa and to investigate seasonal changes (the early, mid- and post-mating seasons) in abnormal morphology of spermatozoa and the characteristics of semen in Japanese black bears. Semen was collected by electroejaculation from 34 captive male Japanese black bears a total of 74 times. Length of head, width of head, length of midpiece and total length of the spermatozoa were 6.3 +/- 0.4, 4.5 +/- 0.3, 10.4 +/- 0.7 and 69.6 +/- 3.1 mum (mean +/- SD; 20 semen, 200 spermatozoa), respectively. In the semen collected during the mid-mating season, ejaculate volume, ejaculate pH, sperm concentration, total sperm count, motility, viability and intact acrosomes were 0.46 +/- 0.36 ml, 7.3 +/- 0.4, 659 +/- 644 x 10(6)/ml, 214 +/- 208 x 10(6), 82.9 +/- 9.6%, 89.3 +/- 9.5% and 97.0 +/- 3.2% (mean +/- SD; n=21, in ejaculate pH n=8), respectively. Sperm motility and viability in the early (n=7) and mid-mating (n=21) seasons were significantly higher than in the post-mating (n=8) season. The rates of detached heads in the early and mid-mating season were significantly lower than in the post-mating season. The main abnormal morphologies observed (mean +/- SD%; n=23) were simply bent tail (19.9 +/- 22.6), distal droplets (13.5 +/- 11.7), proximal droplets (9.6 +/- 7.8), teratoid spermatozoa (6.7 +/- 10.7), knobbed acrosome (4.9 +/- 8.6), acrosome damage (3.7 +/- 2.8) and bent midpiece (3.7 +/- 5.1). The data will be useful for artificial breeding and further research on male reproductive physiology in this species.

Sodium-Hydrogen-Exchanger expression in human sperm and its relationship with semen parameters.

PubMed

Zhang, Zhe; Yang, Yuzhuo; Wu, Han; Zhang, Hongliang; Zhang, Haitao; Mao, Jiaming; Liu, Defeng; Zhao, Lianming; Lin, Haocheng; Tang, Wenhao; Hong, Kai; Jiang, Hui

2017-06-01

Sperm-specific sodium-hydrogen exchanger (sNHE) is essential to maintain sperm normal function in mice; however, its role in human sperm has not been clarified to date. The aim of this study is to investigate the expression pattern of sNHE in human spermatozoa and its relationship with sperm functional parameters. Semen samples from 68 asthenozoospermic and 61 normozoospermic men were analyzed for sperm concentration, motility, and acrosome reaction, and high motile spermatozoa were collected by swim-up method. The expression of sNHE in spermatozoa was detected by Western blot and immunofluorescence staining. The relationship between sNHE expression and sperm parameters was assessed. We identified sNHE is mainly localized to the principal piece of the human sperm tail. The expression of sNHE was positively correlated with sperm concentration, total number, and progressive motility. Moreover, sNHE expression was upregulated in swim-up sperm and associated with most of sperm motility parameters including straight line velocity and curvilinear velocity. Our results also showed that sNHE expression is decreased in sperm from patients with asthenozoospermia compared with that from normal controls. However, no correlation was found between sNHE expression and acrosome reaction in spermatozoa. The expression pattern of sNHE suggested that this protein may be involved in the regulation of sperm motility, and aberration of its expression in sperm may contribute to the pathogenesis of asthenozoospermia.

Sperm-egg penetration assay assessment of the contraceptive effects of glycerol and egg yolk in rooster sperm diluents.

PubMed

Abouelezz, F M K; Castaño, C; Toledano-Díaz, A; Esteso, M C; López-Sebastián, A; Campo, J L; Santiago-Moreno, J

2015-06-01

Glycerol (GLY) and egg yolk (EY) are good cryoprotectants of avian and mammalian sperm, but in birds, they strongly inhibit the eventual fertilization of ova. Using the sperm penetration (SP-holes) assay and fertility trials, the present study investigates (1) the possible mechanism by which this contraceptive effect occurs in chickens and (2) the maximum concentrations of GLY and EY tolerated by fresh rooster sperm. Seventy Black-Barred Andaluza hens (five per treatment) were inseminated four times (twice per week) with 0.1 mL of fresh semen from roosters of the same breed diluted 1:1 (v:v) with Lake and Ravie medium containing different concentrations of GLY or EY. No adverse effects on acrosome integrity, sperm motility, or viability were seen with any concentration of GLY or EY. The number of SP-holes on perivitelline layer samples taken from above the germinal disc became progressively lower at GLY concentrations of 1.5% or greater (P > 0.05). No holes caused by sperms were seen in unfertilized eggs. The corresponding fertility results showed similar reductions when the GLY concentration was 1.5% or greater. No changes in the number of SP-holes were seen with increasing EY concentrations (0%-7.5%), nor were any differences in fertility observed, except for a reduction when 15% EY was used. The results therefore reveal that GLY affects the transit of sperms through the oviduct in their attempt to reach the infundibulum area, limiting their access to the ovum perivitelline layer. Egg yolk had no such effect, nor did it influence acrosome reaction capacity; its mechanism of contraceptive action therefore remains unknown. The maximum GLY and EY concentrations tolerated by the rooster sperm were 0.75% and 7.5%, respectively. Copyright © 2015 Elsevier Inc. All rights reserved.

Influence of temperature and sperm preparation on the quality of spermatozoa.

PubMed

Thijssen, Annelies; Klerkx, Elke; Huyser, Carin; Bosmans, Eugene; Campo, Rudi; Ombelet, Willem

2014-04-01

This study investigated the effects of long-term (24h) in-vitro sperm incubation at room temperature (RT; 23°C) versus testis temperature (35°C) on various sperm-quality parameters. Semen samples (n=41) were prepared both by density-gradient centrifugation (DGC) and the swim-up technique in order to compare the influence of sperm preparation on sperm quality after incubation. Progressive motility and morphology were significantly higher after incubation at RT compared with 35°C (P<0.001 and P<0.01, respectively). The proportions of acrosome-reacted, apoptotic and dead spermatozoa were significantly lower in samples incubated for 24h at RT compared with 35°C (P<0.001, P=0.01 and P<0.001, respectively). The number of motile, morphologically normal, non-acrosome-reacted and nonapoptotic spermatozoa recovered after sperm preparation was significantly higher in DGC compared with swim-up samples (P<0.001). However, spermatozoa prepared by swim-up showed better survival after incubation compared with DGC-prepared spermatozoa, especially when incubated at 35°C. In conclusion, this study indicates a significantly better and longer preservation of sperm quality when incubation is performed at RT. These findings may convince laboratories to change the routinely used sperm storage conditions in order to maximize the quality of the prepared sperm sample. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization

PubMed Central

Jin, Mayuko; Fujiwara, Eiji; Kakiuchi, Yasutaka; Okabe, Masaru; Satouh, Yuhkoh; Baba, Shoji A.; Chiba, Kazuyoshi; Hirohashi, Noritaka

2011-01-01

To fuse with oocytes, spermatozoa of eutherian mammals must pass through extracellular coats, the cumulus cell layer, and the zona pellucida (ZP). It is generally believed that the acrosome reaction (AR) of spermatozoa, essential for zona penetration and fusion with oocytes, is triggered by sperm contact with the zona pellucida. Therefore, in most previous studies of sperm–oocyte interactions in the mouse, the cumulus has been removed before insemination to facilitate the examination of sperm–zona interactions. We used transgenic mouse spermatozoa, which enabled us to detect the onset of the acrosome reaction using fluorescence microscopy. We found that the spermatozoa that began the acrosome reaction before reaching the zona were able to penetrate the zona and fused with the oocyte's plasma membrane. In fact, most fertilizing spermatozoa underwent the acrosome reaction before reaching the zona pellucida of cumulus-enclosed oocytes, at least under the experimental conditions we used. The incidence of in vitro fertilization of cumulus-free oocytes was increased by coincubating oocytes with cumulus cells, suggesting an important role for cumulus cells and their matrix in natural fertilization. PMID:21383182

Season-induced changes in bovine sperm motility following a freeze-thaw procedure.

PubMed

Orgal, Shlomo; Zeron, Yoel; Elior, Nili; Biran, David; Friedman, Eran; Druker, Shaked; Roth, Zvi

2012-01-01

Decreased conception rate of dairy cows in the summer is mainly associated with the deleterious effects of environmental thermal stress on the female reproductive tract. Here, we suggest that decreased reproductive performance might be partially due to inferior-quality semen. Semen from five representative bulls was collected in summer (August to September) and winter (December to January) and evaluated with a computerized sperm-quality analyzer for bulls (SQA-Vb). No seasonal effect was found in fresh ejaculate, but sperm examined post-thawing showed lower velocity, motility and progressive motility (P<0.04) in summer vs. winter samples. Element concentrations in the seminal plasma, determined by inductively coupled plasma-atomic emission spectrometry, differed between seasons, with higher (P<0.01) concentration values of K, Mg, Na and S elements in winter vs. summer samples. Therefore, season-induced alterations in seminal plasma element concentration should be taken into account when using an extender for cryopreservation. Acrosome integrity was assessed by a triple-fluorescence test using Hoechst 33342, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and propidium iodide. Acrosome reaction was examined by a one-step staining method using FITC-PSA. The proportion of sperm cells with a damaged acrosome post-thawing tended to be higher (P<0.07) in semen collected during the summer vs. winter. Such alterations suggest that seasonal reductions in sperm function might also be involved in the decreased conception rate of dairy cows in summer.

Tripeptidyl Peptidase II Regulates Sperm Function by Modulating Intracellular Ca2+ Stores via the Ryanodine Receptor

PubMed Central

Zhou, Yuchuan; Ru, Yanfei; Wang, Chunmei; Wang, Shoulin; Zhou, Zuomin; Zhang, Yonglian

2013-01-01

Recent studies have identified Ca2+ stores in sperm cells; however, it is not clear whether these Ca2+ stores are functional and how they are mobilized. Here, in vitro and in vivo, we determined that tripeptidyl peptidase II antagonists strongly activated the cAMP/PKA signaling pathway that drives sperm capacitation-associated protein tyrosine phosphorylation. We demonstrated that in the absence of Ca2+, TPIII antagonists elevated the intracellular Ca2+ levels in sperm, resulting in a marked improvement in sperm movement, capacitation, acrosome reaction, and the in vitro fertilizing ability. This antagonist-induced release of intracellular Ca2+ could be blocked by the inhibitors of ryanodine receptors (RyRs) which are the main intracellular Ca2+ channels responsible for releasing stored Ca2+. Consistent with these results, indirect immunofluorescence assay using anti-RyR antibodies further validated the presence of RyR3 in the acrosomal region of mature sperm. Thus, TPPII can regulate sperm maturation by modulating intracellular Ca2+ stores via the type 3 RyR. PMID:23818952

Zona-free oocyte fertilized with intracytoplasmic sperm injection and underwent further division: case report and literature review.

PubMed

Hsieh, Y Y; Chang, C C; Tsai, H D

2001-09-01

The zona pellucida (ZP) plays a protective role during fertilization and early embryonic development. It is related to sperm binding, the acrosome reaction, prevention of polyspermic fertilization, and holding blastomeres together before the morular stage. Zona-free oocytes are accidentally encountered. If these oocytes are healthy, they can be fertilized normally by intracytoplasmic sperm injection (ICSI). We reported on a couple with male infertility undergoing oocyte retrieval after ovarian hyperstimulation. Before the ICSI procedure, cumulus cells surrounding the oocytes were removed, which resulted in one oocyte escaping from its ZP. The zona-free oocyte was fertilized normally with ICSI and developed to the 8-cell stage. We observed that the zona-free zygote had the ability to further divide, despite its loose contact. The zona-free embryo was transferred with other zona-intact embryos, but the implantation failed. We conclude that zona-free oocytes can be rescued, fertilized with ICSI, and cultured for further transfer or cryopreservation.

Detection of cooling-induced membrane changes in the response of boar sperm to capacitating conditions.

PubMed

Petrunkina, Anna M; Volker, Gabriele; Weitze, Karl-Fritz; Beyerbach, Martin; Töpfer-Petersen, Edda; Waberski, Dagmar

2005-05-01

There is a need for methods of rapid and sensitive sperm function assessment. As spermatozoa are not able to fertilize an oocyte before having undergone a series of complex physiological changes collectively called capacitation, it is logical to assess sperm function under fertilizing conditions in vitro. In this study, the responsiveness of sperm to capacitating conditions in vitro was monitored by changes in sperm response to ionophore and by changes in the amount of intracellular calcium ions in stored boar semen. Boar semen was diluted at 32 and 20 degrees C and stored for 24 and 72 h at 16 and 10 degrees C. Ionophore-induced changes and increased intracellular calcium ion content in boar spermatozoa were recorded by flow cytometry and found to progress as a function of time during incubation under capacitating conditions. All responsiveness parameters (increases in proportions of membrane-defective spermatozoa, acrosome-reacted spermatozoa, and cells with high intracellular calcium levels) were shown to be sensitive to subtle physiological changes occurring at low storage temperatures. The initial levels of sperm with a high calcium content were higher in semen stored at 10 degrees C, but the accumulation of internal calcium was lower than in semen stored at 16 degrees C. The loss of membrane integrity and increase in the proportion of acrosome-reacted cells were higher in semen stored at 10 degrees C. Dilution at 20 degrees C had no negative effect on membrane integrity or responsiveness to capacitating conditions. There was no significant difference between semen stored for 24 and 72 h in terms of membrane integrity, acrosome reaction, and intracellular calcium after capacitation treatment. However, dynamics of cell death and acrosome reaction in response to capacitating conditions were somewhat accelerated after 72 h storage, especially in semen stored at 10 degrees C. It can be concluded that the simultaneous use of the sperm membrane responsiveness and

Involvement of Fibroblast Growth Factor 2 (FGF2) and its receptors in the regulation of mouse sperm physiology.

PubMed

Saucedo, Lucia; Sobarzo, Cristian; Brukman, Nicolás; Guidobaldi, Hector Alejandro; Lustig, Livia; Giojalas, Laura Cecilia; Buffone, Mariano Gabriel; Vazquez-Levin, Monica Hebe; Marín-Briggiler, Clara

2018-06-04

Fibroblast Growth Factor 2 (FGF2) and its receptors (FGFRs) have been described in several tissues, where they regulate cellular proliferation, differentiation, motility and apoptosis. Although FGF2/FGFRs expression in the male reproductive tract has been reported, there is scarce evidence on their presence in the female reproductive tract and their involvement in the modulation of sperm function. Therefore, the objective of this study was to determine the expression of FGF2 in the female reproductive tract and to assess the role of the FGF2/FGFRs system in the regulation of sperm physiology using the murine model. FGF2 was detected in uterus and oviduct protein extracts, and it was immunolocalized in epithelial cells of the uterus, isthmus and ampulla, as well as in the cumulus oophorus-oocyte complex. The receptors FGFR1, FGFR2, FGFR3 and FGFR4 were immunodetected in the flagellum and acrosomal region of sperm recovered from the cauda epididymis. Analysis of testis sections showed the expression of FGFRs in germ cells at different stages of the spermatogenesis, suggesting the testicular origin of the sperm FGFRs. Sperm incubation with recombinant FGF2 (rFGF2) led to increased sperm motility and velocity, and to enhanced intracellular Ca2+ levels and acrosomal loss compared to the control. In conclusion, this study shows that FGF2 is expressed in tissues of the female reproductive tract. Also, the fact that functional FGFRs are present in mouse sperm and that rFGF2 affects sperm motility and acrosomal exocytosis, suggests the involvement of this system in the in vivo regulation of sperm function.

Quercetin improves the postthaw characteristics of cryopreserved sex-sorted and nonsorted stallion sperm.

PubMed

Gibb, Z; Butler, T J; Morris, L H A; Maxwell, W M C; Grupen, C G

2013-04-01

Excessive reactive oxygen species generation during sex sorting and cryopreservation of stallion sperm leads to DNA fragmentation, lipid peroxidation, and motility loss. In this study we investigated whether antioxidant supplementation during sex sorting and cryopreservation could ameliorate the effects of reactive oxygen species on stallion sperm. In experiment 1, the postthaw characteristics of stallion sperm (N = 9) cryopreserved in the presence or absence of catalase (200 U/mL), cysteine (0.2 mg/mL), or quercetin (0.15 mM) was examined. Motility and acrosome integrity were assessed at 0, 1, and 3 hours after thawing. The sperm chromatin structure assay (SCSA; detectable DNA fragmentation index [DFI], mean DFI, and DFI) was used to assess DNA integrity immediately after thawing. Quercetin increased the total postthaw motility (25.3% vs. 20.9%; P < 0.05), but there was no beneficial effect of catalase or cysteine. Based on these results, the effect of quercetin during cryopreservation on the postthaw zona binding ability of sperm was assessed using a heterologous (bovine) zona binding assay. Quercetin increased the number of sperm bound per oocyte (13.6 vs. 9.2; P < 0.05) compared with the control. In experiment 2, the effect of quercetin (0.15 mM) in the media used during semen storage and transport, Hoechst 33342 staining and cryopreservation of stallion sperm (N = 9) was investigated. Motility, acrosome integrity, and viability were assessed at 0, 1, and 3 hours after thawing and SCSA was performed at 0 hours after thawing. Quercetin supplementation during sex sorting and cryopreservation improved DNA integrity (SCSA; detectable DFI of 54.9% vs. 74.6%, P < 0.05; mean DFI of 270.2 vs. 288.1, P < 0.05; and DFI of 26.3% vs. 28.5%, P < 0.05) compared with control sex-sorted sperm. There was no beneficial effect of quercetin on the motility, acrosome integrity, or viability of sex-sorted sperm. In conclusion, quercetin significantly improved the motility and zona

Interaction of extender composition and freezing method for effective semen cryopreservation in the North American river otter (Lontra canadensis).

PubMed

Bateman, Helen L; Swanson, William F

2017-10-01

Semen cryopreservation and storage in genome resource banks (GRBs), in combination with artificial insemination (AI), could be invaluable for genetic management and conservation of endangered otter species. For any applied conservation benefit, effective methods for otter sperm processing and cryopreservation first must be established. In this study, our objective was to develop an effective semen cryopreservation method for the North American river otter, evaluating the effect of extender composition (i.e., glycerol concentration, Equex STM paste supplementation) and freezing protocol (timing of glycerol addition, pre-freeze cooling rate, freezing/packaging method) on post-thaw sperm motility, longevity and acrosome status. Semen was collected from 14 otters housed at 9 zoos, and following cryopreservation in an egg-yolk based extender, thawed to assess sperm motility and acrosome status immediately post-thaw and during 6 h of in vitro culture. Results indicated that extender containing 4% glycerol was preferable (p < 0.05) to 8% glycerol but the temperature/timing of extender addition containing 4% glycerol did not affect (p > 0.05) post-thaw sperm parameters. Treatments with extender containing Equex and frozen by pelleting on dry ice showed greater (p < 0.05) motility and percentage of intact acrosomes compared to treatments frozen in extender without Equex, regardless of pre-freeze cooling rate. In the absence of Equex, pelleting provided superior post-thaw sperm motility (p < 0.01) and higher (p < 0.001) percentage of sperm with intact acrosomes compared to samples frozen in straws over liquid nitrogen vapor. Results of this study indicate that cryopreservation of otter sperm using an egg-yolk -TEST based extender containing 4% glycerol and 1% Equex, with the pellet freezing method, provided superior post-thaw sperm motility, longevity and acrosomal integrity compared to other combinations. Neither alterations in timing of glycerolated extender

Effect of cryoprotective diluent and method of freeze-thawing on survival and acrosomal integrity of ram spermatozoa.

PubMed

Pontbriand, D; Howard, J G; Schiewe, M C; Stuart, L D; Wildt, D E

1989-08-01

A multifactorial study analyzed the effects of freezing method, cryoprotective diluent, semen to diluent ratio, and thawing velocity on post-thaw motility, progressive status, and acrosomal integrity of ram spermatozoa. Although semen to diluent ratio (1:3 vs 1:6, v/v) had no effect (P greater than 0.05), overall post-thaw spermatozoal viability was highly dependent on freezing method and cryoprotectant. Improved results were obtained by freezing semen in 0.5-ml French straws compared to dry ice pelleting. Manually freezing straws 5 cm above liquid nitrogen (LN2) was comparable to cooling straws in an automated, programmable LN2 unit. Of the two cryoprotective diluents tested, BF5F (containing the surfactant component sodium and triethanolamine lauryl sulfate) yielded approximately 50% fewer (P less than 0.05) spermatozoa with loose acrosomal caps compared to TEST. Thawing straws in a water bath at a higher velocity (60 degrees C for 8 sec) had no effect (P greater than 0.05) on spermatozoal motility, progressive status ratings, or acrosomal integrity when compared to a lower rate (37 degrees C for 20 sec). For the TEST group, thawing pellets in a dry, glass culture tube promoted (P less than 0.05) percentage sperm motility at 3 and 6 hr post-thawing, but for BF5F diluted semen this approach decreased the % of spermatozoa with normal apical ridges. The results suggest that the poor fertility rates often experienced using thawed ram semen likely result not only from reduced sperm motility, but also from compromised ultrastructural integrity. This damage is expressed by an increased loosening of the acrosomal cap, a factor which appears insensitive to freezing method but markedly influenced by the cryoprotective properties of the diluents tested.

In vitro effects of nonylphenol on motility, mitochondrial, acrosomal and chromatin integrity of ram and boar spermatozoa.

PubMed

Uguz, C; Varisli, O; Agca, C; Evans, T; Agca, Y

2015-10-01

The objective of this study was to determine the effects of nonylphenol (NP) on viability of ram and boar sperm in vitro. Ram or boar spermatozoa were exposed to 1, 10, 100, 250 and 500 μg NP ml(-1) for 1, 2, 3 or 4 h. Computer-assisted sperm motility analysis (CASA) system was used to evaluate sperm motility characteristics. Flow cytometry was used to determine mitochondrial membrane potential (MMP) and chromatin integrity, while epifluorescent microscopy was used to determine sperm acrosomal status. Exposure of both species spermatozoa to 250 and 500 μg NP ml(-1) was detrimental to progressive motility (P < 0.05), and its adverse effect was significant at lower (100 μg NP ml(-1) ) concentration (P < 0.05). The percentages of ram and boar spermatozoa with high MMP declined drastically after exposures to ≥250 μg ml(-1) NP (P < 0.05). Unlike chromatin integrity, which did not appear to be altered by NP exposure, there were dose-dependent NP effects (P < 0.05) on acrosomal integrity of both species at as low as 1 μg ml(-1) NP for boar spermatozoa and 10 μg ml(-1) NP for ram spermatozoa. These data show adverse effects of NP on ram and boar spermatozoa and thus its potential harmful effects on male reproduction as NP is found in fruits, vegetables, human milk, fish and livestock products. © 2014 Blackwell Verlag GmbH.

[Molecules involved in sperm-zona pellucida interaction in mammals. Role in human fertility].

PubMed

Serres, Catherine; Auer, Jana; Petit, François; Patrat, Catherine; Jouannet, Pierre

2008-01-01

Fertilization in mammals requires an initial interaction of sperm with the oocyte envelope, the zona pellucida (ZP), before it reaches the oocyte. ZP is a highly glycosylated structure, composed of three (mouse) or four (rabbit, boar, bovine, humans...) glycoproteins. The presence of ZP around the oocyte does not allow heterospecific fertilization. This barrier is principally due to the presence of species-specific glycosylations on ZP proteins. Sperm bind ZP by means of membrane receptors which recognize carbohydrate moieties on ZP glycoproteins according to a well-precised sequential process. Upon initial attachment, spermatozoa bind ZP3/ZP4 which induces the sperm acrosome exocytosis followed by a secondary binding of acrosome reacted spermatozoa to ZP2 and by ZP penetration. The sperm receptors are adhesive proteins or integral plasma membrane proteins linked to intraspermatic signalling pathways activating the acrosome reaction. Over the last twenty years, numerous studies have been carried out to identify sperm receptors to ZP in several species, but the data in humans are still incomplete. Work initiated in our research group has identified several proteins interacting with recombinant human ZP2, ZP3 and ZP4, among which are glycolytic enzymes. These enzymes are involved in the gamete interaction by means of their affinity to sugars and not by their catalytic properties. From a clinical point of view, an observed lack or weak expression of some sperm receptors to ZP3 in cases of idiopathic infertility associated with in vitro fertilization failure suggests that knowing the molecular mechanism driving the gamete recognition can be important at the diagnostic level. Furthermore, it has been shown that proteins that mediate gamete recognition diverge rapidly, as a result of positive darwinian selection. A sexual conflict can drive co-evolution of reproductive molecules in both sexes resulting in reproductive isolation and species emergence.

Improved cryopreservability of stallion sperm using a sorbitol-based freezing extender.

PubMed

Pojprasath, T; Lohachit, C; Techakumphu, M; Stout, T; Tharasanit, T

2011-06-01

Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. Sugars are among the important components of a freezing extender and act as non-permeating cryoprotectants. This study aimed to compare the quality of stallion sperm frozen with glucose, fructose or sorbitol-containing freezing extenders. Semen was collected from six stallions of proven fertility and cryopreserved using a freezing extender containing different types of monosaccharide sugars (glucose, fructose or sorbitol). After thawing, the semen was examined for sperm motility, viability, acrosome integrity, plasma membrane functionality and sperm longevity. The fertility of semen frozen in the presence of sorbitol was also tested by artificial insemination. Sperm quality was significantly decreased following freezing and thawing (P < 0.05). Fructose was inferior for protecting sperm during cryopreservation when compared to sorbitol and glucose (P < 0.05). Although the viability, motility and acrosome integrity of sperm cryopreserved with a glucose-containing extender did not significantly differ from sperm frozen in the sorbitol-based extender when examined at 2 and 4 h post-thaw, all of these parameters plus plasma membrane functionality were improved for sperm frozen in the sorbitol extender than in the glucose extender when examined 10 min post-thaw. Two of four mares (50%) inseminated with semen frozen with a sorbitol-containing freezing extender became pregnant. It is concluded that different sugars have different abilities to protect against cryoinjury during freezing and thawing of stallion sperm. This study demonstrated that an extender containing sorbitol as primary sugar can be used to successfully cryopreserve equine sperm; moreover, the quality of frozen-thawed sperm appeared to be better than when glucose or fructose was the principle sugar in the freezing extender. Copyright © 2011 Elsevier Inc. All rights reserved.

Ascorbic acid, catalase and chlorpromazine reduce cryopreservation-induced damages to crossbred bull spermatozoa.

PubMed

Paudel, K P; Kumar, S; Meur, S K; Kumaresan, A

2010-04-01

The present study evaluated the effectiveness of ascorbic acid, catalase, chlorpromazine and their combinations in reducing the cryodamages to crossbred bull (Bos taurus x Bos indicus) spermatozoa. A total of 32 ejaculates (eight each from four bulls) were diluted in Tris-citric acid-fructose-egg yolk-glycerol extender. Each ejaculate was split into six parts (five treatment and one control). Treatment groups included 10 mm ascorbic acid, 0.1 mm chlorpromazine, 200 IU/ml catalase, 10 mm ascorbic acid + 0.1 mm chlorpromazine or 200 IU/ml catalase + 0.1 mm chlorpromazine in the extender. Fluorescent probes (Fluorescein isothiocyanate--Pisum sativum agglutinin + Propidium iodide) were used for the assessment of spermatozoa viability and acrosomal status. The proportion of acrosome intact live (AIL), acrosome intact dead, acrosome reacted live and acrosome reacted dead sperm was assessed in fresh, equilibrated and frozen-thawed semen. The functional status of the sperm was assessed using hypo-osmotic sperm swelling test (HOSST). Activities of acrosin and hyaluronidase enzyme were also determined. Lipid peroxidation level was assayed based on the melonaldehyde (MDA) production. In cryopreserved semen, the values of AIL spermatozoa, HOSST response, hyaluronidase and acrosin activity were reduced by 53%, 47%, 34% and 54%, respectively from their initial values in fresh semen. However, MDA level was threefold higher in the frozen-thawed sperm compared with fresh sperm. Significant (p < 0.05) improvement in motility, viability, HOSST response, retention of hyaluonidase and acrosin and reduction in MDA was recorded in ascorbic acid, catalase, ascorbic acid + chlorpromazine and catalase + chlorpromazine incorporated groups. The percentage of AIL sperm was significantly (p < 0.05) higher in ascorbic acid, catalase and ascorbic acid + chlorpromazine incorporated groups compared with the control. Chlorpromazine alone did not improve the post-thaw semen quality but when combined

Sperm morphology in four species of African platypleurine cicadas (Hemiptera: Cicadomorpha: Cicadidae).

PubMed

Chawanji, A S; Hodgson, A N; Villet, M H

2005-08-01

Mature spermatozoa from four species of platypleurine cicadas (Albanycada albigera, Azanicada zuluensis, Platypleura capensis and P. hirtipennis) were examined by light and electron microscopy. The filiform sperm have a similar ultrastructure in all species but notable variations were found in sperm dimensions. All species produce more than one discrete length of nucleated, motile sperm, a form of polymorphism termed polymegaly. Polymegaly is expressed in two ways: sperm have bi- or trimodal head and tail lengths. The anterior parts of sperm heads are embedded in an elongate homogenous matrix forming a spermatodesm. The conical acrosome is deeply invaginated posteriorly, and sits on top of the nucleus. The acrosomal contents are differentiated internally with an electron-lucent central medulla and a denser cortex. The homogenously electron-dense nucleus is pointed anteriorly and is generally cylindrical, although posteriorly there is a lateral invagination that extends part-way along the nucleus. This invagination houses fine granular material of the centriolar adjunct. Vesicle-like elements that are associated with both the posterior nucleus and the centriolar adjunct are also found within the invagination. Immediately posterior of and adjoining the centriolar adjunct is a pair of mitochondrial derivatives that are elongated and extend for almost the entire length of the tail. The absence of accessory bodies in cicada sperm suggests that within the Cicadomorpha, the families Cicadidae and Cercopidae are closely affiliated.

Comparative morphology of zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussel sperm: Light and electron microscopy

USGS Publications Warehouse

Walker, G.K.; Black, M.G.; Edwards, C.A.

1996-01-01

Adult zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussels were induced to release large quantities of live spermatozoa by the administration of 5-hydroxytryptamine (serotonin). Sperm were photographed alive using phase-contrast microscopy and were fixed subsequently with glutaraldehyde followed by osmium tetroxide for eventual examination by transmission or scanning electron microscopy. The sperm of both genera are of the ect-aquasperm type. Their overall dimensions and shape allow for easy discrimination at the light and scanning electron microscopy level. Transmission electron microscopy of the cells reveals a barrel-shaped nucleus in zebra mussel sperm and an elongated nucleus in quagga mussel sperm. In both species, an acrosome is cradled in a nuclear fossa. The ultrastructure of the acrosome and axial body, however, is distinctive for each species. The structures of the midpiece are shown, including a unique mitochondrial "skirt" that includes densely packed parallel cristae and extends in a narrow sheet from the mitochondria.

Red wolf (Canis rufus) sperm quality and quantity is affected by semen collection method, extender components, and post-thaw holding temperature.

PubMed

Franklin, Ashley D; Waddell, William T; Goodrowe, Karen L

2018-08-01

using EE compared to UC (P = 0.041), and sperm samples collected using EE also had greater motility and FPS (P < 0.05). Additionally, though no gross morphological differences were observed, there were fewer sperm with intact acrosomes in the samples collected via UC (P = 0.0443). Thus, UC should not be considered sufficient for semen collection in red wolves when the desired fate of sperm is cryopreservation and/or AI. However, UC does provide an opportunity for a basic reproductive evaluation of a red wolf male. Copyright © 2018 Elsevier Inc. All rights reserved.

Improved quality of cryopreserved cheetah (Acinonyx jubatus) spermatozoa after centrifugation through Accudenz.

PubMed

Crosier, Adrienne E; Henghali, Josephine N; Howard, Jogayle; Pukazhenthi, Budhan S; Terrell, Kimberly A; Marker, Laurie L; Wildt, David E

2009-01-01

Sperm cryopreservation, in combination with assisted reproductive techniques, is a valuable tool for the genetic management of endangered felids. However, the acrosome of the cheetah spermatozoon is especially sensitive to cryopreservation, with approximately 40% of spermatozoa experiencing acrosomal damage immediately after thawing and then another approximately 15% loss during the next 4 hours in vitro. Additionally, thawing causes a reduction in sperm motility by approximately 20% with another decrease of approximately 12% during subsequent incubation in vitro. We hypothesized that slow removal of glycerol from cryopreserved cheetah spermatozoa using an Accudenz gradient would improve acrosomal integrity, sperm motility longevity, and structural morphology. Accudenz was compared with traditional cheetah sperm processing methods for glycerol removal that involves washing, multistep resuspension, and swim-up processing. Electroejaculates (n = 21 total from 8 males) were washed in Ham F10 medium, and sperm pellets were resuspended in TEST-yolk buffer with 0% glycerol. Samples were cryopreserved in straws in 4% final glycerol, thawed, and assessed for percent intact acrosomes (% IA), percent motility (% M), and forward progressive status (FPS; scale, 0-5). Sperm motility index (SMI) was calculated as (% M + [FPS x 20]) / 2. In study 1, glycerol removal by centrifugation through an Accudenz gradient (4%, 10%) was compared with traditional sperm washing (control) and multistep resuspension protocols. At each time after centrifugation (hourly for 4 hours), % IA was improved (P < .05) for Accudenz (range, 36%-39%) compared with control (30%-33%) and multistep (29%-33%) treatments. In study 2, a modified Accudenz protocol was compared with traditional washing and was found to improve (P < .05) SMI (range, 52-64) compared with controls (range, 41-52) at each time postthaw after centrifugation. In study 3, swim-up processed sperm were compared with those treated by

Sperm quality and fertility of boar seminal doses after 2 days of storage: does the type of extender really matter?

PubMed

Pinart, Elisabeth; Yeste, Marc; Prieto-Martínez, Noelia; Reixach, Josep; Bonet, Sergi

2015-06-01

The present approach was designed to evaluate the extender effects on sperm quality and fertility of short-term refrigerated seminal doses from Landrace boars lodged in husbandry-controlled conditions. For this purpose, we analyzed the sperm quality of seminal doses diluted in short-term (Beltsville Thawing Solution) and extra-long-term (Duragen) extenders from Days 0 to 2 of storage at 17 °C during an 8-month period. Pregnancy rates and litter size were evaluated from double inseminations within an interval of 12 hours (36 and 48 hours of refrigeration) of multiparous females using seminal doses diluted in each extender type. Sperm quality was assessed from the analyses of sperm motility and kinetics, sperm viability, expressed as plasma and acrosome membrane integrity, membrane lipid disorder, intracellular calcium levels, and acrosin activity. Results indicated significant differences between the extenders in the sperm quality of seminal doses. Therefore, the seminal doses diluted in Duragen had higher percentages of progressive motile spermatozoa and membrane-intact spermatozoa than those diluted in Beltsville Thawing Solution throughout all the experimental months. Nevertheless, despite these differences in preserving the sperm quality, pregnancy rates (>90%) and litter sizes (>10 piglets born per litter) were similar between the extenders. Our results had great relevance from a practical point of view because they reported lack of an extender effect on the reproductive performance of seminal doses during short-tem storage. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of pH during liquid storage of goat semen on sperm viability and fertilizing potential.

PubMed

Liu, Chang-He; Dong, Hai-Bo; Ma, Dong-Li; Li, You-Wei; Han, Dong; Luo, Ming-Jiu; Chang, Zhong-Le; Tan, Jing-He

2016-01-01

A specific problem in goat semen preservation is the detrimental effect of seminal plasma on sperm viability in extenders containing yolk or milk. Thus, the use of chemically defined extenders will have obvious advantages. Although previous studies indicate that the initial pH of an extender is crucial to sustain high sperm motility, changes in extender pH during long-term semen storage have not been observed. Monitoring extender pH at different times of semen storage and modeling its variation according to nonlinear models is thus important for protocol optimization for long-term liquid semen preservation. The present results showed that during long-term liquid storage of goat semen, both sperm motility and semen pH decreased gradually, and a strong correlation was observed between the two. Whereas increasing the initial extender pH from 6.04 to 6.25 or storage with stabilized pH improved, storage with artificially lowered pH impaired sperm motility. Extender renewal improved sperm motility by maintaining a stable pH. Sperm coating with chicken (Gallus gallus) egg yolk improved motility by increasing tolerance to pH decline. A new extender (n-mZAP) with a higher buffering capacity was formulated, and n-mZAP maintained higher sperm motility, membrane integrity and acrosome intactness than the currently used mZAP extender did. Goat semen liquid-stored for 12 d in n-mZAP produced pregnancy and kidding rates similar to those obtained with freshly collected semen following artificial insemination. In conclusion, maintenance of a stable pH during liquid semen storage dramatically improved sperm viability and fertilizing potential. Copyright © 2015 Elsevier B.V. All rights reserved.

Impaired sperm fertilizing ability in mice lacking Cysteine-RIch Secretory Protein 1 (CRISP1)

PubMed Central

Da Ros, Vanina G.; Maldera, Julieta A.; Willis, William D.; Cohen, Débora J.; Goulding, Eugenia H.; Gelman, Diego M.; Rubinstein, Marcelo; Eddy, Edward M.; Cuasnicu, Patricia S.

2008-01-01

Mammalian fertilization is a complex multi-step process mediated by different molecules present on both gametes. Epididymal protein CRISP1, a member of the Cysteine-RIch Secretory Protein (CRISP) family, was identified by our laboratory and postulated to participate in both sperm-zona pellucida (ZP) interaction and gamete fusion by binding to egg-complementary sites. To elucidate the functional role of CRISP1 in vivo, we disrupted the Crisp1 gene and evaluated the effect on animal fertility and several sperm parameters. Male and female Crisp1−/− animals exhibited no differences in fertility compared to controls. Sperm motility and the ability to undergo a spontaneous or progesterone-induced acrosome reaction were neither affected in Crisp1−/− mice. However, the level of protein tyrosine phosphorylation during capacitation was clearly lower in mutant sperm than in controls. In vitro fertilization assays showed that Crisp1−/− sperm also exhibited a significantly reduced ability to penetrate both ZP-intact and ZP-free eggs. Moreover, when ZP-free eggs were simultaneously inseminated with Crisp1+/+ and Crisp1−/− sperm in a competition assay, the mutant sperm exhibited a greater disadvantage in their fusion ability. Finally, the finding that the fusion ability of Crisp1−/− sperm was further inhibited by the presence of CRISP1 or CRISP2 during gamete co-incubation, supports that another CRISP cooperates with CRISP1 during fertilization and might compensate for its lack in the mutant mice. Together, these results indicate that CRISP proteins are players in the mammalian fertilization process. To our knowledge this is the first knockout mice generated for a CRISP protein. The information obtained might have important functional implications for other members of the widely distributed and evolutionarily conserved CRISP family. PMID:18571638

Cryopreservation of epididymal stallion sperm.

PubMed

Olaciregui, M; Gil, L; Montón, A; Luño, V; Jerez, R A; Martí, J I

2014-02-01

Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallion's breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from the epididymis, and frozen for future use. However, the fertility of frozen-thawed epididymal sperm has been found to be lower than that of ejaculated sperm. Therefore, this study aimed to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of different cryoprotectants and freezing protocols on sperm quality. Dimethylformamide was tested alone or combination with pasteurized egg yolk as substitute of fresh egg yolk. In addition, the effect of the pre-freeze stabilization on sperm quality was analyzed. Heterospermic samples obtained from stallion epididymis were collected and cryopreserved in lactose-egg-yolk extender or in the same extender with varying content of cryoprotectant and content of egg yolk, stabilized and no-stabilized. Sperm motility, viability, hypoosmotic swelling test (HOST) and acrosome integrity were evaluated post-thawing. No improvement was observed on the replacement of fresh yolk by pasteurized egg yolk, whereas the results suggest that dimethylformamide is a cryoprotectant suitable for cryopreservation of equine epididymal semen, even better than glycerol. In addition, we found that the stabilization before freezing on epididymal stallion sperm, can improve sperm quality parameters. Copyright © 2014 Elsevier Inc. All rights reserved.

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 (IP 3 R), 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.

Effects of genetic captive-breeding protocols on sperm quality and fertility in the white-footed mouse.

PubMed

Malo, Aurelio F; Martinez-Pastor, Felipe; Alaks, Glen; Dubach, Jean; Lacy, Robert C

2010-10-01

Mice (Peromyscus leucopus noveboracensis) from a captive-breeding program were used to test the effects of three genetic breeding protocols (minimizing mean kinship [MK], random breeding, and selection for docility [DOC]) and inbreeding levels on sperm traits and fertility. Earlier, in generation 8, one DOC replicate went extinct because of poor reproductive success. By generation 10, spermatozoa from DOC mice had more acrosome and midpiece abnormalities, which were shown to be strong determinants of fertility, as well as lower sperm production and resistance to osmotic stress. In addition, determinants of fertility, including male and female components, were assessed in a comprehensive manner. Results showed that the probability (P) of siring litters is determined by sperm number, sperm viability, and midpiece and acrosome abnormalities; that the P of siring one versus two litters is determined by tail abnormalities; and that the total number of offspring is influenced by female size and proportion of normal sperm, showing the relative importance of different sperm traits on fertility. On average, males with 20% normal sperm sired one pup per litter, and males with 70% normal sperm sired eight pups per litter. Interestingly, the proportion of normal sperm was affected by docility but not by relatively low inbreeding. However, inbreeding depression in sperm motility was detected. In the MK group, inbreeding depression not only affected sperm motility but also fertility: An increase in the coefficient of inbreeding (f) of 0.03 reduced sperm motility by 30% and translated into an offspring reduction of three pups in second litters. A genetic load of 48 fecundity equivalents was calculated.

Use of antioxidants reduce lipid peroxidation and improve quality of crossbred ram sperm during its cryopreservation.

PubMed

Banday, Mohamad Naiem; Lone, Farooz Ahmad; Rasool, Fabiha; Rashid, Muzamil; Shikari, Arif

2017-02-01

Ram sperm are subjected to extreme oxidative stress during their preservation at -196 °C resulting in reduced quality at post thaw. Therefore, the main objective of this study was to evaluate the effect of antioxidants taurine, quercetin and reduced glutathione on the post thaw quality of crossbred ram sperm. A total of twenty four ejaculates from six crossbred rams were collected and extended with tris-based extender with no antioxidant (Control), with taurine (40 mM), quercetin (5 μg/ml) and reduced glutathione (5 mM). The post thaw sperm quality was determined by percent sperm motility, live sperm count, intact acrosome and hypo-osmotic swelling test (HOST) reacted spermatozoa and lipid peroxidation was measured in terms of malondialdehyde (MDA) level both in seminal plasma and sperm cell. At post thaw, percent sperm motility and live sperm count were significantly (p < 0.05) higher for taurine than control and reduced glutathione but did not differ significantly (p > 0.05) from quercetin. The percent HOST reacted spermatozoa were significantly higher for taurine than control, quercetin and reduced glutathione. Seminal plasma MDA level was significantly (p < 0.05) lower for taurine than control and non-significantly lower than quercetin and reduced glutathione. However, spermatic MDA level did not differ significantly (p > 0.05) among the control and antioxidants. In conclusion, taurine at 40 mM reduced lipid peroxidation and improved post thaw sperm quality of cryopreserved crossbred ram semen. Further, transportation time of semen samples in an ice chest at 4-5 °C may be included as a part of equilibration period, when collection shed and frozen semen unit are located at a distance. Copyright © 2016 Elsevier Inc. All rights reserved.

Discovery of human posterior head 20 (hPH20) and homo sapiens sperm acrosome associated 1 (hSPACA1) immunocontraceptive epitopes and their effects on fertility in male and female mice.

PubMed

Chen, Xuemei; Liu, Xiaodong; Ren, Xiuhua; Li, Xuewu; Wang, Li; Zang, Weidong

2016-03-01

The key goals of immunocontraception research are to obtain full contraceptive effects using vaccines administered to both males and females. Current research concerning human anti-sperm contraceptive vaccines is focused on delineating infertility-related epitopes to avoid autoimmune disease. We constructed phage-display peptide libraries to select epitope peptides derived from human posterior head 20 (hPH20) and homo sapiens sperm acrosome associated 1 (hSPACA1) using sera collected from infertile women harbouring anti-sperm antibodies. Following five rounds of selection, positive colonies were reconfirmed for reactivity with the immunoinfertile sera. We biopanned and analysed the chemical properties of four epitope peptides, named P82, Sa6, Sa37 and Sa76. Synthetic peptides were made and coupled to either bovine serum albumin (BSA) or ovalbumin. We used the BSA-conjugated peptides to immunise BALB/c mice and examined the effects on fertility in female and male mice. The synthetic peptides generated a sperm-specific antibody response in female and male mice that caused a contraceptive state. The immunocontraceptive effect was reversible and, with the disappearance of peptide-specific antibodies, there was complete restoration of fertility. Vaccinations using P82, Sa6 and Sa76 peptides resulted in no apparent side effects. Thus, it is efficient and practical to identify epitope peptide candidates by phage display. These peptides may find clinical application in the specific diagnosis and treatment of male and female infertility and contraceptive vaccine development.

Coiled-coil domain containing 42 (Ccdc42) is necessary for proper sperm development and male fertility in the mouse.

PubMed

Pasek, Raymond C; Malarkey, Erik; Berbari, Nicolas F; Sharma, Neeraj; Kesterson, Robert A; Tres, Laura L; Kierszenbaum, Abraham L; Yoder, Bradley K

2016-04-15

Spermiogenesis is the differentiation of spermatids into motile sperm consisting of a head and a tail. The head harbors a condensed elongated nucleus partially covered by the acrosome-acroplaxome complex. Defects in the acrosome-acroplaxome complex are associated with abnormalities in sperm head shaping. The head-tail coupling apparatus (HTCA), a complex structure consisting of two cylindrical microtubule-based centrioles and associated components, connects the tail or flagellum to the sperm head. Defects in the development of the HTCA cause sperm decapitation and disrupt sperm motility, two major contributors to male infertility. Here, we provide data indicating that mutations in the gene Coiled-coil domain containing 42 (Ccdc42) is associated with malformation of the mouse sperm flagella. In contrast to many other flagella and motile cilia genes, Ccdc42 expression is only observed in the brain and developing sperm. Male mice homozygous for a loss-of-function Ccdc42 allele (Ccdc42(KO)) display defects in the number and location of the HTCA, lack flagellated sperm, and are sterile. The testes enriched expression of Ccdc42 and lack of other phenotypes in mutant mice make it an ideal candidate for screening cases of azoospermia in humans. Copyright © 2016 Elsevier Inc. All rights reserved.

EFFECT OF SOLID MEDIUM DURING COOLED STORAGE ON STALLION SPERM PARAMETERS.

PubMed

Santos, F C; Corcini, C D; Costa, V G; Gheller, S M; Nogueira, C E; da Rosa Curcio, B; Varel, A S

2015-01-01

Solid storage medium prevents cellular sedimentation, reduces metabolic demand via limiting movement, and avoids the modification of an extender composition in the sedimentary microenvironment. It has been proven to prolong spermatozoa viability in mammalians. This experiment aims to evaluate the effect of cool storage in solid phase extender on stallion sperms. Semen was collected from 10 Crioulo stallions (n=30) and submitted to treatments: control group (semen extender) and groups with gelatin addition in different concentrations (semen extender + 1%, 2% and 3%). Seminal analyses included motility, mitochondrial functionality, plasma membrane integrity, DNA and acrosome at 0; 24; 48 and 72 hours during cooled storage at 5 degree C. Motility, mitochondrial functionality, plasma membrane and acrosome integrity declined during storage time, with no statistical difference between treatments. DNA integrity did not significantly change during storage period. Solid medium was not harmful and did not improved stallion sperm parameters during cooled storage.

Protein expression pattern of PAWP in bull spermatozoa is associated with sperm quality and fertility following artificial insemination.

PubMed

Kennedy, Chelsey E; Krieger, Kari Beth; Sutovsky, Miriam; Xu, Wei; Vargovič, Peter; Didion, Bradley A; Ellersieck, Mark R; Hennessy, Madison E; Verstegen, John; Oko, Richard; Sutovsky, Peter

2014-05-01

Post-acrosomal WW-domain binding protein (PAWP) is a signaling molecule located in the post-acrosomal sheath (PAS) of mammalian spermatozoa. We hypothesized that the proper integration of PAWP in the sperm PAS is reflective of bull-sperm quality and fertility. Cryopreserved semen samples from 298 sires of acceptable, but varied, fertility used in artificial insemination services were analyzed using immunofluorescence microscopy and flow cytometry for PAWP protein. In normal spermatozoa, PAWP fluorescence formed a regular band around the proximal PAS. Anomalies of PAWP labeling in defective spermatozoa were reflected in flow cytometry by varied intensities of PAWP-induced fluorescence. Distinct sperm phenotypes were also identified, including morphologically normal and some defective spermatozoa with moderate levels of PAWP; grossly defective spermatozoa with low/no PAWP; and defective spermatozoa with high PAWP. Analysis by ImageStream flow cytometry confirmed the prevalence of abnormal sperm phenotypes in the spermatozoa with abnormal PAWP content. Live/dead staining and video recording showed that some abnormal spermatozoa are viable and capable of progressive motility. Conventional flow-cytometric measurements of PAWP correlated significantly with semen quality and fertility parameters that reflect the sires' artificial insemination fertility, including secondary sperm morphology, conception rate, non-return rate, and residual value. A multiplex, flow-cytometric test detecting PAWP, aggresomes (ubiquitinated protein aggregates), and acrosomal integrity (peanut-agglutinin-lectin labeling) had a predictive value for conception rate, as demonstrated by step-wise regression analysis. We conclude that PAWP correlates with semen/fertility parameters used in the cattle artificial insemination industry, making PAWP a potential biomarker of bull fertility. © 2014 Wiley Periodicals, Inc.

Toxic effects of 2,4-dichlorophenoxyacetic acid on human sperm function in vitro.

PubMed

Tan, Zhengyu; Zhou, Jun; Chen, Houyang; Zou, Qianxing; Weng, Shiqi; Luo, Tao; Tang, Yuxin

2016-01-01

The herbicide 2,4-Dichlorophenoxyacetic acid (2,4-D) is globally used in agriculture and has been linked to human sperm abnormalities in vivo. However, its effects on ejaculated human spermatozoa in vitro have not been characterized. Therefore, we examined the effects of 2,4-D on the functions of ejaculated human spermatozoa in vitro, including: sperm motility, the ability to move through a viscous medium, capacitation, and the acrosome reaction. Different doses of 2,4-D (10 nM, 100 nM, 1 µM, 10 µM, 100 µM, and 200 µM) were applied to human spermatozoa prepared from normal fresh semen samples. The results indicated that 2,4-D did not affect the viability, capacitation, or spontaneous acrosome reactions of human spermatozoa, but it dose-dependently inhibited the total motility, progressive motility, ability to penetrate viscous medium, and progesterone-induced capacitation and acrosome reaction rates. These results suggest that exposure to 2,4-D and its accumulation in the seminal plasma and follicular fluid might increase the risk of infertility. Our findings provide new insights for understanding the male reproductive toxicity of 2,4-D.

Decoding mechanisms of loss of fertilization ability of cryopreserved mouse sperm

NASA Astrophysics Data System (ADS)

Gray, Jeffrey Earl

Cryopreservation of mouse sperm is an important technology for management of biomedical research resources. Dramatic progress has been made recently in the development of protocols that combat mouse-strain specific reduction of IVF after cryopreservation. Equal emphasis, however, has not been placed on investigating the biological mechanisms underlying these improvements to IVF. This dissertation broadly investigates the basic question of how mouse-strain specific reduction of IVF occurs after cryopreservation, and how recently developed protocols prevent this process. My research investigated the effects of antioxidants, the cholesterol-acceptor CD, reduced calcium media, and TYH capacitation media on sperm function and oxidative stress after cryopreservation in a variety of mouse strains. I found that reduced IVF was associated with loss of capacitation-dependent sperm function in three strains, B6/J, B6/N, and 129X1, and CD improved sperm function and IVF in all three strains. These findings suggest that cryopreservation inhibits cholesterol efflux resulting in reduced IVF of many mouse strains. I also found that cryopreservation induces uniquely high production of mitochondrial H2O2 by B6/J sperm. H2O2 present in other cellular compartments of B6/J sperm was not elevated compared to other strains. High levels of mitochondrial H2O2 were associated with lipid peroxidation of the sperm head and inability to acrosome react. Antioxidants reduced mitochondrial H2O2 production, decreased sperm head lipid peroxidation, and improved acrosome reaction. The cryopreservation-induced increase in mitochondrial H2O2 production of B6/J and B6129XF1 sperm was associated with elevation of intracellular calcium after cryopreservation and dependent on mitochondrial metabolic substrates. Reducing intracellular calcium levels or removing mitochondrial metabolic substrates decreased mitochondrial H2O2 production and increased IVF rates of cryopreserved B6/J sperm. Many of the strains

Effect of rapid warming of boar semen on sperm morphology and physiology.

PubMed

Bamba, K; Cran, D G

1985-09-01

The effect of rapid dilution (1:8 with BTS or 1:6.5 with KRP) and temperature change on sperm morphology and physiology were studied using boar spermatozoa pre-diluted in BF5 diluent. Rapid dilution of cold semen (5 degrees C) with a warm solution (37 degrees C) caused marked acrosomal changes which were most prominent in the anterior region. The acrosomal damage appeared to be caused mainly by rapid warming. In contrast to rapid cooling, rapid warming had little effect upon motility, glutamic-oxaloacetic transaminase release and respiration.

Inhibition of capacitation-associated tyrosine phosphorylation signaling in rat sperm by epididymal protein Crisp-1.

PubMed

Roberts, Kenneth P; Wamstad, Joseph A; Ensrud, Kathy M; Hamilton, David W

2003-08-01

Ejaculated sperm are unable to fertilize an egg until they undergo capacitation. Capacitation results in the acquisition of hyperactivated motility, changes in the properties of the plasma membrane, including changes in proteins and glycoproteins, and acquisition of the ability to undergo the acrosome reaction. In all mammalian species examined, capacitation requires removal of cholesterol from the plasma membrane and the presence of extracellular Ca2+ and HCO3-. We designed experiments to elucidate the conditions required for in vitro capacitation of rat spermatozoa and the effects of Crisp-1, an epididymal secretory protein, on capacitation. Protein tyrosine phosphorylation, a hallmark of capacitation in sperm of other species, occurs during 5 h of in vitro incubation, and this phosphorylation is dependent upon HCO3-, Ca2+, and the removal of cholesterol from the membrane. Crisp-1, which is added to the sperm surface in the epididymis in vivo, is lost during capacitation, and addition of exogenous Crisp-1 to the incubation medium inhibits tyrosine phosphorylation in a dose-dependent manner, thus inhibiting capacitation and ultimately the acrosome reaction. Inhibition of capacitation by Crisp-1 occurs upstream of the production of cAMP by the sperm.

Anion channels in the sea urchin sperm plasma membrane.

PubMed

Morales, E; de la Torre, L; Moy, G W; Vacquier, V D; Darszon, A

1993-10-01

Ionic fluxes in sea urchin sperm plasma membrane regulate cell motility and the acrosome reaction (AR). Although cationic channels mediate some of the ionic movements, little is known about anion channels in these cells. The fusion of sperm plasma membranes into lipid bilayers allowed identification of a 150 pS anion channel. This anion channel was enriched from detergent-solubilized sperm plasma membranes using a wheat germ agglutinin Sepharose column. Vesicles formed from this preparation were fused into black lipid membranes (BLM), yielding single channel anion-selective activity with the properties of those found in the sperm membranes. The following anion selectivity sequence was found: NO3- > CNS- > Br- > Cl-. This anion channel has a high open probability at the holding potentials tested, it is partially blocked by 4,4'-diisothiocyano-2,2'-stilbendisulfonic acid (DIDS), and it often displays substates. The sperm AR was also inhibited by DIDS.

The effects of trypanosomosis on sperm morphology in Zebu x Friesian crossbred bulls.

PubMed

Sekoni, V O; Rekwot, P I; Bawa, E K

2004-01-01

Detailed studies of sperm morphological abnormalities were carried out on 12 Zebu x Friesian crossbred bulls used in a study of the effects of trypanosomosis. Four bulls were infected with T. vivax, another four with T. congolense, while four served as controls. The infected bulls developed chronic trypanosomosis. All the bulls initially had very low sperm morphological abnormalities that were within acceptable limits for fertile animals. After infection there was a rapid and progressive increase in all sperm abnormalities. Spermatozoa of infected bulls were highly deformed with multiple morphological defects. Mean percentage pre-infection baseline values prior to infection for acrosomal, sperm-head, detached heads, proximal cytoplasmic droplets, distal cytoplasmic droplets, sperm-tail, midpiece and total sperm morphological defects ranged between 0.1 +/- 0.1 for acrosomal and 8.3 +/- 3.2 for total morphological abnormalities in the semen of the bulls. All the infected bulls developed sperm morphological abnormalities of more than a mean of 40.0% from the 4th week after infection until the end of the investigation and were considered unfit for breeding. At 7 weeks post-infection (PI) until the end of the study (12 weeks PI), the controls had a mean of less than 5% sperm morphological defects, while the infected bulls had 100%. Mean percentage values of sperm morphological defects throughout the duration of the investigation for control bulls were low and within the normal range for fertile bulls. These values differed significantly (p<0.001) from the elevated values of the infected bulls. The results show that trypanosomosis due to T. vivax or T. congolense infection can render Zebu x Friesian crossbred bulls unfit for breeding within a very short time. The resultant infertility could be of economic importance in trypanosomosis-endemic sub-Saharan Africa where Zebu x Friesian crossbred bulls are kept.

Acidic hyaluronidase activity is present in mouse sperm and is reduced in the absence of SPAM1: Evidence for a Role for Hyaluronidase 3 in mouse and human sperm

PubMed Central

Reese, Kristen L.; Aravindan, Rolands G.; Griffiths, Genevieve S.; Shao, Minghai; Wang, Yipei; Galileo, Deni S.; Atmuri, Vasantha; Triggs-Raine, Barbara L.; Martin-DeLeon, Patricia A.

2010-01-01

The molecular mechanisms underlying sperm penetration of the physical barriers surrounding the oocyte have not been completely delineated. Although neutral-active or “reproductive” hyaluronidases (hyases), exemplified by Sperm Adhesion Molecule 1 (SPAM1), are thought to be responsible for hyaluronan digestion in the egg vestments and for sperm-zona binding, their roles in mouse sperm have been recently questioned. Here we report that acidic “somatic” Hyaluronidase 3 (HYAL3) exists in two isoforms in human (~47 kDa, ~55 kDa) and mouse (~44, ~47kDa) sperm where it resides on the plasma membrane over the head and midpiece. Mouse isoforms are differentially distributed in the soluble (SAP), membrane (MBP), and acrosome-reacted (AR) fraction where they are most abundant. Comparisons of zymography of Hyal3 null and wild-type (WT) AR and MBP fractions show significant HYAL3 activity at pH 3 and 4, and less at 7. At pH 4, a second acid-active hyase band at ~57 kDa is present in the AR fraction. HYAL3 activity was confirmed using immunoprecipitated HYAL3 and spectrophotometry. In total proteins, hyase activity was higher at pH 6 than at 4 where Spam1 nulls had significantly (P<0.01) diminished activity, indicating that murine SPAM1 has acidic activity. Although fully fertile, Hyal3 null sperm showed delayed cumulus penetration and reduced acrosomal exocytosis. HYAL3, similar to SPAM1 with which it shares 74.6% structural similarity, exists in epididymal tissue/fluid from which it is acquired by caudal mouse sperm in vitro. Our results indicate for the first time the concerted activity of both neutral- and acid-active hyaluronidases in sperm. PMID:20586096

Improvements in human sperm quality by long-term in vitro co-culture with isolated porcine Sertoli cells.

PubMed

Menegazzo, Massimo; Zuccarello, Daniela; Luca, Giovanni; Ferlin, Alberto; Calvitti, Mario; Mancuso, Francesca; Calafiore, Riccardo; Foresta, Carlo

2011-10-01

Spermatogenesis is a complex process where spermatogonial germ cells become spermatozoa with the indispensable support of Sertoli cells (SCs), which provide 'ad hoc' structural and nutritional support. Unfortunately, for most sperm dysfunctions, no therapies are yet available except assisted reproductive technologies (ART) that are based on the use of different culture media to preserve sperm in vitro. However, sperm culture is only possible for short periods of time, since long-term culture would invariably and irreversibly damage the cells with negative impact on their fertilization potential. Fresh sperm cells (5 ml of 20 × 10(6)/ml) were co-cultured with SCs layers, derived from prepubertal pig testes or incubated in cell free SC medium or BWW (Biggers, Whitten and Whittingham) medium for 2, 4 or 7 days. Sperm viability, motility, mitochondrial status, DNA fragmentation, chromatin integrity, intracellular calcium and acrosome status were assessed after every co-culture or incubation time, but capacitation and induction of acrosome reaction (AR) with progesterone was only evaluated after 7 days. SCs layers derived from prepubertal pig testes (co-culture of sperm and SC feeder, CCSCF) were able to preserve normal sperm viability, motility and normal mitochondrial function, after 7 days of culture; CCSCF did not induce AR or hyperactivation of spermatozoa, keeping the sperm in a quiescent state for 7 days of culture. Nevertheless, the sperm were readily able to initiate AR after stimulation with progesterone. CCSCF maintained good sperm viability and motility for 7 days. This approach could improve retention of sperm viability and motility during ART procedures and maintain sperm viability, during transfer between two distant Centres, avoiding the need for cryopreservation.

Binder of Sperm Proteins 1 and 5 have contrasting effects on the capacitation of ram spermatozoa.

PubMed

Pini, Taylor; de Graaf, Simon P; Druart, Xavier; Tsikis, Guillaume; Labas, Valerie; Teixeira-Gomes, Ana Paula; Gadella, Barend M; Leahy, Tamara

2018-06-01

Binder of Sperm Proteins (BSPs) are the most abundant seminal plasma protein family in the ram and bull. They have been extensively studied in the bull but less is known about their function in ovine seminal plasma and current knowledge suggests that BSPs may have different effects in these two species. In the bull, they facilitate capacitation and destabilize the sperm membrane during in vitro handling, whereas in the ram, they appear to stabilize the sperm membrane and prevent cryopreservation-induced capacitation-like changes. Further investigation into the effects of BSPs on ram spermatozoa under capacitating conditions is required to further clarify their physiological roles in the ram. We investigated the effects of Binder of Sperm Proteins 1 and 5 on epididymal ram spermatozoa in conditions of low, moderate, and high cAMP. BSPs had minimal effects on sperm function in low-cAMP conditions, but caused significant changes under cAMP upregulation. BSP1 stabilized the membrane and qualitatively reduced protein tyrosine phosphorylation, but significantly increased cholesterol efflux and induced spontaneous acrosome reactions. BSP5 slightly increased spontaneous acrosome reactions and caused sperm necrosis. However, BSP5 had minimal effects on membrane lipid order and cholesterol efflux and did not inhibit protein tyrosine phosphorylation. These findings demonstrate that under maximal cAMP upregulation, BSP1 affected ram spermatozoa in a manner comparable to bull spermatozoa, while BSP5 did not.

Group III secreted phospholipase A2 regulates epididymal sperm maturation and fertility in mice

PubMed Central

Sato, Hiroyasu; Taketomi, Yoshitaka; Isogai, Yuki; Miki, Yoshimi; Yamamoto, Kei; Masuda, Seiko; Hosono, Tomohiko; Arata, Satoru; Ishikawa, Yukio; Ishii, Toshiharu; Kobayashi, Tetsuyuki; Nakanishi, Hiroki; Ikeda, Kazutaka; Taguchi, Ryo; Hara, Shuntaro; Kudo, Ichiro; Murakami, Makoto

2010-01-01

Although lipid metabolism is thought to be important for the proper maturation and function of spermatozoa, the molecular mechanisms that underlie this dynamic process in the gonads remains incompletely understood. Here, we show that group III phospholipase A2 (sPLA2-III), a member of the secreted phospholipase A2 (sPLA2) family, is expressed in the mouse proximal epididymal epithelium and that targeted disruption of the gene encoding this protein (Pla2g3) leads to defects in sperm maturation and fertility. Although testicular spermatogenesis in Pla2g3–/– mice was grossly normal, spermatozoa isolated from the cauda epididymidis displayed hypomotility, and their ability to fertilize intact eggs was markedly impaired. Transmission EM further revealed that epididymal spermatozoa in Pla2g3–/– mice had both flagella with abnormal axonemes and aberrant acrosomal structures. During epididymal transit, phosphatidylcholine in the membrane of Pla2g3+/+ sperm underwent a dramatic shift in its acyl groups from oleic, linoleic, and arachidonic acids to docosapentaenoic and docosahexaenoic acids, whereas this membrane lipid remodeling event was compromised in sperm from Pla2g3–/– mice. Moreover, the gonads of Pla2g3–/– mice contained less 12/15-lipoxygenase metabolites than did those of Pla2g3+/+ mice. Together, our results reveal a role for the atypical sPLA2 family member sPLA2-III in epididymal lipid homeostasis and indicate that its perturbation may lead to sperm dysfunction. PMID:20424323

Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer.

PubMed

Arias, María Elena; Sánchez-Villalba, Esther; Delgado, Andrea; Felmer, Ricardo

2017-02-01

Sperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

Effects of feeding omega-3-fatty acids on fatty acid composition and quality of bovine sperm and on antioxidative capacity of bovine seminal plasma.

PubMed

Gürler, Hakan; Calisici, Oguz; Calisici, Duygu; Bollwein, Heinrich

2015-09-01

The aim of the present study was to examine the effects of feeding alpha-linolenic (ALA) acid on fatty acid composition and quality of bovine sperm and on antioxidative capacity of seminal plasma. Nine bulls (ALA bulls) were fed with 800 g rumen-resistant linseed oil with a content of 50% linolenic acid and eight bulls with 400 g palmitic acid (PA bulls). Sperm quality was evaluated for plasma membrane and acrosome intact sperm (PMAI), the amount of membrane lipid peroxidation (LPO), and the percentage of sperm with a high DNA fragmentation index (DFI). Fatty acid content of sperm was determined using gas chromatography. Total antioxidant capacity, glutathione peroxidase, and superoxide dismutase activity were determined in seminal plasma. Feeding ALA increased (P < 0.05) the docosahexaenoic acid (DHA) content in bulls whereas in PA bulls did not change. PMAI increased after cryopreservation in ALA bulls as well as in PA bulls during the experiment period (P < 0.005). LPO of sperm directly after thawing did not change during the study period in ALA group, but decreased in PA group (P < 0.006). After 3h of incubation LPO increased in the ALA group (P < 0.02), while LPO did not differ between phases within groups. In conclusion, feeding of neither saturated nor polyunsaturated fatty acids affect the antioxidant levels in seminal plasma. Both saturated as well as polyunsaturated fatty acids had positive effects on quality of cryopreserved bovine sperm, although the content of docosahexaenoic acid in sperm membranes increased only in ALA bulls. Copyright © 2015 Elsevier B.V. All rights reserved.

Sperm morphology in five species of cicadettine cicadas (Hemiptera: Cicadomorpha: Cicadidae).

PubMed

Chawanji, Abraham S; Hodgson, Alan N; Villet, Martin H

2006-12-01

Mature spermatozoa from five species of cicadas of the subfamily Cicadettinae (Quintilia wealei, Melampsalta leucoptera, Stagira simplex, Xosopsaltria thunbergi and Monomatapa matoposa) were examined by light and electron microscopy. In each species sperm are elongate, aggregated into organized bundles with their heads embedded in a homogenous matrix to form spermatodesmata, and exhibit polymegaly. The head of the sperm consist of an anteriorly positioned conical acrosome that has a tubular substructure and a deep, posterior invagination that forms the subacrosomal space (eccentrically positioned anteriorly). The acrosome is flattened anteriorly; posteriorly it extends along either side of the nucleus as two tubular processes that gradually decrease in diameter. The filiform nucleus tapers anteriorly and intrudes into the subscrosomal space. Posteriorly the nucleus has a lateral invagination that houses material of the so-called centriolar adjunct. Posterior to the centriolar adjuct and the nucleus are two crystalline mitochondrial derivatives and a centriole, respectively, the latter giving rise to the axoneme, which has a 9 + 9 + 2 arrangement of microtubules. In these respects the sperm are similar to those of platypleurine cicadas. However, some features seem unique to cicadettines, including the structural organization of an enlarged centriolar adjunct and the dimensions of the tails. The enlarged centriolar adjunct has a lamella-like substructure and can be considered a synapomorphic character in the Cicadettinae. It is, therefore, potentially useful in the separation of this subfamily from the Cicadinae. In addition, the great length of the sperm nucleus of long-headed sperm in M. matoposa could be a synapomorphy of this genus and related taphurine and cicadettine species.

Low levels of PRSS37 protein in sperm are associated with many cases of unexplained male infertility.

PubMed

Liu, Jianbing; Shen, Chunling; Fan, Weimin; Chen, Yan; Zhang, Aijun; Feng, Yun; Li, Zheng; Kuang, Ying; Wang, Zhugang

2016-11-01

PRSS37, a putative trypsin-like serine protease, is highly conserved during mammalian evolution as revealed by multiple sequence alignment. Mice deficient for Prss37 gene exhibit male infertility, but their mating behavior, spermatogenesis, sperm morphology, and motility remain unaffected, similar to a situation called unexplained male infertility (UMI) in men (human being). Here, we demonstrated that PRSS37 is restrictively expressed in human testis, where it is mainly located in the elongating and elongated spermatids during spermiogenesis as shown by immunohistochemical analysis of normal human testicular sections. In mature sperm, PRSS37 appears in the acrosome region and diminishes during acrosome reaction. Further examination reveals that PRSS37 contents in sperm from patients with UMI are dramatically lower than those in sperm from men with proven fertility or from sperm donors. Sperm with low PRSS37 contents exhibit abnormal activation of the proacrosin/acrosin system and premature proteolysis of ADAM2, which may impair the functional competence of human sperm in vivo However, the in vitro fertilization outcomes of sperm with low PRSS37 contents are not affected. Together, these data implicate an important role of PRSS37 for male fertility. PRSS37 can be used as a potential molecular biomarker for evaluating sperm fertilization capability in vivo but not in vitro. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Field fertility of liquid stored and cryopreserved flow cytometrically sex-sorted stallion sperm.

PubMed

Gibb, Z; Grupen, C G; Maxwell, W M C; Morris, L H A

2017-03-01

The fertility of sex-sorted, cryopreserved stallion sperm must be improved for the sex-sorting technology to be applied commercially. To optimise the conditions used to liquid store stallion sperm prior to sex-sorting and assess the fertility of sperm following sex-sorting and cryopreservation. Both in vitro experiment and randomised controlled trial in healthy, client-owned mares. Stallion ejaculates (n = 9) were diluted in either a skimmed milk (KMT) or BSA (I-BSA) based media to 25 × 10 6 sperm/ml directly (+SP25) or washed to remove seminal plasma and diluted to 25 or 111 × 10 6 sperm/ml (-SP25 and -SP111). Sperm were stored for 18 h at 10 to 15°C and -SP25 and +SP25 treatments were centrifuged and resuspended to 111 × 10 6 sperm/ml. Sperm were incubated under H33342 staining conditions and motility, viability and acrosome integrity assessed. Semen was collected from stallions (n = 4), liquid stored at 10-15°C for up to 5 h and sperm either cryopreserved directly, sex-sorted and cryopreserved, or sex-sorted and returned to liquid storage until insemination. Low-dose hysteroscopic insemination was performed in 23 mares randomly allocated to the semen preparation group and pregnancy determined following embryo flushing on Day 9 after ovulation, or via transrectal ultrasonography on Day 14 after ovulation. Skimmed milk was superior to I-BSA in maintaining motility, viability and acrosome integrity. Seminal plasma removal did not affect the parameters measured at the concentrations examined. Conception rates did not differ significantly between the groups, although a high incidence of pregnancy loss was observed in both the cryopreserved groups. While the conception rates achieved are among the highest yet reported for sex-sorted, cryopreserved stallion sperm, the high incidence of pregnancy loss suggests that the development of the resulting embryos was significantly impaired by the sperm processing treatments. © 2016 EVJ Ltd.

Effect of hen's egg yolk on capacitation and acrosome reaction of diluted canine spermatozoa.

PubMed

Witte, T S; Schäfer-Somi, S; Kuchar, A; Möstl, E; Iben, C; Aurich, C

2009-02-01

The aim of this study was to investigate the influence of progesterone, cholesterol and calcium (Ca(2+)) in an egg-yolk-containing extender on capacitation and acrosome reactions (AR) of diluted canine spermatozoa during 4 days of cooled-storage. For this purpose, we first investigated the effect of supplementation of a Tris-citrate-fructose buffer (TCF) with progesterone in a final concentration of 0.1, 0.2 and 1.0 microg progesterone/ml TCF-diluted semen. We then compared the effects of TCF and the same buffer-containing 20% egg yolk (TCF-EY). In egg yolks and the TCF-EY, progesterone was measured by enzyme immunoassay, cholesterol by enzymatic colorimetry and Ca(2+) by flame atomic absorption spectrophotometry. For both experiments, ejaculates from eight dogs were used. For the comparison of diluents, one ejaculate was divided and one half diluted with TCF, the other with TCF-EY. One half of each TCF- and TCF-EY-diluted sample was evaluated immediately (D1), the other after storage for 4 days at +4 degrees C (D4). In diluted semen, motility and viability were measured by a computer assisted sperm analyzer (CASA; Sperm Vision, Minitüb, Germany), capacitation and AR were evaluated with a modified chlortetracycline assay (CTC) and the AR additionally by flow cytometry. Supplementation of progesterone revealed, that between D1 and D4, total and progressive motility decreased with all progesterone concentrations, while viability as well as percentage of capacitated and acrosome reacted spermatozoa stayed constant. Progesterone-, cholesterol- and Ca(2+) concentrations in egg yolks were 524.8+/-131.4 ng/g, 13.9+/-2.03 mg/g and 1.27+/-0.17 mg/g, respectively. In the TCF-EY-diluent, the respective values were 210.9 ng/g, 2.52 mg/g and 1.1mg/g. In TCF-semen, at D1, motility and viability were significantly higher than in TCF-EY-samples (p<0.05), however at D4, no significant differences were detectable. Further, in TCF-semen, percentages of spermatozoa with intact

Semen Quality and Sperm Function Loss by Hypercholesterolemic Diet Was Recovered by Addition of Olive Oil to Diet in Rabbit

PubMed Central

Romero, Aida A.; Funes, Abi K.; Cid-Barria, Macarena; Cabrillana, María E.; Monclus, María A.; Simón, Layla; Vicenti, Amanda E.; Fornés, Miguel W.

2013-01-01

Fat increment (0.05% cholesterol, chol) in standard diet promoted a significant increase in serum and sperm membrane chol, which ultimately altered membrane-coupled sperm specific functions: osmotic resistance, acrosomal reaction, and sperm capacitation in White New Zealand rabbits. These changes were also associated with a reduction in motility percentage and appearance of abnormal sperm morphology. The present study was carried out to evaluate the effect of dietary olive oil (OO, 7% v/w) administration to several male hypercholesterolemic rabbits (hypercholesterolemic rabbits, HCR) with altered fertility parameters. These HCR males were achieved by feeding normal rabbits with a high-fat diet (0.05% chol). HCR were associated with a modest non-significant increase in body weight (standard diet, 4.08±0.17 Kg, versus high-fat diet, 4.37±0.24 Kg). Hypercholesterolemic rabbits presented a marked decrease in semen volume, sperm cell count, and percentage of sperm motility, associated with a significant increase in sperm cell abnormalities. Moreover, sperm capacitation measured by the characteristic phosphorylated protein pattern in and induced acrosomal reaction were also altered suggesting sperm dysfunction. However, the administration of OO (for 16 weeks) to rabbits that were fed with 50% of the high-fat diet normalized serum chol. Curiously, OO supply succeeded to attenuate the seminal and sperm alterations observed in HCR group. Administration of OO alone did not cause any significant changes in above mentioned parameters. These data suggest that OO administration to HCR male rabbits recovers the loss of semen quality and sperm functionality. PMID:23326331

The effects on boar sperm quality of dietary supplementation with omega-3 polyunsaturated fatty acids differ among porcine breeds.

PubMed

Yeste, Marc; Barrera, Xavier; Coll, David; Bonet, Sergi

2011-07-01

The present study was undertaken to shed light on the relationship between boar sperm quality and dietary supplementation with omega-3 polyunsaturated fatty acids, which has been reported inconsistently in the literature. With this aim, such effects were evaluated and compared among three different porcine breeds: Duroc, Large-White, and Pietrain. Animals were randomly separated into two groups and fed either with a control diet or with a diet supplemented with omega-3. Sperm quality of these boar (ejaculate volume, sperm concentration, sperm viability, acrosome and mitochondrial sheath integrity, sperm motility, sperm morphology, and osmotic resistance of spermatozoa) was assessed every week for a 26-week period. Supplementing boar's diet with omega-3 did not affect ejaculate volume, sperm concentration, sperm motility, sperm viability, and acrosome and mitochondrial sheath integrity. In contrast, supplemented diet positively affected both sperm morphology in Large-White and Pietrain breeds and the osmotic resistance of Pietrain spermatozoa. No effects were seen for the same sperm parameters in Duroc breed. These breed-differences in boar fed with the supplemented diet could explain the contradictions in literature and might be related with differences in the composition of plasma membrane among breeds reported by other authors. Because no harmful effects were observed in the three evaluated breeds, but positive effects in Large-White and Pietrain boar, we can conclude that omega-3 fatty acids may be added to boar's diet at the levels used in this study to improve their sperm quality. More research is, however, needed to determine how these fatty acids differently affect the morphology and the osmotic resistance of the spermatozoa in these breeds. Copyright © 2011 Elsevier Inc. All rights reserved.

Variation of rabbit sperm physiologic parameters after the X-ray exposure.

PubMed

Klepko, A V; Gorban, L V; Motryna, O A; Kondratova, Y A; Chernyshov, A V; Andreychenko, S V

2013-01-01

To study the impact of X-irradiation on the morpho-functional characteristics of rabbit spermatozoa and the contents of fructose, L-carnitine, α-tocopherol, ascorbate and citrate in the animal sperm. The total body irradiation of rabbits (Soviet Shinchilla) was performed at the RUM-17 device in the dose range of 1.0-7.0 Gy with 2.8·10-3 Gy/sec intensity. The animal sperm was collected by the artificial vagina and then separated on seminal plasma and spermatozoa through centrifugation. Sperm concentration, motility, morphology and linear velocity were identified using the "MBI-6" light microscope. The quantitative determination of ascorbate, α-tocopherol and L-carnitine in seminal plasma was made on the "Agilent 1200" liquid chromatograph, while the concentration of fructose and citrate was measured on a spectrophotometer. The X-ray irradiation was shown to cause a dose-dependent suppressing effect on the prostate, seminal vesicles and epididymices expresed through the decreased content of fructose, citrate, L-carnitine, α-tocopherol and ascorbate in a rabbit sperm. Simultaneously the exacerbation of morphologic anomalies (damage of acrosomes, heads and tails) in spermatozoa along with spermatozoid motility and linear velocity decrease were elucidated. The run-time pattern of morpho-functional changes in spermatozoa together with component content of rabbit sperm in response to the total body irradiation was studied. Consequently the dose dependent raise of morphologic anomalies (damage of acrosomes, heads and tails) in spermatozoa and the slowing of spermatozoid linear velocity was found out. The sperm capacity to fulfill the post-radiation recovery was proven up. The effectiveness of the latter was shown to be dependent upon the radiation dose absorbed and the duration of post-irradiation period. Klepko A. V., Gorban L. V., Motryna O. A., Kondratova Ju. A., Chernyshov A. V., Andrejchenko S. V., 2013.

Spermatozoa in the sperm-peak-fraction of the boar ejaculate show a lower flow of Ca(2+) under capacitation conditions post-thaw which might account for their higher membrane stability after cryopreservation.

PubMed

Hossain, Md Sharoare; Johannisson, Anders; Siqueira, Amanda Pimenta; Wallgren, Margareta; Rodriguez-Martinez, Heriberto

2011-10-01

Boar spermatozoa collected in the ejaculate sperm peak-portion (P1, first 10 mL of the sperm-rich fraction, SRF), had shown a higher resilience to freezing and thawing compared to spermatozoa from the rest of the ejaculate (2nd portion of the SRF plus the post-sperm-rich fraction, PSRF), even when using a simplified freezing technique, as long as spermatozoa were incubated in their own seminal plasma (SP). This experiment studied the stability of P1- and SRF-P1 boar spermatozoa frozen in MiniFlatPacks (MFP), post-thaw, using flow cytometry. Since spermatozoa from either portion showed similar cryosurvival and low proportions of unstable membranes (<3%, annexin-V/propidium iodide staining), and only a tendency for SRF-P1 live spermatozoa to depict acrosome exocytosis (FITC-PNA/PI/H33342); they were explored for Ca(2+) contents using a Fluo-4 probe under in vitro capacitating conditions (mBO+ medium), as well they were tested for their ability to sustain a short Ca(2+)-ionophore (A23187) in vitro challenge. The proportions of live spermatozoa depicting high Ca(2+)-levels were initially <2% but increased over incubation time, particularly in SRF-P1(P<0.05), while proportions of live spermatozoa with low Ca(2+)-levels were basically constant over incubation time (~11-14%), for either portion. Incubation in capacitation medium did not modify the proportions of low-Ca(2+) but dramatically increased the proportions of high-Ca(2+) spermatozoa (P<0.001) already after 15 min exposure, highest for SRF-P1 spermatozoa. While the proportion of live spermatozoa with intact acrosome was significantly decreased among SRF-P1 (P<0.001), that of P1-spermatozoa remained unchanged, probably owing to the lowest relative content of cytosolic Ca(2+). The results suggest that spermatozoa in the P1-portion are more resilient to express acrosome exocytosis post-thaw compared to those bathing in the rest of the SRF-fraction when cryopreserved using a simplified technique, in MFPs. Copyright �

Combining reduced glutathione and ascorbic acid has supplementary beneficial effects on boar sperm cryotolerance.

PubMed

Giaretta, Elisa; Estrada, Efrén; Bucci, Diego; Spinaci, Marcella; Rodríguez-Gil, Joan E; Yeste, Marc

2015-02-01

The main aim of this work was to evaluate how supplementing freezing and thawing media with reduced glutathione (GSH) and l-ascorbic acid (AA) affected the quality parameters of frozen-thawed boar spermatozoa. With this purpose, semen samples of 12 ejaculates coming from 12 boars were used. Each ejaculate was split into seven aliquots to which 5 mM of GSH and 100 μM of AA were added separately or together at two different steps of freeze-thawing. Various sperm parameters (levels of free cysteine residues in sperm nucleoproteins, sperm viability, acrosome membrane integrity, intracellular peroxide and superoxide levels [ROS], and total and progressive motility) were evaluated before freezing and at 30 and 240 minutes after thawing. Both GSH and AA significantly improved boar sperm cryotolerance when they were separately added to freezing and thawing media. However, the highest improvement was recorded when both freezing and thawing media were supplemented with 5 mM of GSH plus 100 μM of AA. This improvement was observed in sperm viability and acrosome integrity, sperm motility, and nucleoprotein structure. Although ROS levels were not much increased by freeze-thawing procedures, the addition of GSH and AA to both freezing and thawing extenders significantly decreased intracellular peroxide levels and had no impact on superoxide levels. According to our results, we can conclude that supplementation of freezing and thawing media with both GSH and AA has a combined, beneficial effect on frozen-thawed boar sperm, which is greater than that obtained with the separate addition of either GSH or AA. Copyright © 2015 Elsevier Inc. All rights reserved.

A proton pump ATPase with testis-specific E1-subunit isoform required for acrosome acidification.

PubMed

Sun-Wada, Ge-Hong; Imai-Senga, Yoko; Yamamoto, Akitsugu; Murata, Yoshiko; Hirata, Tomoyuki; Wada, Yoh; Futai, Masamitsu

2002-05-17

The vacuolar-type H(+)-ATPases (V-ATPases) are a family of multimeric proton pumps involved in a wide variety of physiological processes. We have identified two novel mouse genes, Atp6e1 and Atp6e2, encoding testis-specific (E1) and ubiquitous (E2) V-ATPase subunit E isoforms, respectively. The E1 transcript appears about 3 weeks after birth, corresponding to the start of meiosis, and is expressed specifically in round spermatids in seminiferous tubules. Immunohistochemistry with isoform-specific antibodies revealed that the V-ATPase with E1 and a2 isoforms is located specifically in developing acrosomes of spermatids and acrosomes in mature sperm. In contrast, the E2 isoform was expressed in all tissues examined and present in the perinuclear compartments of spermatocytes. The E1 isoform exhibits 70% identity with the E2, and both isoforms functionally complemented a null mutation of the yeast counterpart VMA4, indicating that they are bona fide V-ATPase subunits. The chimeric enzymes showed slightly lower K(m)(ATP) than yeast V-ATPase. Consistent with the temperature-sensitive growth of Deltavma4-expressing E1 isoform, vacuolar membrane vesicles exhibited temperature-sensitive coupling between ATP hydrolysis and proton transport. These results suggest that E1 isoform is essential for energy coupling involved in acidification of acrosome.

Confocal fluorescence microscopy in a murine model of microdissection testicular sperm extraction to improve sperm retrieval.

PubMed

Smith, Ryan P; Lowe, Greg J; Kavoussi, Parviz K; Steers, William D; Costabile, Raymond A; Herr, John C; Shetty, Jagathpala; Lysiak, Jeffrey J

2012-05-01

Microdissection testicular sperm extraction markedly improves the sperm retrieval rates in men with nonobstructive azoospermia. However, localizing sperm foci can be time-consuming and it is not always successful. Fiberoptic confocal fluorescent microscopy offers the advantage of rapid in vivo detection of fluorescently labeled sperm in the seminiferous tubules. After establishing the feasibility of fiberoptic confocal fluorescent microscopy to identify antibody labeled sperm in vivo C57/B6 mice underwent intraperitoneal injection of busulfan to induce azoospermia. During spermatogenesis reestablishment at approximately 16 weeks the mice were anesthetized and the testes were delivered through a low midline incision. Fluorescein isothiocyanate labeled antibody to intra-acrosomal protein Hs-14 was injected retrograde into a single murine rete testis. The testes were imaged in vivo with fiberoptic confocal fluorescent microscopy and sperm foci were detected. The respective seminiferous tubules were excised and squash prepared for immunofluorescence microscopy. Sperm foci were identified in the testis injected with fluorescently tagged antibody by in vivo fiberoptic confocal fluorescence microscopy. The contralateral control testis of each mouse showed no specific signal. Immunofluorescence microscopy of the excised tubules provided morphological confirmation of the presence of labeled sperm with an absence in controls. Findings were consistent in the feasibility portion of the study and in the busulfan model of nonobstructive azoospermia. Fiberoptic confocal fluorescent microscopy was feasible during microdissection testicular sperm extraction in an azoospermic mouse model to identify fluorescently labeled sperm in vivo. Translation to the clinical setting could decrease operative time and improve the sperm harvest rate. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

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

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.

[Impact of mobile phone radiation on the quality and DNA methylation of human sperm in vitro].

PubMed

Wang, Dong; Li, Bo; Liu, Yuan; Ma, Ye-fei; Chen, Shu-qiang; Sun, Hui-jun; Dong, Jie; Ma, Xu-hui; Zhou, Jing; Wang, Xiao-hong

2015-06-01

To investigate the influences of mobile phone radiation on the quality and DNA methylation of human sperm in vitro. According to the fifth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, we randomly selected 97 male volunteers with normal semen parameters and divided each semen sample from the subjects into two equal parts, one exposed to mobile phone radiation at 1950 M Hz, SAR3. 0 W/kg for 3 hours while the other left untreated as the control. We obtained routine semen parameters as well as the acrosomal reaction ability, apoptosis and DNA methylation of sperm, and compared them between the two groups. Compared with the control, the radiation group showed significantly decreased progressive sperm motility ([36.64 ± 16.93] vs [27.56 ± 16.92]%, P < 0.01) and sperm viability ([63.72 ± 16.35] vs [54.31 ± 17.35]%, P < 0.01) and increased sperm head defects ([69.92 ± 4.46] vs [71.17 ± 4.89]%, P < 0.05), but no significant differences in sperm acrosomal reaction ([66.20 ± 6.75] vs [64.50 ± 3.47]%, P > 0.05). The early apoptosis rate of sperm cells was remarkably higher in the radiation group ([6.89 ± 9.84]%) than in the control ([4.44 ± 5.89]%) (P < 0.05). However, no statistically significant differences were found between the control and radiation groups in the DNA methylation patterns of the paternal imprinting gene H19 ICR ([0.60 ± 0.02] vs [1.40 ± 0.03]%, P > 0.05) or the maternal imprinting gene KvDMR1 ([0.00 ± 0.00] vs [1.80 ± 0.031%, P > 0.05). Mobile phone radiation reduces the progressive motility and viability of human sperm and increases sperm head defects and early apoptosis of sperm cells.

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. © 2015 Blackwell Verlag GmbH.

SPINK2 deficiency causes infertility by inducing sperm defects in heterozygotes and azoospermia in homozygotes.

PubMed

Kherraf, Zine-Eddine; Christou-Kent, Marie; Karaouzene, Thomas; Amiri-Yekta, Amir; Martinez, Guillaume; Vargas, Alexandra S; Lambert, Emeline; Borel, Christelle; Dorphin, Béatrice; Aknin-Seifer, Isabelle; Mitchell, Michael J; Metzler-Guillemain, Catherine; Escoffier, Jessica; Nef, Serge; Grepillat, Mariane; Thierry-Mieg, Nicolas; Satre, Véronique; Bailly, Marc; Boitrelle, Florence; Pernet-Gallay, Karin; Hennebicq, Sylviane; Fauré, Julien; Bottari, Serge P; Coutton, Charles; Ray, Pierre F; Arnoult, Christophe

2017-08-01

Azoospermia, characterized by the absence of spermatozoa in the ejaculate, is a common cause of male infertility with a poorly characterized etiology. Exome sequencing analysis of two azoospermic brothers allowed the identification of a homozygous splice mutation in SPINK2, encoding a serine protease inhibitor believed to target acrosin, the main sperm acrosomal protease. In accord with these findings, we observed that homozygous Spink2 KO male mice had azoospermia. Moreover, despite normal fertility, heterozygous male mice had a high rate of morphologically abnormal spermatozoa and a reduced sperm motility. Further analysis demonstrated that in the absence of Spink2, protease-induced stress initiates Golgi fragmentation and prevents acrosome biogenesis leading to spermatid differentiation arrest. We also observed a deleterious effect of acrosin overexpression in HEK cells, effect that was alleviated by SPINK2 coexpression confirming its role as acrosin inhibitor. These results demonstrate that SPINK2 is necessary to neutralize proteases during their cellular transit toward the acrosome and that its deficiency induces a pathological continuum ranging from oligoasthenoteratozoospermia in heterozygotes to azoospermia in homozygotes. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Antioxidative effects of magnetized extender containing bovine serum albumin on sperm oxidative stress during long-term liquid preservation of boar semen.

PubMed

Lee, Sang-Hee; Park, Choon-Keun

2015-08-21

Magnetized water is defined as water that has passed through a magnet and shows increased permeability into cells and electron-donating characteristics. These attributes can protect against membrane damage and remove reactive oxygen species (ROS) in mammalian cells. We explored the effects of improved magnetized semen extenders containing bovine serum albumin (BSA) as antioxidants on apoptosis in boar sperm. Ejaculated semen was diluted in magnetized extender (0G and 6000G) with or without BSA (0G + BSA and 6000G + BSA), and sperm were analyzed based on viability, acrosome reaction, and H2O2 level of live sperm using flow cytometry. Sperm were then preserved for 11 days at 18 °C. We found that viability was significantly higher in 6000G + BSA than under the other treatments (P < 0.05). The acrosome reaction was significantly lower in the 6000G + BSA group compared with the other treatments (P < 0.05). Live sperm with high intracellular H2O2 level were significantly lower in the 6000G + BSA group than under other treatments (P < 0.05). Based on our results, magnetized extenders have antioxidative effects on the liquid preservation of boar sperm. Copyright © 2015 Elsevier Inc. All rights reserved.

Stress and dietary factors modify boar sperm for processing.

PubMed

Radomil, L; Pettitt, M J; Merkies, K M; Hickey, K D; Buhr, M M

2011-09-01

This paper reviews stresses boar sperm undergo during processing and presents preliminary results of dietary modification that minimize this damage. Processing for artificial insemination (AI) stresses boar sperm by osmotic effects; altering cell size, shape and membranes; intracellular ice formation; and production of reactive oxygen species (ROS). Sperm response to ROS is concentration-dependent, with low levels activating the ERK pathway to stimulate tyrosine phosphorylation (Tyr-P) and capacitation, but high concentrations or inappropriately timed onset of ROS pathways can harm sperm. Fresh boar sperm exposed to ROS increased intracellular hydrogen peroxide (H(2) O(2) ) phospholipase and lipid peroxidation, maintained viability but lost motility and underwent acrosome reactions (AR). Direct incorporation of lipids ± the antioxidant Vitamin E improves the survival of liquid- and frozen-stored semen. Boars fed dietary flaxseed for 8 weeks to increase n-3 fatty acids displayed improved sperm morphology (p < 0.05), increased membrane fluidity (p < 0.05) and better retention of motility and viability during 5-7 day storage (p < 0.05). Processes reducing oxidative damage to stored sperm should be evaluated. © 2011 Blackwell Verlag GmbH.

Toxic effects of Pb2+ entering sperm through Ca2+ channels in the freshwater crab Sinopotamon henanense.

PubMed

Li, Na; Xu, Peng; Jing, Wei-Xin; Hwang, Jiang-Shiou; Wang, Lan

2017-11-01

Lead (Pb) is a heavy metal that can damage animal sperm. To study the effects of Pb on calcium homeostasis and calcium channel in the sperm of freshwater crab Sinopotamon henanense, the induction of acrosome reaction (AR) and acrosin activity were investigated when crabs were exposed to different Pb concentrations (0, 3.675, 7.35, 14.7, 29.4 and 58.8mg/L) for 3, 5 and 7 d separately. Fluorescent probe Fluo-3/AM was loaded into the sperm, and [Ca 2+ ] in the sperm was measured by fluorescence microscopy and using microplate reader. The calmodulin (CaM) concentration was measured by ELISA method. Verapamil (VRP), a calcium channel blocker, was used to evaluate whether Pb can enter the sperm through calcium channels leading to sperm damage. After sperm were exposed at 50μg/L VRP, 100μg/L Pb, 50μg/L VRP+100μg/L Pb, 1000μg/L Pb and 50μg/L VRP+1000μg/L Pb for 1h in vitro，sperm quality parameters (sperm survival and sperm DNA integrity) and levels of parameters indicating oxidative stress (protein carbonylation [PCO] and malondialdehyde [MDA]) were measured. Our data showed that Pb reduced the induction of acrosome reaction (AR), down-regulated the acrosin activity, decreased the intracellular concentration of Ca 2+ and elevated CaM concentration. Compared to controls, Pb alone induced significant stress, as reflected by decreasing sperm survival and sperm DNA integrity, and increasing PCO and MDA contents. In the presence of VRP, 100μg/L Pb-induced stresses were reduced, all the measured parameters in the sperm exposed at 100μg/L Pb returned to control levels. Our results indicate that Pb enters the sperm of the crab S. henanense through calcium channels, the inhibition of which blocks Pb-induced stresses such as sperm quality decline and oxidative damage. Copyright © 2017 Elsevier B.V. All rights reserved.

Hyaluronidase 2: A Novel Germ Cell Hyaluronidase with Epididymal Expression and Functional Roles in Mammalian Sperm1

PubMed Central

Modelski, Mark J.; Menlah, Gladys; Wang, Yipei; Dash, Soma; Wu, Kathie; Galileo, Deni S.; Martin-DeLeon, Patricia A.

2014-01-01

ABSTRACT To initiate the crucial cell adhesion events necessary for fertilization, sperm must penetrate extracellular matrix barriers containing hyaluronic acid (HA), a task thought to be accomplished by neutral-active hyaluronidases. Here we report that the ∼57 kDa hyaluronidase 2 (HYAL2) that in somatic tissues has been highly characterized to be acid-active is present in mouse and human sperm, as detected by Western blot, flow cytometric, and immunoprecipitation assays. Immunofluorescence revealed its presence on the plasma membrane over the acrosome, the midpiece, and proximal principal piece in mice where protein fractionation demonstrated a differential distribution in subcellular compartments. It is significantly more abundant in the acrosome-reacted (P = 0.04) and soluble acrosomal fractions (P = 0.006) (microenvironments where acid-active hyaluronidases function) compared to that of the plasma membrane where neutral hyaluronidases mediate cumulus penetration. Using HA substrate gel electrophoresis, immunoprecipitated HYAL 2 was shown to have catalytic activity at pH 4.0. Colocalization and coimmunoprecipitation assays reveal that HYAL2 is associated with its cofactor, CD44, consistent with CD44-dependent HYAL2 activity. HYAL2 is also present throughout the epididymis, where Hyal2 transcripts were detected, and in the epididymal luminal fluids. In vitro assays demonstrated that HYAL2 can be acquired on the sperm membrane from epididymal luminal fluids, suggesting that it plays a role in epididymal maturation. Because similar biphasic kinetics are seen for HYAL2 and SPAM1 (Sperm adhesion molecule 1), it is likely that HYAL2 plays a redundant role in the catalysis of megadalton HA to its 20 kDa intermediate during fertilization. PMID:25232017

Variability in in vitro fertilization outcomes of prepubertal goat oocytes explained by basic semen analyses.

PubMed

Palomo, M J; Quintanilla, R; Izquierdo, M D; Mogas, T; Paramio, M T

2016-12-01

This work analyses the changes that caprine spermatozoa undergo during in vitro fertilization (IVF) of in vitro matured prepubertal goat oocytes and their relationship with IVF outcome, in order to obtain an effective model that allows prediction of in vitro fertility on the basis of semen assessment. The evolution of several sperm parameters (motility, viability and acrosomal integrity) during IVF and their relationship with three IVF outcome criteria (total penetration, normal penetration and cleavage rates) were studied in a total of 56 IVF replicates. Moderate correlation coefficients between some sperm parameters and IVF outcome were observed. In addition, stepwise multiple regression analyses were conducted that considered three grouping of sperm parameters as potential explanatory variables of the three IVF outcome criteria. The proportion of IVF outcome variation that can be explained by the fitted models ranged from 0.62 to 0.86, depending upon the trait analysed and the variables considered. Seven out of 32 sperm parameters were selected as partial covariates in at least one of the nine multiple regression models. Among these, progressive sperm motility assessed immediately after swim-up, the percentage of dead sperm with intact acrosome and the incidence of acrosome reaction both determined just before the gamete co-culture, and finally the proportion of viable spermatozoa at 17 h post-insemination were the most frequently selected sperm parameters. Nevertheless, the predictive ability of these models must be confirmed in a larger sample size experiment.

Cryopreservation of human spermatozoa. I. Effects of holding procedure and seeding on motility, fertilizability, and acrosome reaction.

PubMed

Critser, J K; Huse-Benda, A R; Aaker, D V; Arneson, B W; Ball, G D

1987-04-01

Three experiments were conducted to evaluate effects of holding semen at +5.0 degrees C for 30 minutes or -5.0 degrees C for 10 minutes and ice crystal induction (seeding) on frozen-thawed human spermatozoa. In experiment 1, spermatozoa were frozen, and postthaw motility was evaluated immediately (0 hour) and 24 hours later. At both 0 and 24 hours, nonfrozen control samples had higher motility than all other treatment groups. At 0 hour postthaw, motility was higher in samples held at -5.0 degrees C for 10 minutes with no significant effect of seeding. At 24 hours, samples held at -5.0 degrees C for 10 minutes and seeded, but not samples held at -5.0 degrees C and not seeded, had higher motility than samples held at +5.0 degrees C. In experiment 2, semen samples were frozen, and fertilizability was evaluated in a zona-free hamster egg penetration assay. Seeded samples had a higher frequency of sperm penetration than either nonfrozen or nonseeded samples. In experiment 3, nonfrozen controls and frozen treatment groups were evaluated for the frequency of survival and acrosomal integrity. Seeded samples had higher frequencies of survival and loss of acrosomal integrity than nonseeded samples. All frozen-thawed samples had a lower frequency of survival and a higher frequency of loss of acrosomal integrity than nonfrozen controls. Although altered patterns of fertilizability and acrosomal integrity are induced, collectively these data suggest that incorporating a holding temperature of -5.0 degrees C for 10 minutes and seeding may result in a superior protocol for freezing human spermatozoa.

Atrial natriuretic peptide induces acrosomal exocytosis in bovine spermatozoa.

PubMed

Zamir, N; Barkan, D; Keynan, N; Naor, Z; Breitbart, H

1995-08-01

The induction of acrosomal exocytosis in capacitated bull spermatozoa by atrial natriuretic peptide (ANP) was studied in vitro. ANP markedly stimulated acrosomal exocytosis in a calcium-dependent manner. Typically, ANP exerts its action via activation of the ANP receptor (ANPR-A), a particulate guanylyl cyclase-linked receptor, and subsequent formation of guanosine 3',5'-cyclic monophosphate (cGMP). We found that the ANP-induced acrosome reaction was inhibited by the competitive ANPR-A receptor antagonist-anantin, indicating a receptor-mediated effect. We could mimic the effect of ANP on the acrosome reaction by using 8-bromo-cGMP, suggesting that cGMP may serve as a signal transducer mediating the acrosome reaction. Indeed, the ANP-induced acrosome reaction was associated with elevation of cGMP levels. cGMP can also be formed by activation of the soluble form of guanylyl cyclase. Sodium nitroprusside (SNP) stimulated cGMP accumulation and acrosome reaction of capacitated spermatozoa. Thus ANP and the nitric oxide-releasing compound SNP, via activation of guanylyl cyclase (the former activating the particulate and the latter activating the soluble form of the enzyme), may play a significant role in the induction of the acrosome reaction.

Lipid raft dynamics linked to sperm competency for fertilization in mice.

PubMed

Watanabe, Hitomi; Takeda, Rie; Hirota, Keiji; Kondoh, Gen

2017-05-01

It is well known that mammalian sperm acquires fertilization ability after several maturation processes, particularly within the female reproductive tract. In a previous study, we found that both glycosylphosphatidylinositol (GPI)-anchored protein (GPI-AP) release and lipid raft movement occur during the sperm maturation process. In several genetic studies, release of GPI-AP is a crucial step for sperm fertilization ability in the mouse. Here, we show that lipid raft movement is also fundamental for sperm to be competent for fertilization by comparing the sperm maturation process of two mouse inbred strains, C57BL/6 and BALB/c. We found that ganglioside GM1 movement was exclusively reduced in BALB/c compared with C57BL/6 among other examined sperm maturation parameters, such as GPI-AP release, sperm migration to the oviduct, cholesterol efflux, protein tyrosine phosphorylation and acrosome reaction, and was strongly linked to sperm fertility phenotype. The relationship between GM1 movement and in vitro fertilization ability was confirmed in other mouse strains, suggesting that lipid raft movement is one of the important steps for completing the sperm maturation process. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Murine sperm capacitation, oocyte penetration and decondensation following moderate alcohol intake.

PubMed

Sánchez, Melisa C; Fontana, Vanina A; Galotto, Camila; Cambiasso, Maite Y; Sobarzo, Cristian M A; Calvo, Lucrecia; Calvo, Juan C; Cebral, Elisa

2018-06-01

Male chronic alcohol abuse causes testicular failure and infertility. We analyzed the effects of moderate sub-chronic alcohol intake on sperm morphology, capacitation, fertilization and sperm head decondensation. CF-1 male mice were administered 15% ethanol in drinking water for 15 days; control mice received ethanol-free water. Similar patterns of tyrosine phosphorylation were observed in capacitated spermatozoa of control and treated males. Percentage of hyperactivation (H) and spontaneous (SAR) and progesterone-induced (IAR) acrosome reaction significantly decreased at 120 and 150 min of capacitation in treated males compared to controls (H: 14.1 ± 2.5 vs 23.7 ± 2.6, P  < 0.05; SAR-T120 min: 17.9 ± 2.5 vs 32.9 ± 4.1, P  < 0.01; IAR-150 min: 43.3 ± 3.5 vs 73.1 ± 1.1, P  < 0.001, n  = 6). During in vitro fertilization (2.5, 3.5 and 4.5 h post-insemination), there was an increased percentage of fertilized oocytes (with a decondensed sperm head and one or two pronuclei) in treated males ( P  < 0.001, n  = 7). After 60 min of in vitro decondensation with glutathione plus heparin, the percentage of decondensed sperm heads was significantly higher in treated males than in controls (mean ± s.d.: 57.1 ± 5.6 vs 48.3 ± 4.5, P  < 0.05, n  = 5). The percentage of morphologically normal sperm heads was significantly decreased in treated males with respect to controls ( P  < 0.001, n  = 9). These results show that short-term moderate alcohol consumption in outbred mice affect sperm morphology, hyperactivation, acrosomal exocytosis, and the dynamics of in vitro fertilization and in vitro sperm nuclear decondensation. © 2018 Society for Reproduction and Fertility.

Progesterone Accelerates the Completion of Sperm Capacitation and Activates CatSper Channel in Spermatozoa from the Rhesus Macaque1

PubMed Central

Sumigama, Shiho; Mansell, Steven; Miller, Melissa; Lishko, Polina V.; Cherr, Gary N.; Meyers, Stuart A.; Tollner, Theodore

2015-01-01

During transit through the female reproductive tract, mammalian spermatozoa are exposed to increasing concentrations of progesterone (P4) released by the cumulus oophorus. P4 triggers massive calcium influx into human sperm through activation of the sperm-specific calcium channel CatSper. These properties of human spermatozoa are thought to be unique since CatSper is not progesterone sensitive in rodent sperm. Here, by performing patch clamp recording from spermatozoa from rhesus macaque for the first time, we report that they express P4-sensitive CatSper channel identically to human sperm and react to P4 by inducing responsiveness to zona pellucida, unlike human sperm, which respond directly to P4. We have also determined the physiologic levels of P4 capable of inducing capacitation-associated changes in macaque sperm. Progesterone (1 μM) induced up to a 3-fold increase in the percentage of sperm undergoing the zona pellucida-induced acrosome reaction with the lowest threshold as low as 10 nM of P4. Submicromolar levels of P4 induced a dose-dependent increase in curvilinear velocity and lateral head displacement, while sperm protein tyrosine phosphorylation was not altered. Macaque spermatozoa exposed to 10 μM of P4 developed fully hyperactivated motility. Similar to human sperm, on approaching cumulus mass and binding to zona pellucida, macaque spermatozoa display hyperactivation and undergo an acrosome reaction that coincides with the rise in the sperm intracellular calcium. Taken together, these data indicate that P4 accelerates the completion of capacitation and provides evidence of spermatozoa “priming” as they move into a gradient of progesterone in search for the oocyte. PMID:26490839

AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility.

PubMed

Calle-Guisado, Violeta; de Llera, Ana Hurtado; Martin-Hidalgo, David; Mijares, Jose; Gil, Maria C; Alvarez, Ignacio S; Bragado, Maria J; Garcia-Marin, Luis J

2017-01-01

AMP-activated kinase (AMPK), a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work's aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%-80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS) in the presence or absence of the AMPK inhibitor compound C (CC). AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied.

AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility

PubMed Central

Calle-Guisado, Violeta; de Llera, Ana Hurtado; Martin-Hidalgo, David; Mijares, Jose; Gil, Maria C; Alvarez, Ignacio S; Bragado, Maria J; Garcia-Marin, Luis J

2017-01-01

AMP-activated kinase (AMPK), a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work's aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%–80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS) in the presence or absence of the AMPK inhibitor compound C (CC). AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied. PMID:27678462

The secretory products of Trichomonas vaginalis decrease fertilizing capacity of mice sperm in vitro

PubMed Central

Roh, Jaesook; Lim, Young-Su; Seo, Min-Young; Choi, Yuri; Ryu, Jae-Sook

2015-01-01

Trichomonas vaginalis infection is one of the most prevalent sexually transmitted infections in humans and is now recognized as an important cause of infertility in men. There is little information about the effect of extracellular polymeric substances (EPS) from T. vaginalis on sperm, but previous reports do not provide a conclusive description of the functional integrity of the sperm. To investigate the impact of EPS on the fertilizing capacity of sperm, we assessed sperm motility, acrosomal status, hypo-osmotic swelling, and in vitro fertilization rate after incubating the sperm with EPS in vitro using mice. The incubation of sperm with EPS significantly decreased sperm motility, viability, and functional integrity in a concentration and time-dependent manner. These effects on sperm quality also resulted in a decreased fertilization rate in vitro. This is the first report that demonstrates the direct negative impact of the EPS of T. vaginalis on the fertilization rate of sperm in vitro. However, further study should be performed using human sperm to determine if EPS has similar negative impact on human sperm fertilizing capacity in vitro. PMID:25578937

Sperm function and assisted reproduction technology

PubMed Central

MAAß, GESA; BÖDEKER, ROLF‐HASSO; SCHEIBELHUT, CHRISTINE; STALF, THOMAS; MEHNERT, CLAAS; SCHUPPE, HANS‐CHRISTIAN; JUNG, ANDREAS; SCHILL, WOLF‐BERNHARD

2005-01-01

The evaluation of different functional sperm parameters has become a tool in andrological diagnosis. These assays determine the sperm's capability to fertilize an oocyte. It also appears that sperm functions and semen parameters are interrelated and interdependent. Therefore, the question arose whether a given laboratory test or a battery of tests can predict the outcome in in vitro fertilization (IVF). One‐hundred and sixty‐one patients who underwent an IVF treatment were selected from a database of 4178 patients who had been examined for male infertility 3 months before or after IVF. Sperm concentration, motility, acrosin activity, acrosome reaction, sperm morphology, maternal age, number of transferred embryos, embryo score, fertilization rate and pregnancy rate were determined. In addition, logistic regression models to describe fertilization rate and pregnancy were developed. All the parameters in the models were dichotomized and intra‐ and interindividual variability of the parameters were assessed. Although the sperm parameters showed good correlations with IVF when correlated separately, the only essential parameter in the multivariate model was morphology. The enormous intra‐ and interindividual variability of the values was striking. In conclusion, our data indicate that the andrological status at the end of the respective treatment does not necessarily represent the status at the time of IVF. Despite a relatively low correlation coefficient in the logistic regression model, it appears that among the parameters tested, the most reliable parameter to predict fertilization is normal sperm morphology. (Reprod Med Biol 2005; 4: 7–30) PMID:29699207

Hepatitis B Virus S Protein Enhances Sperm Apoptosis and Reduces Sperm Fertilizing Capacity In Vitro

PubMed Central

Huang, JiHua; Zhong, Ying; Fang, XiaoWu; Xie, QingDong; Kang, XiangJin; Wu, RiRan; Li, FangZheng; Xu, XiaoQin; Lu, Hui; Xu, Lan; Huang, TianHua

2013-01-01

Objective Studying the impact of Hepatitis B virus S protein (HBs) on early apoptotic events in human spermatozoa and sperm fertilizing capacity. Methodology/Principal Findings Spermatozoa were exposed to HBs (0, 25, 50, 100 µg/ml) for 3 h, and then fluo-4 AM calcium assay, Calcein/Co2+ assay, protein extraction and ELISA, ADP/ATP ratio assay, sperm motility and hyperactivation and sperm-zona pellucida (ZP) binding and ZP-induced acrosome reaction (ZPIAR) tests were performed. The results showed that in the spermatozoa, with increasing concentration of HBs, (1) average cytosolic free Ca2+ concentration ([Ca2+]i) rose; (2) fluorescence intensity of Cal-AM declined; (3) average levels of cytochrome c decreased in mitochondrial fraction and increased in cytosolic fraction; (4) ADP/ATP ratios rose; (5) average rates of total motility and mean hyperactivation declined; (6) average rate of ZPIAR declined. In the above groups the effects of HBs exhibited dose dependency. However, there was no significant difference in the number of sperms bound to ZP between the control and all test groups. Conclusion HBs could induce early events in the apoptotic cascade in human spermatozoa, such as elevation of [Ca2+]i, opening of mitochondrial permeability transition pore (MPTP), release of cytochrome c (cyt c) and increase of ADP/ATP ratio, but exerted a negative impact on sperm fertilizing capacity. PMID:23874723

Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs) Effects on AMP-Activated Protein Kinase (AMPK) Regulation of Chicken Sperm Functions.

PubMed

Nguyen, Thi Mong Diep; Combarnous, Yves; Praud, Christophe; Duittoz, Anne; Blesbois, Elisabeth

2016-01-01

Sperm require high levels of energy to ensure motility and acrosome reaction (AR) accomplishment. The AMP-activated protein kinase (AMPK) has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca(2+)/calmodulin-dependent protein kinase kinases (CaMKKs) mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca(2+), or of CaMKKs inhibitor (STO-609). Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β), CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca(2+) but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca(2+) than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca(2+). Our results show for the first time the presence of CaMKKs (α and β) and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca(2+) entry in sperm through the Ca(2+)/CaM/CaMKKs/CaMKI pathway. The Ca(2+)/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca(2+) entry

Effect of cooling rate on sperm quality of cryopreserved Andalusian donkey spermatozoa.

PubMed

Demyda-Peyrás, S; Bottrel, M; Acha, D; Ortiz, I; Hidalgo, M; Carrasco, J J; Gómez-Arrones, V; Gósalvez, J; Dorado, J

2018-06-01

The aim of this study was to evaluate the effect of different cooling rates on post-thaw quality of cryopreserved donkey spermatozoa. Eighteen ejaculates from six adult Andalusian donkeys (three ejaculates per donkey) were collected using an artificial vagina. Pooled semen samples (two ejaculates per pool) were divided into three aliquots, and frozen in Gent freezing extender using three different cryopreservation protocols (P): P1 (conventional slow freezing, as control): semen pre-cooled in an Equitainer for 2 h and frozen in liquid nitrogen (LN 2 ) vapour; P2 (controlled pre-freeze cooling rate): semen pre-cooled at a controlled rate for 73 min and frozen in LN 2 vapour; and P3 (rapid freezing) semen frozen immediately in LN 2 vapour. After thawing at 37 °C for 30 s, semen samples were assessed for motility, morphology, acrosome and plasma membrane integrity; spermatozoa were also tested for DNA integrity. Significant (P < 0.01) differences were found between the cryopreservation protocols for all sperm parameters evaluated, except for DNA integrity. Semen samples frozen using P2 showed significantly (P < 0.01) higher values for sperm motility, morphology, sperm membrane integrity, and acrosome integrity. On the contrary, P3 reduced sperm motility (P < 0.01) and increased the percentage of spermatozoa with damaged plasma membrane (P < 0.001). In our study, we demonstrated that the sperm of Andalusian donkey is particularly sensitive to the cooling rate used before freezing. Furthermore, Andalusian donkey semen can be successfully cryopreserved using controlled cooling rates combined with freezing in LN 2 vapour. Copyright © 2018 Elsevier B.V. All rights reserved.

p,p′-DDE activates CatSper and compromises human sperm function at environmentally relevant concentrations

PubMed Central

Tavares, Renata S.; Mansell, Steven; Barratt, Christopher L.R.; Wilson, Stuart M.; Publicover, Stephen J.; Ramalho-Santos, João

2013-01-01

STUDY QUESTION Is the environmental endocrine disruptor p,p′-dichlorodiphenyldichloroethylene (p,p′-DDE) able to induce non-genomic changes in human sperm and consequently affect functional sperm parameters? SUMMARY ANSWER p,p′-DDE promoted Ca2+ flux into human sperm by activating CatSper channels even at doses found in human reproductive fluids, ultimately compromising sperm parameters important for fertilization. WHAT IS KNOWN ALREADY p,p′-DDE may promote non-genomic actions and interact directly with pre-existing signaling pathways, as already observed in other cell types. However, although often found in both male and female reproductive fluids, its effects on human spermatozoa function are not known. STUDY DESIGN, SIZE, DURATION Normozoospermic sperm samples from healthy individuals were included in this study. Samples were exposed to several p,p′-DDE concentrations for 3 days at 37°C and 5% CO2 in vitro to mimic the putative continuous exposure to this toxicant in the female reproductive tract in vivo. Shorter p,p′-DDE incubation periods were also performed in order to monitor sperm rapid Ca2+ responses. All experiments were repeated on a minimum of five sperm samples from different individuals. PARTICIPANTS/MATERIALS, SETTING, METHODS All healthy individuals were recruited at the Biosciences School, University of Birmingham, the Medical Research Institute, University of Dundee and in the Human Reproduction Service at University Hospitals of Coimbra. Intracellular Ca2+ concentration ([Ca2+]i) was monitored by imaging single spermatozoa loaded with Oregon Green BAPTA-1AM and further whole-cell patch-clamp recordings were performed to validate our results. Sperm viability and acrosomal integrity were assessed using the LIVE/DEAD sperm vitality kit and the acrosomal content marker PSA-FITC, respectively. MAIN RESULTS AND THE ROLE OF CHANCE p,p′-DDE rapidly increased [Ca2+]i (P < 0.05) even at extremely low doses (1 pM and 1 nM), with magnitudes of

Atrial natriuretic peptide induces acrosomal exocytosis of human spermatozoa.

PubMed

Rotem, R; Zamir, N; Keynan, N; Barkan, D; Breitbart, H; Naor, Z

1998-02-01

Acrosomal exocytosis in mammalian spermatozoa is a process essential for fertilization. We report here that atrial natriuretic peptide (ANP) markedly stimulates acrosomal exocytosis of capacitated human spermatozoa. Typically, ANP exerts some of its actions via activation of the ANP receptor (ANPR-A), a particulate guanylyl cyclase-linked receptor, and subsequent formation of guanosine 3',5'-cyclic monophosphate (cGMP). We found that ANP-stimulated acrosome reaction was inhibited by the competitive ANPR-A antagonist anantin, indicating a receptor-mediated process. A linear fragment of ANP, ANP-(13-28), and another ANP-like compound, brain natriuretic peptide, were inactive. The stimulatory effect of ANP on acrosome reaction was mimicked by the permeable cGMP analog, 8-bromo-cGMP (8-BrcGMP). Addition of the protein kinase C (PKC) inhibitors, staurosporine and GF-109203X, resulted in a dose-related inhibition of ANP-induced acrosome reaction. Also, downregulation of endogeneous PKC activity resulted in inhibition of ANP- but not 8-BrcGMP-induced acrosome reaction. Removal of extracellular Ca2+ abolished ANP-induced acrosome reaction. Thus ANP via Ca2+ influx, PKC activation, and stimulation of particulate guanylyl cyclase may play a role in the induction of acrosome reaction of human spermatozoa.

Optimization of conditions for long-term prefreezing storage of brown bear sperm before cryopreservation.

PubMed

López-Urueña, E; Alvarez, M; Gomes-Alves, S; Anel-López, L; Martínez-Rodríguez, C; Manrique, P; Borragan, S; Anel, L; de Paz, P

2015-10-15

Brown bear ejaculates are usually collected in field conditions and may need to be shipped to a laboratory for the application of reproductive biotechnologies before cryopreservation. The aim of this study was to extend the prefreezing step to 48 hours (1 hour vs. long-term storage [LS] to 24 and 48 hours) to enable the sample to be transported. The effects of storage temperature (experiment 1), glycerol concentration (experiment 2), and dilution rate (experiment 3) on sperm were evaluated. Electroejaculates from brown bears were stored under different experimental conditions and cryopreserved. The sperm motility and viability, apoptotic status, and acrosomal status of sperm were assessed before freezing (prefreezing), after thawing, and after 2-hour incubation at 37 °C (thermal stress test). In all experiments, one control sample was frozen using a standard protocol (control). In experiment 1, three temperatures during LS with 6% glycerol were tested: 5 °C (T5), 15 °C (T15), and room temperature (RT). The LS-T5 sample yielded the highest postthawing results for viability (42.4%), progressive motility (15.6%), and intact acrosome (83.1%) after 24 hours in comparison with the other temperatures (P < 0.05); for 48 hours, the LS-T5 sample reached higher total and progressive motility (25.9% and 9%, respectively) and nonapoptotic values (36.5%). Recovery rates revealed susceptibility to freezing at LS-15 or LS-RT samples at 24 hours (viability) or 48 hours (viability and motility). In experiment 2, samples were stored at 5 °C up to 48 hours and three glycerol concentrations were evaluated: 0% (0Gly), 3% (3Gly), and 6% (6Gly). Postthawing viability and motility increased progressively with the percentage of glycerol for 24 hours at 5 °C; 6% glycerol during 48-hour storage had beneficial effects on sperm cryopreservation. Besides, 6% glycerol had a clearly superior freezability for viability (42.7% and 40.8% for 24 hours and 48 hours, respectively) and motility (24

Retention of membrane charge attributes by cryopreserved-thawed sperm and zeta selection.

PubMed

Kam, Tricia L; Jacobson, John D; Patton, William C; Corselli, Johannah U; Chan, Philip J

2007-09-01

Mature sperm can be selected based on their negative zeta electrokinetic potential. The zeta selection of cryopreserved sperm is unknown. The objective was to study the effect of zeta processing on the morphology and kinematic parameters of cryopreserved-thawed sperm. Colloid-washed sperm (N = 9 cases) were cryopreserved for 24 h, thawed and diluted in serum-free medium in positive-charged tubes. After centrifugation, the tubes were decanted, serum-supplemented medium was added and the resuspended sperm were analyzed. Untreated sperm and fresh sperm served as the controls. There were improvements in strict normal morphology in fresh (11.8 +/- 0.3 versus control 8.8 +/- 0.3 %, mean +/- SEM) and thawed (8.7 +/- 0.2 versus control 5.4 +/- 0.2%) sperm after zeta processing. Percent sperm necrosis was reduced after zeta processing (66.0 +/- 0.6 versus unprocessed 74.6 +/- 0.3%). Progression decreased by 50% but not total motility after zeta processing of thawed sperm. The results suggested that the cryopreservation process did not impact the sperm membrane net zeta potential and higher percentages of sperm with normal strict morphology, acrosome integrity and reduced necrosis were recovered. The zeta method was simple and improved the selection of quality sperm after cryopreservation but more studies would be needed before routine clinical application.

Effect of selenium and vitamin E on acrosome reaction in porcine spermatozoa.

PubMed

Tareq, K M A; Akter, Quzi Sharmin; Takagi, Yuji; Hamano, Koh-Ichi; Sawada, Tomio; Tsujii, Hirotada

2010-06-01

Selenium (Se) and vitamin E (Vit-E), as an integral part of antioxidant systems, play an important role in the motility and acrosome reaction (AR) of mammalian spermatozoa. The purpose of this study was to investigate the effect of Se and Vit-E on motility, viability, AR and accumulation of ammonia in the culture medium during different incubation periods in porcine sperm. Sperm samples were washed, swum-up and incubated at 37°C for 1 and 3 h in Sp-TALP medium supplemented with sodium selenite (SS), seleon l-methionine (SeMet) and Vit-E in the presence or absence of ammonia. Sperm motility was determined on the basis of movement quality examined by phase microscopy. Viability and AR of spermatozoa were assessed by Hoechst 33258 and chlortetracycline (CTC) staining technique, and accumulation of ammonia was measured by the indophenol method. The incorporation of 14 C(U)-glucose was assessed with a liquid scintillation counter. In experiment 1, the sperm motility, viability, AR and incorporation of 14 C(U)-glucose increased significantly ( P < 0.05) in SS, SeMet and Vit-E (5, 5 μg/l and 1.0 mM, respectively) compared with the control. In experiment 2, treatment of the sperm with SeMet and SeMet + Vit-E in the presence of 300 μM ammonia also resulted in a significant increase ( P < 0.05) in the rate of motility, viability, AR and incorporation of 14 C(U)-glucose. In contrast, the accumulation of ammonia was reduced by SeMet and SeMet + Vit-E compared with the other treatments. These findings indicate that SeMet and SeMet + Vit-E may play an important role in reducing the accumulation of ammonia and subsequently in increasing the rate of AR and the utilization of glucose in porcine spermatozoa.

Rooibos (Aspalathus linearis) extract enhances boar sperm velocity up to 96 hours of semen storage.

PubMed

Ros-Santaella, José Luis; Pintus, Eliana

2017-01-01

Rooibos (Aspalathus linearis) is a native shrub from South African fynbos and has become very popular in the last decades for its antioxidant and medicinal attributes. Several studies have shown its beneficial properties in numerous cell lines, but to date, the in vitro effects of rooibos extract on sperm cells are still unknown. In this study, boar semen was supplemented with four concentrations both of fermented and unfermented rooibos extracts during 96 h of liquid storage at 17°C. The effects of rooibos extracts on sperm velocity, membrane integrity, and acrosomal status were evaluated at 2 h, 48 h, and 96 h of semen storage. Overall our results indicate that rooibos extract enhances sperm velocity, protects the acrosome structure, and tends to preserve the membrane integrity during semen storage. Although the unfermented rooibos showed higher total polyphenol content and total antioxidant capacity than the fermented one, the latter had better effects on sperm velocity leading to, for instance, an increase of 30% in the rectilinear velocity (VSL) at 48 h compared to the control group. Taking into account the different storage times, we established a suitable range of extracts concentrations to be used in boar semen. The rooibos extract ought to be considered as a powerful and natural source of antioxidants for the preservation of boar semen.

Rooibos (Aspalathus linearis) extract enhances boar sperm velocity up to 96 hours of semen storage

PubMed Central

2017-01-01

Rooibos (Aspalathus linearis) is a native shrub from South African fynbos and has become very popular in the last decades for its antioxidant and medicinal attributes. Several studies have shown its beneficial properties in numerous cell lines, but to date, the in vitro effects of rooibos extract on sperm cells are still unknown. In this study, boar semen was supplemented with four concentrations both of fermented and unfermented rooibos extracts during 96 h of liquid storage at 17°C. The effects of rooibos extracts on sperm velocity, membrane integrity, and acrosomal status were evaluated at 2 h, 48 h, and 96 h of semen storage. Overall our results indicate that rooibos extract enhances sperm velocity, protects the acrosome structure, and tends to preserve the membrane integrity during semen storage. Although the unfermented rooibos showed higher total polyphenol content and total antioxidant capacity than the fermented one, the latter had better effects on sperm velocity leading to, for instance, an increase of 30% in the rectilinear velocity (VSL) at 48 h compared to the control group. Taking into account the different storage times, we established a suitable range of extracts concentrations to be used in boar semen. The rooibos extract ought to be considered as a powerful and natural source of antioxidants for the preservation of boar semen. PMID:28837611

Human spermatozoa selected by Percoll gradient or swim-up are equally capable of binding to the human zona pellucida and undergoing the acrosome reaction.

PubMed

Morales, P; Vantman, D; Barros, C; Vigil, P

1991-03-01

Several techniques have been used for selecting motile spermatozoa including Percoll and albumin gradients, swim-up, and glass wool filtration. A high yield of motile spermatozoa as well as an enhancement of motility are the most desirable features of a practical method. An equally important consideration is whether or not these techniques select functionally normal spermatozoa. In this study we have compared two methods for separation of motile cells, swim-up and Percoll gradient. Normal semen samples from 12 different men were used in this study. Each sample was simultaneously processed by swim-up and Percoll gradient using modified Tyrode's medium. After the sperm concentration was adjusted to 1 x 10(7) spermatozoa/ml, the suspensions were incubated at 37 degrees C, 5% CO2 in air. In each suspension the percentage of sperm recovery, percentage of motile spermatozoa, percentage of acrosome reacted spermatozoa (either spontaneously or stimulated with human follicular fluid), percentage of zona-free hamster oocytes penetrated, and number of spermatozoa bound to the human zona pellucida were determined. The results obtained indicated that the percentage of sperm recovery was higher with the Percoll gradient than with the swim-up procedure (P less than 0.001). However, no significant differences were found between these two sperm populations in the percentage of motile cells, in the percentage of acrosome reacted spermatozoa, and in the percentage of zona-free hamster oocytes penetrated. In addition, the number of spermatozoa bound per zona pellucida was similar for spermatozoa selected by Percoll or swim-up. We conclude that there were no functional differences between the spermatozoa selected by either method.

Spermiogenesis and sperm ultrastructure of Machilontus sp (Insecta: Archaeognatha) with phylogenetic consideration.

PubMed

Fanciulli, Pietro Paolo; Mercati, David; Machida, Ryuichiro; Dallai, Romano

2015-06-01

The sperm structure of the jumping bristletail Machilontus sp has been described. The species shares several sperm characteristics with other genera of the same order Archaeognatha. During late spermiogenesis the spermatid bends at half of its length with the two limbs closely apposed within the same plasma membrane. The sperm has a helicoidal bi-layered acrosome with a filamentous perforatorium and a long nucleus. The elongated flagellum consists of an axoneme with 9 accessory microtubules external to the 9+2, two rows of conventional mitochondria and two accessory bodies. The accessory bodies are located lateral to the axoneme and are probably responsible for the shifting of the accessory tubules in two opposite groups of 5 and 4 tubules, respectively. These sperm characteristics represent common traits of all Archaeognatha. Copyright © 2015 Elsevier Ltd. All rights reserved.

Peroxiredoxins prevent oxidative stress during human sperm capacitation

PubMed Central

Lee, Donghyun; Moawad, Adel R.; Morielli, Tania; Fernandez, Maria C.

2017-01-01

Abstract STUDY QUESTION Do peroxiredoxins (PRDXs) control reactive oxygen species (ROS) levels during human sperm capacitation? SUMMARY ANSWER PRDXs are necessary to control the levels of ROS generated during capacitation allowing spermatozoa to achieve fertilizing ability. WHAT IS KNOWN ALREADY Sperm capacitation is an oxidative event that requires low and controlled amounts of ROS to trigger phosphorylation events. PRDXs are antioxidant enzymes that not only act as scavengers but also control ROS action in somatic cells. Spermatozoa from infertile men have lower levels of PRDXs (particularly of PRDX6), which are thiol-oxidized and therefore inactive. STUDY DESIGN, SIZE, DURATION Semen samples were obtained from a cohort of 20 healthy nonsmoker volunteers aged 22–30 years old over a period of 1 year. PARTICIPANTS/MATERIALS, SETTINGS, METHODS Sperm from healthy donors was capacitated with fetal cord serum ultrafiltrate (FCSu) in the absence or presence of thiostrepton (TSP), inhibitor of 2-Cys PRDXs or 1-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol lithium (MJ33), inhibitor of calcium independent-phospholipase A2 (Ca2+-iPLA2) activity of PRDX6, added at different times of incubation. Capacitation was also induced by the dibutyryl cAMP+3-isobuty1-1-methylxanthine system. Sperm viability and motility were determined by the hypo-osmotic swelling test and computer-assisted semen analysis system, respectively. Capacitation was determined by the ability of spermatozoa to undergo the acrosome reaction triggered by lysophosphatidylcholine. Percentages of acrosome reaction were obtained using the FITC-conjugated Pisum sativum agglutinin assay. Phosphorylation of tyrosine residues and of protein kinase A (PKA) substrates were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblotting with specific antibodies. Actin polymerization was determined by phalloidin labeling. MAIN RESULTS AND THE ROLE OF CHANCE TSP and MJ33 prevented sperm

Seminal plasma applied post-thawing affects boar sperm physiology: a flow cytometry study.

PubMed

Fernández-Gago, Rocío; Domínguez, Juan Carlos; Martínez-Pastor, Felipe

2013-09-01

Cryopreservation induces extensive biophysical and biochemical changes in the sperm. In the present study, we used flow cytometry to assess the capacitation-like status of frozen-thawed boar spermatozoa and its relationship with intracellular calcium, assessment of membrane fluidity, modification of thiol groups in plasma membrane proteins, reactive oxygen species (ROS) levels, viability, acrosomal status, and mitochondrial activity. This experiment was performed to verify the effect of adding seminal plasma on post-thaw sperm functions. To determine these effects after cryopreservation, frozen-thawed semen from seven boars was examined after supplementation with different concentrations of pooled seminal plasma (0%, 10%, and 50%) at various times of incubation from 0 to 4 hours. Incubation caused a decrease in membrane integrity and an increase in acrosomal damage, with small changes in other parameters (P > 0.05). Although 10% seminal plasma showed few differences with 0% (ROS increase at 4 hours, P < 0.05), 50% seminal plasma caused important changes. Membrane fluidity increased considerably from the beginning of the experiment, and ROS and free thiols in the cell surface increased by 2 hours of incubation. By the end of the experiment, viability decreased and acrosomal damage increased in the 50% seminal plasma samples. The addition of 50% of seminal plasma seems to modify the physiology of thawed boar spermatozoa, possibly through membrane changes and ROS increase. Although some effects were detrimental, the stimulatory effect of 50% seminal plasma could favor the performance of post-thawed boar semen, as showed in the field (García JC, Domínguez JC, Peña FJ, Alegre B, Gonzalez R, Castro MJ, Habing GG, Kirkwood RN. Thawing boar semen in the presence of seminal plasma: effects on sperm quality and fertility. Anim Reprod Sci 2010;119:160-5). Copyright © 2013 Elsevier Inc. All rights reserved.

Acrosome reaction in spermatozoa from the amphioxus Branchiostoma belcheri (Cephalochordata, Chordata).

PubMed

Morisawa, Sachiko; Mizuta, Takanobu; Kubokawa, Kaoru; Tanaka, Hiroyuki; Morisawa, Masaaki

2004-11-01

The formation of an acrosomal process at acrosomal exocytosis in spermatozoa of the amphioxus was described in the present report for the first time. A non-reacted acrosome was located in front of the nucleus, where a cup-shaped acrosomal vesicle covered a conical accumulation of subacrosomal material. When naturally spawned spermatozoa were treated with a calcium ionophore, ionomycin, the acrosomal vesicle opened at the apex and an acrosomal process was projected. The process exhibited a filamentous structure. The reaction followed the mode typically seen in marine invertebrates. These observations suggest that the features and function of the acrosome of amphioxus, whose position is on the border between invertebrates and vertebrates, reflect their ecological adaptation and phylogenic position.

Reversibility of Vasalgel™ male contraceptive in a rabbit model.

PubMed

Waller, Donald; Bolick, David; Lissner, Elaine; Premanandan, Christopher; Gamerman, Gary

2017-01-01

Development of a non-hormonal long-acting reversible contraceptive for men could have a significant impact on reducing unintended pregnancies. Vasalgel™ is a high molecular weight polymer consisting of styrene-alt-maleic acid (SMA) dissolved in dimethyl sulfoxide being developed as a reversible male contraceptive device. It forms a hydrogel when implanted into the vasa deferentia, which prevents the passage of sperm. Previous studies in the rabbit have proven its efficacy, durability and rapid onset. This study evaluates the capacity to restore sperm concentrations in ejaculates after a reversal procedure. Sodium bicarbonate was injected into the vasa deferentia after fourteen months of azoospermia following the injection of two device variations (Vasalgel 100 and Vasalgel 80). Semen samples were then collected for six months and sperm characteristics were compared to baseline levels. Samples of vasa deferentia were obtained for histological examination. Spermatozoa were present in all subject ejaculates after the reversal procedure. Sperm concentration and sperm motility were similar to baseline levels after reversal, while sperm forward progression was significantly lower and normal acrosomes were not observed. Forward progression percentages increased linearly during six months of semen collection, however, normal acrosomes were not observed at the conclusion of the study. Histologically, several vasa deferentia were clear of the device and contained an intact epithelial lining. A smaller proportion of tissues contained residual test material. A secondary intraluminal inflammatory response was seen occasionally in the tissues containing residual material. There was no difference between the two device variations for studied parameters. Vasalgel's prevention of sperm transport for 14 months was reversed through an intravasal injection of sodium bicarbonate. Post-reversal sperm concentrations and motility returned to baseline levels during the six-month follow

Quality of canine spermatozoa retrieved by percutaneous epididymal sperm aspiration.

PubMed

Varesi, S; Vernocchi, V; Faustini, M; Luvoni, G C

2013-02-01

To investigate the feasibility of percutaneous epididymal sperm aspiration in dogs and whether it might provide a population of epididymal spermatozoa similar to the population that can be obtained by processing isolated epididymis caudae. Concentration and total sperm number, motility, morphology and acrosomal integrity of spermatozoa retrieved by percutaneous epididymal sperm aspiration, in vitro aspiration and mincing of the cauda of the epididymis were compared. Percutaneous epididymal sperm aspiration is a feasible procedure to retrieve a population of spermatozoa in dogs. Quality is similar to that of spermatozoa collected in vitro, although a wide variation amongst animals was observed. In case of ejaculation failure due to pathological conditions in dogs, the collection of spermatozoa from the cauda of the epididymis could be an option for providing gametes for assisted reproductive technologies. Percutaneous epididymal sperm aspiration can be used in dogs with compromised reproductive performance, in which orchiectomy cannot be performed for medical or owner reasons. Further studies aimed to investigate whether the percutaneous epididymal sperm aspiration technique might be feasible for repeated semen collection and to accurately evaluate side effects are required. © 2013 British Small Animal Veterinary Association.

Partial deoxygenation of extender improves sperm quality, reduces lipid peroxidation and reactive oxygen species during cryopreservation of buffalo (Bubalus bubalis) semen.

PubMed

Balamurugan, B; Ghosh, S K; Lone, S A; Prasad, J K; Das, G K; Katiyar, R; Mustapha, Abdul Rahman; Kumar, Ajay; Verma, M R

2018-02-01

The present study was designed to investigate the effect of partial deoxygenation of extender on sperm quality, lipid peroxidation (LPO) and reactive oxygen species (ROS) in buffalo (Bubalus bubalis) during cryopreservation of semen. Semen extender was prepared freshly and split into three sub-extenders [Extender I: control (non-deoxygenated), Extender II (partially deoxygenated by using LN 2 flushing) and Extender III (partially deoxygenated mechanically by vacuum pump)]. Amounts of dissolved oxygen (DO) were determined in all the three extenders and also in post-thaw semen. Ejaculates with mass motility of  ≥3+ and individual progressive motility of 70% or greater were collected from Murrah buffalo bulls and utilized in the study. Each semen sample was divided into Groups I (diluted with Extender I), II (diluted with Extender II) and III (diluted Extender III) with a maximum of 60 × 10 6 sperm/mL. French mini straws (0.25 mL) were filled with the extended semen samples, sealed with polyvinyl alcohol powder, kept for 3 h at 5 °C for equilibration and then stored in an automatic programmable freezer until the temperature of straws reached -145 °C followed by plunging the straws into liquid nitrogen (-196 °C). Semen samples were evaluated at pre-freeze and post-thaw stages for various variables [sperm motility, live sperm count, acrosomal integrity, hypo-osmotic swelling (HOS) response, LPO and ROS concentrations]. The mean DO was less (P < 0.05) in Extender II as compared to I and III. The DO was less (P < 0.05) in Group II (semen extended with Extender II) as compared with III (semen extended with Extender III) and I (semen extended with Extender I). The percentages for sperm motility, viability and intact acrosomes (PIA) were greater (P < 0.05) in Groups II and III as compared to the control group at the pre-freeze stage, while at the post-thaw stage, percentages of sperm motility, viability, PIA and HOS response were greater (P�

Pig sperm preincubation and gamete coincubation with glutamate enhance sperm-oocyte binding and in vitro fertilization.

PubMed

Spinaci, M; Bucci, D; Gadani, B; Porcu, E; Tamanini, C; Galeati, G

2017-06-01

As the taste receptor for monosodium glutamate (umami) is expressed in both murine and human spermatozoa and the presence of α-gustducin and α-transducin, G proteins involved in the umami taste signaling, has been described in boar germ cells, the aim of this study was to evaluate if monosodium glutamate (MSG) would exert any effect on sperm-oocyte binding, in vitro fertilization (IVF) and sperm parameters during in vitro induced capacitation. For sperm-zona pellucida binding assay, boar spermatozoa were preincubated for 1 h and then coincubated for 1 h with denuded in vitro matured oocytes in presence of different concentrations of MSG (0, 0.1, 1, 10 mM). MSG 1 and 10 mM significantly (P < 0.05) increased the mean number of sperm bound to ZP compared with control (12.3 ± 9.0, 17.8 ± 11.3, 17.6 ± 10.8, MSG 0, 1 and 10 mM respectively). For in vitro fertilization trials, both sperm preicubation (1 h) and gamete coincubation (1 h) were performed in presence of different concentrations of MSG (0, 0.1, 1, 10 mM). After 19 h of culture in fresh IVF medium, oocytes were fixed. MSG 1 mM significantly (P < 0.05) increased the penetration rate compared with control (53.7 ± 20.4 vs. 36.8 ± 16.2). The addition of MSG during in vitro induced capacitation of boar spermatozoa did not cause any significant difference, compared with control, on the percentage of viable cells, spermatozoa with intact acrosome and the percentage of spermatozoa displaying tyrosine-phosphorylation of sperm tail proteins. In order to evaluate whether the effect elicited by MSG could be due to glutamate uptake in boar spermatozoa, fertilization trials were performed in presence of either 1 mM MSG or 1 mM MSG + 100 μM DL-threo-beta-hydroxyaspartic acid (THA), a non selective inhibitor of glutamate uptake. A significant increase (P < 0.05) in the penetration rate in both MSG and MSG + THA groups compared to control was recorded (39.8 ± 15.7, 53.7 ± 22

Effect of controlled and uncontrolled rate of cooling, prior to controlled rate of freezing, on motion characteristics and acrosomal integrity of cryopreserved ram spermatozoa.

PubMed

Joshi, Anil; Kumar, Davendra; Naqvi, S M K; Maurya, V P

2008-12-01

A programmable cell freezer provides ideal cryobiological conditions for controlled-rate cooling and freezing of ram spermatozoa. The purpose of this study was to investigate the effects of controlled (Group 1) and uncontrolled (Group 2) cooling conditions prior to programmable freezing of ram semen on post-thaw sperm motion characteristics and acrosomal integrity of ram spermatozoa. Semen samples of good initial motility obtained from adult Malpura rams were pooled, diluted to 1 × 10(9) spermatozoa per milliliter with Egg yolk-TEST-glycerol extender, and packaged in 0.25 mL straws. Straws representing Group 1 were cooled in a programmable cell freezer from 25°C to 5°C at the rate of -0.15°C per minute followed by a holding time of 2 h for equilibration, while straws of Group 2 were allowed to cool slowly up to 5°C and equilibrate for 2 h in the cold cabinet. After equilibration, straws of Group 2 were also loaded in the cell freezer for freezing straws of both the treatment groups simultaneously from 5°C to -125°C at the rate of -25°C per minute. Thawing of straws was done at 50°C for 10 s and the quality of frozen-thawed spermatozoa was objectively assessed by using sperm motility analyzer. Thawed samples were also evaluated for acrosomal integrity after staining the dried semen smears with Giemsa stain. The average post-thaw motility of straws was significantly higher (P < 0.05) in samples frozen after controlled cooling, compared with samples frozen after uncontrolled rate of cooling. The percent of spermatozoa with normal acrosome was also significantly (P < 0.05) higher in Group 1, compared to Group 2. The results indicate that controlled-rate cooling has a significant effect on post-thaw motility and acrosomal integrity of frozen-thawed ram spermatozoa, compared to uncontrolled-rate cooling prior to programmable freezing.

Post-thaw sperm characteristics following long-term storage of boar semen in liquid nitrogen.

PubMed

Fraser, L; Strzeżek, J; Kordan, W

2014-06-30

This study investigated the effect of long-term liquid nitrogen storage of semen from individual boars on post-thaw sperm characteristics. Ejaculates, collected from five Polish large white (PLW) and five Polish landrace (PLR) boars, were frozen using a standard cryopreservation protocol. Post-thaw analysis was performed within a week (Period 1) and 42-48 months (Period 2) of semen storage in liquid nitrogen. Post-thaw sperm assessments included total motility, mitochondrial function (JC-1/PI assay), plasma membrane integrity (SYBR-14/PI assay), osmotic resistance test (ORT), lipid peroxidation (LPO) status and DNA fragmentation, analysed by the neutral Comet assay. Individual boar variability within breed and cryostorage periods had significant effects on the analysed parameters of frozen-thawed spermatozoa. Prolonged semen storage in liquid nitrogen (Period 2) induced a marked reduction in post-thaw sperm motility, mitochondrial function and plasma membrane integrity in most of the boars. Post-thaw semen of eight boars exhibited a marked decrease in osmotic resistance of the sperm acrosomal membrane, whereas a significant increase in the sperm cryo-susceptibility to induced LPO and DNA fragmentation was observed only in three boars after long-term semen storage. Additionally, frozen-thawed spermatozoa of PLR boars exhibited significantly lower osmotic resistance of the acrosomal membrane than PLW boars following prolonged semen storage in liquid nitrogen. The results of this study provide evidence of ageing processes in frozen-thawed boar spermatozoa following prolonged cryostorage. It seems that, even though cryopreservation allows long-term semen storage in liquid nitrogen, spermatozoa from individual boars are more susceptible to cryo-induced damage. Copyright © 2014 Elsevier B.V. All rights reserved.

Immunization against lysozyme-like proteins affect sperm function and fertility in the rat.

PubMed

Narmadha, Ganapathy; Yenugu, Suresh

2016-11-01

Proteins of the epididymal and testicular mileu contribute to sperm maturation and a vast majority of them remain uncharacterised. In this study, the role of three Lysozyme-like (LYZL) proteins, namely LYZL1, LYZL4 and LYZL6 in sperm function was assessed using in vitro neutralization and auto antibodies generation model. Rats immunized with LYZL1, LYZL4 and LYZL6 proteins had a litter size of 5.93, 8.47 and 2.10 respectively compared to 9.96 in the control rats. The litter size was further reduced to 4.53, 7.67 and 1.23 for the corresponding proteins in the second mating conducted 14 weeks after immunization. Epididymal and testicular fluids obtained from the immunized rats displayed a very high antibody titre against all the three proteins. Sperm count was significantly reduced in rats immunized with LYZL1 or LYZL6 and to a lower extent in LYZL4 group. Acrosome reaction associated calcium release was inhibited in spermatozoa obtained from LYZL1 or LYZL4 or LYZL6 immunized rats as well as in spermatozoa incubated with antiserum against the three proteins. Impairment in path velocity, progressive velocity and track speed were observed in spermatozoa obtained from LYZL6 immunized rats. Treatment of spermatozoa with LYZL6 recombinant protein did not potentiate calcium release and acrosome reaction. Results of this study indicate a role for LYZL proteins in sperm function and further studies are warranted to explore them as potential contraceptive agents. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Use of heterospermic inseminations and paternity testing to evaluate the relative contributions of common sperm traits and seminal plasma proteins in boar fertility.

PubMed

Flowers, W L; Deller, F; Stewart, K R

2016-11-01

The objective of this study was to evaluate relationships between common semen quality estimates including sperm motility, sperm morphology, spontaneous capacitation status and seminal plasma proteins and boar fertility using heterospermic inseminations and subsequent paternity testing. All boars (n=12) used in the study had excellent semen quality (≥70% normal sperm) that resulted in average farrowing rates and litter sizes of 88.9±0.7% and 11.7±0.1 pigs, respectively. Their ejaculates were combined to make heterospermic insemination doses in such a way that each boar was tested against all of his contemporaries. The proportion of piglets sired by each individual was used to separate boars into three fertility groups: High (71.6±4.8%; n=3); Medium (51.6±3.8%; n=6); and Low (25.2%±5.3%; n=3). Ejaculates from High fertility boars had more motile sperm with normal acrosomes that moved faster in a straight-line and were more likely to undergo an acrosome reaction (p≤0.05) compared with their counterparts in the Low fertility group. Ejaculates from High fertility boars contained the greatest concentrations of three seminal plasma proteins (25.9kD/5.9pI; 55.1kD/4.8pI; and 70.1kD/5.2pI; p≤0.05), whereas concentrations of a 19.1kD/6.8pI were highest in semen from Low fertility boars (p≤0.05). Multiple regression analyses indicated that concentrations of the 25.9kD/5.9pI seminal plasma protein explained 66% of the variation observed in the proportion of pigs sired within a litter among boars (p≤0.00001). These results demonstrate that heterospermic inseminations and subsequent paternity testing is an effective technique for defining relationships between common semen quality tests and fertility, especially in situations where reproductive performance of all the boars is high. Motility, normal acrosome morphology, average linear velocity of motile sperm, and the proportion of sperm capable of an acrosome reaction were all positively associated with boar

Peroxisome proliferator-activated receptor gamma signaling in human sperm physiology

PubMed Central

Liu, Li-Li; Xian, Hua; Cao, Jing-Chen; Zhang, Chong; Zhang, Yong-Hui; Chen, Miao-Miao; Qian, Yi; Jiang, Ming

2015-01-01

Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the PPARs, which are transcription factors of the steroid receptor superfamily. PPARγ acts as an important molecule for regulating energy homeostasis, modulates the hypothalamic-pituitary-gonadal (HPG) axis, and is reciprocally regulated by HPG. In the human, PPARγ protein is highly expressed in ejaculated spermatozoa, implying a possible role of PPARγ signaling in regulating sperm energy dissipation. PPARγ protein is also expressed in Sertoli cells and germ cells (spermatocytes). Its activation can be induced during capacitation and the acrosome reaction. This mini-review will focus on how PPARγ signaling may affect fertility and sperm quality and the potential reversibility of these adverse effects. PMID:25851655

Cysteine-rich secretory protein 4 is an inhibitor of transient receptor potential M8 with a role in establishing sperm function

PubMed Central

Gibbs, Gerard M.; Orta, Gerardo; Reddy, Thulasimala; Koppers, Adam J.; Martínez-López, Pablo; Luis de la Vega-Beltràn, José; Lo, Jennifer C. Y.; Veldhuis, Nicholas; Jamsai, Duangporn; McIntyre, Peter; Darszon, Alberto; O'Bryan, Moira K.

2011-01-01

The cysteine-rich secretory proteins (CRISPs) are a group of four proteins in the mouse that are expressed abundantly in the male reproductive tract, and to a lesser extent in other tissues. Analysis of reptile CRISPs and mouse CRISP2 has shown that CRISPs can regulate cellular homeostasis via ion channels. With the exception of the ability of CRISP2 to regulate ryanodine receptors, the in vivo targets of mammalian CRISPs function are unknown. In this study, we have characterized the ion channel regulatory activity of epididymal CRISP4 using electrophysiology, cell assays, and mouse models. Through patch-clamping of testicular sperm, the CRISP4 CRISP domain was shown to inhibit the transient receptor potential (TRP) ion channel TRPM8. These data were confirmed using a stably transfected CHO cell line. TRPM8 is a major cold receptor in the body, but is found in other tissues, including the testis and on the tail and head of mouse and human sperm. Functional assays using sperm from wild-type mice showed that TRPM8 activation significantly reduced the number of sperm undergoing the progesterone-induced acrosome reaction following capacitation, and that this response was reversed by the coaddition of CRISP4. In accordance, sperm from Crisp4 null mice had a compromised ability to undergo to the progesterone-induced acrosome reaction. Collectively, these data identify CRISP4 as an endogenous regulator of TRPM8 with a role in normal sperm function. PMID:21482758

Effects of aqueous crude extract of Echeveria gibbiflora on mouse sperm function.

PubMed

Cordero-Martínez, Joaquín; Aguirre-Alvarado, Charmina; Guzmán-Soriano, Jessica Gabriela; Sánchez-Arroyo, Cinthia Erika; Flores-Alonso, Juan Carlos; Rodríguez-Páez, Lorena

2016-10-01

The present study evaluates the possible antifertility effect of aqueous crude extract (OBACE) of Echeveria gibbiflora, a plant that belongs to the crassulaceae family, used in traditional Mexican medicine as a vaginal post coital rinse to prevent pregnancy and shown to have an immobilization/agglutination effect on sperm of different mammal species. We evaluated the effect of OBACE on functional parameters of mouse sperm, such as viability, capacitation, and acrosome reaction. In addition, due to the high concentrations of calcium bis-(hydrogen-1-malate) hexahydrate [Ca (C4H5O5)2•6H2O] present in this plant extract, we evaluated its effect on Ca(2+) influx in mouse sperm under capacitating conditions. Moreover, we determined the acute toxicity of OBACE and its in vivo effect in mouse sperm motility administering a single daily dose of 50 and 100 mg/kg during seven days, intraperitoneally. The sperm viability was not affected by the presence of different concentrations of OBACE, however, the capacitation and acrosome reaction suffered a significant decrease in a concentration-dependent manner, coinciding with the reduction of Ca(2+) influx. Furthermore, OBACE displayed an LD50 of 3,784.42 mg/kg and can be classified as a low toxic substance. Also, in vivo OBACE showed an inhibition of total and progressive motility on mouse sperm alongside a significant decrease of motility kinematic parameters and IVF rates. The results confirm the antifertility effect of this plant used in Mexican folk medicine. Further study on OBACE as a possible contraceptive treatment is warranted because of its activity and low in vivo toxicity. ALH: lateral amplitude; AP: acid phosphatase; BCF: beat frequency; BSA: bovine serum albumine; CTC: chlortetracycline; FDA: fluorescein diacetate; Fura-2 AM: fura-2-acetoxymethyl ester; HIV: human immunodeficiency virus; IVF: in vitro fertilization; OBACE: aqueous crude extract of Echeveria gibbiflora; PI: propidum iodide; SN: supernatant; VAP

Uterosome-like vesicles prompt human sperm fertilizing capability.

PubMed

Franchi, A; Cubilla, M; Guidobaldi, H A; Bravo, A A; Giojalas, L C

2016-12-01

Does the rapid transit through the uterine environment modulate the sperm physiological state? The uterosome-like vesicles (ULVs) secreted by endometrial epithelial cells (EECs) in vitro are able to fuse with human spermatozoa, prompting their fertilizing capacity. Early studies suggest that sperm capacitation begins in the uterus and ends in the oviduct, and that a synergistic effect of both female organs may accelerate this process. Although it has been reported that co-incubation of human spermatozoa with endometrial cell-conditioned medium (CM) stimulates sperm capacitation, the mechanism mediating this communication is unknown. Human ULVs secreted by EECs were characterized and their effect on human sperm physiology was analysed. Spermatozoa were incubated with EEC-derived CM or ULV, after which sperm capacitation was evaluated at different time points. In addition, the interaction of spermatozoa with ULV was analysed. ULVs were isolated by ultracentrifugation and identified using electron microscopy and Western blotting to assess the presence of specific protein markers. Following seminal plasma removal, human spermatozoa were incubated CM or ULV, after which sperm capacitation was evaluated as the ability of the sperm to undergo the induced acrosome reaction and the level of protein tyrosine phosphorylation (PY) determined by Western blot and immunocytochemistry. The interaction of spermatozoa with labelled ULV was analysed by fluorescence microscopy. In all cases, at least three biological replicates from different sperm donors were performed for each set of experiments. Significant differences between mean values were determined by one-way ANOVA followed by Tukey's post hoc test. Differences between treatments were considered statistically significant at P ≤ 0.05. The level of capacitated spermatozoa and those recruited by chemotaxis increased 3- to 4-fold when spermatozoa were incubated in the presence of CM for 4 h. Even a 15 min incubation of

Antioxidative effects of magnetized extender containing bovine serum albumin on sperm oxidative stress during long-term liquid preservation of boar semen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Sang-Hee; Park, Choon-Keun, E-mail: parkck@kangwon.ac.kr

Magnetized water is defined as water that has passed through a magnet and shows increased permeability into cells and electron-donating characteristics. These attributes can protect against membrane damage and remove reactive oxygen species (ROS) in mammalian cells. We explored the effects of improved magnetized semen extenders containing bovine serum albumin (BSA) as antioxidants on apoptosis in boar sperm. Ejaculated semen was diluted in magnetized extender (0G and 6000G) with or without BSA (0G + BSA and 6000G + BSA), and sperm were analyzed based on viability, acrosome reaction, and H{sub 2}O{sub 2} level of live sperm using flow cytometry. Sperm were then preserved formore » 11 days at 18 °C. We found that viability was significantly higher in 6000G + BSA than under the other treatments (P < 0.05). The acrosome reaction was significantly lower in the 6000G + BSA group compared with the other treatments (P < 0.05). Live sperm with high intracellular H{sub 2}O{sub 2} level were significantly lower in the 6000G + BSA group than under other treatments (P < 0.05). Based on our results, magnetized extenders have antioxidative effects on the liquid preservation of boar sperm. - Highlights: • Magnetized water is water that has been passed through a magnetic field. • Magnetized extender improve viability and decrease oxidative stress of boar sperm for preservation. • Ejaculated semen diluted with magnetized extender can improve liquid preservation period.« less

Identification of Peroxiredoxin-5 in Bovine Cauda Epididymal Sperm

PubMed Central

Nagdas, Subir K; Buchanan, Teresa; Raychoudhury, Samir

2013-01-01

Developing spermatozoa require a series of post-testicular modifications within the luminal environment of the epididymis to achieve maturation; this involves several surface modifications including changes in plasma membrane lipids, proteins, carbohydrates, and alterations in the outer acrosomal membrane. Epididymal maturation can therefore allow sperm to gain forward motility and fertilization capabilities. The objective of this study was to identify maturation dependent protein(s) and to investigate their role with the production of functionally competent spermatozoa. Lectin blot analyses of caput and cauda sperm plasma membrane fractions identified a 17.5kDa Wheat Germ Agglutinin (WGA) binding polypeptide present in the cauda sperm plasma membrane not in the caput sperm plasma membrane. Among the several WGA stained bands, the presence of a 17.5kDa WGA binding polypeptide band was detected only in cauda epididymal fluid not in caput epididymal fluid suggesting that the 17.5kDa WGA-binding polypeptide is secreted from the cauda epididymis and binds to the cauda sperm plasma membrane during epididymal transit. Proteomic identification of the 17.5kDa polypeptide yielded 13 peptides that matched the sequence of peroxiredoxin-5 (PRDX5) protein (Bos Taurus). We propose that bovine cauda sperm PRDX5 acts as an antioxidant enzyme in the epididymal environment, which is crucial in protecting the viable sperm population against the damage caused by endogeneous or exogeneous peroxide. PMID:24186847

Lower blastocyst quality after conventional vs. Piezo ICSI in the horse reflects delayed sperm component remodeling and oocyte activation.

PubMed

Salgado, R M; Brom-de-Luna, J G; Resende, H L; Canesin, H S; Hinrichs, Katrin

2018-04-10

The aim of this study was to evaluate the differential effects of conventional and Piezo-driven ICSI on blastocyst development, and on sperm component remodeling and oocyte activation, in an equine model. In vitro-matured equine oocytes underwent conventional (Conv) or Piezo ICSI, the latter utilizing fluorocarbon ballast. Blastocyst development was compared between treatments to validate the model. Then, oocytes were fixed at 0, 6, or 18 h after injection, and stained for the sperm tail, acrosome, oocyte cortical granules, and chromatin. These parameters were compared between injection techniques and between sham-injected and sperm-injected oocytes among time periods. Blastocyst rates were 39 and 40%. The nucleus number was lower, and the nuclear fragmentation rate was higher, in blastocysts produced by Conv. Cortical granule loss started at 0H after both sperm and sham injection. The acrosome was present at 0H in both ICSI treatments, and persisted to 18H in significantly more Conv than Piezo oocytes (72 vs. 21%). Sperm head area was unchanged at 6H in Conv but significantly increased at this time in Piezo; correspondingly, at 6H significantly more Conv than Piezo oocytes remained at MII (80 vs. 9.5%). Sham injection did not induce significant meiotic resumption. These data show that Piezo ICSI is associated with more rapid sperm component remodeling and oocyte meiotic resumption after sperm injection than is conventional ICSI, and with higher embryo quality at the blastocyst stage. This suggests that there is value in exploring the Piezo technique, utilized with a non-toxic fluorocarbon ballast, for use in clinical human ICSI.

Selenium in blood, semen, seminal plasma and spermatozoa of stallions and its relationship to sperm quality.

PubMed

Bertelsmann, H; Keppler, S; Höltershinken, M; Bollwein, H; Behne, D; Alber, D; Bukalis, G; Kyriakopoulos, A; Sieme, H

2010-01-01

The essential trace element selenium is indispensable for male fertility in mammals. Until now, little data existed regarding the relationship between selenium and sperm quality in the stallion. Selenium, or selenium-dependent glutathione peroxidase activity, was determined in red blood cells, semen, seminal plasma and spermatozoa, and the percentages of spermatozoa with progressive motility (PMS), intact membranes (PMI), altered (positive) acrosomal status (PAS) and detectable DNA damage, determined by the sperm chromatin structure assay, were evaluated in 41 healthy stallions (three samples each). The pregnancy rate per oestrus cycle (PRC) served as an estimation of fertility. An adverse effect on stallion fertility caused by low dietary selenium intake was excluded, as all stallions had sufficient selenium levels in their blood. Interestingly, no significant correlations (P > 0.05) between the selenium level in blood and the selenium level in seminal plasma or spermatozoa were found, suggesting that the selenium level in blood is no indicator of an adequate selenium supply for spermatogenesis. The selenium level in spermatozoa (nmol billion(-1)) was correlated with PMI, PMS and PAS (r = 0.40, r = 0.31 and r = -0.42, respectively; P sperm quality and fertility in the stallion.

Curcumin Inhibits The Adverse Effects of Sodium Arsenite in Mouse Epididymal Sperm

PubMed Central

Momeni, Hamid Reza; Eskandari, Najmeh

2016-01-01

Background The aim of this study was to investigate the effects of curcumin on epididy- mal sperm parameters in adult male Navel Medical Research Institute (NMRI) mice ex- posed to sodium arsenite. Materials and Methods In this experimental study, we divided the animals into four groups: control, sodium arsenite (5 mg/kg), curcumin (100 mg/kg) and curcumin+sodium arsenite. Exposures were performed by intraperitoneal injections for a 5-week period. After the exposure period, we recorded the animals’ body and left testes weights. The left caudal epididymis was used to count the sperm number and analyze motility, viability, morphological abnormalities, acrosome reaction, DNA integrity, and histone-protamine replacement in the spermatozoa. One-way analysis of variance (ANOVA) followed by the Tukey’s test was used to assess the statistical significance of the data with SPSS 16.0. P<0.05 was considered significant. Results Mice exposed to sodium arsenite showed a significant decrease in the num- ber, motility, viability, normal sperm morphology and acrosome integrity of spermato- zoa compared to the control group. In the curcumin+sodium arsenite group, curcumin significantly reversed these adverse effects to the point where they approximated the control. In addition, the application of curcumin alone had no significant difference in these parameters compared to the control and curcumin+sodium arsenite groups. However, we observed no significant differences in the body and the testis weight as well as the DNA integrity and histone-protamine replacement in the spermatozoa of the four groups. Conclusion Curcumin compensated for the toxic effects of sodium arsenite on a number of sperm parameters in adult mice. PMID:27441059

In-vitro effects of Thymus munbyanus essential oil and thymol on human sperm motility and function.

PubMed

Chikhoune, Amirouche; Stouvenel, Laurence; Iguer-Ouada, Mokrane; Hazzit, Mohamed; Schmitt, Alain; Lorès, Patrick; Wolf, Jean Philippe; Aissat, Kamel; Auger, Jacques; Vaiman, Daniel; Touré, Aminata

2015-09-01

Traditional medicine has been used worldwide for centuries to cure or prevent disease and for male or female contraception. Only a few studies have directly investigated the effects of herbal compounds on spermatozoa. In this study, essential oil from Thymus munbyanus was extracted and its effect on human spermatozoa in vitro was analysed. Gas chromatography and Gas chromatography-mass spectrometry analyses identified 64 components, accounting for 98.9% of the composition of the oil. The principal components were thymol (52.0%), γ-terpinene (11.0%), ρ-cymene (8.5%) and carvacrol (5.2%). Freshly ejaculated spermatozoa was exposed from control individuals to various doses of the essential oil for different time periods, and recorded the vitality, the mean motility, the movement characteristics (computer-aided sperm analysis), the morphology and the ability to undergo protein hyperphosphorylation and acrosomal reaction, which constitute two markers of sperm capacitation and fertilizing ability. In vitro, both the essential oil extracted from T. munbyanus and thymol, the principal compound present in this oil, impaired human sperm motility and its capacity to undergo hyperphosphorylation and acrosome reaction. These compounds may, therefore, be of interest in the field of reproductive biology, as potential anti-spermatic agents. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Quality of boar spermatozoa from the sperm-peak portion of the ejaculate after simplified freezing in MiniFlatpacks compared to the remaining spermatozoa of the sperm-rich fraction.

PubMed

Siqueira, A P; Wallgren, M; Hossain, M S; Johannisson, A; Sanz, L; Calvete, J J; Rodríguez-Martínez, H

2011-04-15

Boar sperm viability post-thaw differs depending on the ejaculate fraction used, with spermatozoa present in the first 10 mL of the sperm-rich fraction (SRF) (portion 1, P1, sperm-peak portion) displaying the best cryosurvival in vitro compared with that of spermatozoa from the rest of the ejaculate (portion 2 of the SRF plus the post-spermatic fraction), even when using simplified freezing routines. This viability apparently relates to the specific profile of seminal plasma in P1 (i.e., glycoprotein and bicarbonate concentrations, and pH). However, spermatozoa from P1 have not been compared with spermatozoa from the rest of the SRF (SRF-P1, usually 30-40 mL of the SRF), which is routinely used for freezing. We compared P1 with SRF-P1 in terms of sperm kinematics (using the QualiSperm™ system), while membrane integrity (SYBR-14/PI), acrosome integrity (FITC PNA/PI), and sperm membrane stability (Annexin-V) were explored using flow cytometry. As well, total protein concentration and the proteomics of the seminal plasma (SP) of both portions of the SRF were studied using two-dimensional electrophoresis (2DE), mass fingerprinting (MALDI-TOF), and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) on selected peptides. The SRF portions were collected weekly from four mature boars (4-5 replicates per boar, sperm concentration: P1, 1.86 ± 0.20; SRF-P1, 1.25 ± 0.14 × 10(9) spz/mL) and processed using a quick freezing method in MiniFlatPacks. Post-thaw sperm motility reached 50%, without differences between SRF portions, but with clear inter-boar variation. Neither plasma membrane nor acrosome integrity differed (ns) between fractions. These results indicate that there are no differences in cryosurvival after quick freezing of boar spermatozoa derived from either of the two SRF portions. While P1 and SRF-P1 clearly differed in relative total protein contents, as expected, they displayed very similar protein profiles as assessed using 2DE and mass

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

Xenoestrogenic chemicals effectively alter sperm functional behavior in mice.

PubMed

Park, Yoo-Jin; Mohamed, El-Sayed A; Kwon, Woo-Sung; You, Young-Ah; Ryu, Buom-Yong; Pang, Myung-Geol

2011-12-01

Xenoestrogenic compounds (XCs) can disrupt endogenous hormone function and affect sperm function by binding to receptors on sperm membrane. Albeit spermatozoa are potentially a useful model for screening estrogenic activities of endocrine disruptors, high-quality in vitro test system that examination of the XCs effects on sperm function is required. The objective of this study was to compare the effects of XCs (genistein and 4-tert-octylphenol) to those of steroids (estrogen and progesterone) and heparin on in vitro capacitation and acrosome reaction (AR) in mouse spermatozoa. Mouse spermatozoa were incubated with various concentrations (0.001-100 μM) of each chemical for 15 or 30 min, and then capacitation and AR were assessed using chlortetracycline. All chemicals studied effectively alter capacitation and/or AR in mouse spermatozoa with different manner. Therefore, we believed that our system will provide a good in vitro model system to characterize the physiological effect of XCs especially when compared with steroids. Copyright © 2011 Elsevier Inc. All rights reserved.

Resveratrol and Epigallocatechin-3-gallate addition to thawed boar sperm improves in vitro fertilization.

PubMed

Gadani, B; Bucci, D; Spinaci, M; Tamanini, C; Galeati, G

2017-03-01

Thawing is one of the most delicate process after semen cryopreservation as spermatozoa pass from a dormant metabolic stage to a sudden awakening in cellular metabolism. The rapid oxygen utilization leads to an overproduction of reactive oxygen species that can damage sperm cells, thus causing a significant decrease of fertilizing potential of frozen-thawed spermatozoa. Resveratrol (Res) is a natural grape-derived phytoalexin and Epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis); both molecules are known to possess high levels of antioxidant activity. The objective of the present study was to assess the effect of different concentrations of Res (0.5, 1 or 2 mM; Experiment 1) or EGCG (25, 50 or 100 μM; Experiment 2) supplementation to thawing boar semen extender on sperm quality parameters (viability and acrosome integrity) and in vitro fertilization (IVF). Semen after thawing and dilution with three volumes of Beltsville Thawing Solution (BTS), was immediately divided in control group without antioxidants addition (CTR) and either Res or EGCG groups. Sperm viability and acrosome integrity were evaluated in CTR, Res or EGCG groups after 1 h of incubation at 37 °C. The addition of different doses of Res or EGCG to thawing extender for 1 h did not induce any effect on boar sperm viability and acrosome integrity. However, both Res and EGCG treated samples exhibited a significantly higher penetration rate compared with CTR when used for IVF. In particular the treatment with all the EGCG concentrations increased the penetration rate (P < 0.01) while only Res 2 mM induced a significant increase of this parameter (P < 0.01). In addition, EGCG 25 and 50 μM supplementation significantly increased total fertilization efficiency as compared to control (EGCG 25 μM: 40.3 ± 8.2 vs 26.8 ± 9.5, P < 0.05; EGCG 50 μM: 40.4 ± 7.8 vs 26.8 ± 9.5, P < 0.01). The same effect was observed with Res 2 mM (51.0�

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. Copyright © 2016 Elsevier B.V. All rights reserved.

The Molecular Chaperone HSPA2 Plays a Key Role in Regulating the Expression of Sperm Surface Receptors That Mediate Sperm-Egg Recognition

PubMed Central

Redgrove, Kate A.; Nixon, Brett; Baker, Mark A.; Hetherington, Louise; Baker, Gordon; Liu, De-Yi; Aitken, R. John

2012-01-01

A common defect encountered in the spermatozoa of male infertility patients is an idiopathic failure of sperm–egg recognition. In order to resolve the molecular basis of this condition we have compared the proteomic profiles of spermatozoa exhibiting an impaired capacity for sperm-egg recognition with normal cells using label free mass spectrometry (MS)-based quantification. This analysis indicated that impaired sperm–zona binding was associated with reduced expression of the molecular chaperone, heat shock 70 kDa protein 2 (HSPA2), from the sperm proteome. Western blot analysis confirmed this observation in independent patients and demonstrated that the defect did not extend to other members of the HSP70 family. HSPA2 was present in the acrosomal domain of human spermatozoa as a major component of 5 large molecular mass complexes, the most dominant of which was found to contain HSPA2 in close association with just two other proteins, sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), both of which that have previously been implicated in sperm-egg interaction. The interaction between SPAM1, ARSA and HSPA2 in a multimeric complex mediating sperm-egg interaction, coupled with the complete failure of this process when HSPA2 is depleted in infertile patients, provides new insights into the mechanisms by which sperm function is impaired in cases of male infertility. PMID:23209833

Ultrastructural Morphology of Sperm from Human Globozoospermia

PubMed Central

Ricci, Giuseppe; Andolfi, Laura; Luppi, Stefania; Boscolo, Rita; Zweyer, Marina; Trevisan, Elisa

2015-01-01

Globozoospermia is a rare disorder characterized by the presence of sperm with round head, lacking acrosome. Coiling tail around the nucleus has been reported since early human studies, but no specific significance has conferred it. By contrast, studies on animal models suggest that coiling tail around the nucleus could represent a crucial step of defective spermatogenesis, resulting in round-headed sperm. No observations, so far, support the transfer of this hypothesis to human globozoospermia. The purpose of this work was to compare ultrastructural morphology of human and mouse model globozoospermic sperm. Sperm have been investigated by using scanning and transmission electron microscopy. The images that we obtained show significant similarities to those described in GOPC knockout mice, an animal model of globozoospermia. By using this model as reference, we were able to identify the probable steps of the tail coiling process in human globozoospermia. Although we have no evidence that there is the same pathophysiology in man and knocked-out mouse, the similarities between these ultrastructural observations in human and those in the experimental model are very suggestive. This is the first demonstration of the existence of relevant morphological homologies between the tail coiling in animal model and human globozoospermia. PMID:26436098

Effect of maca supplementation on bovine sperm quantity and quality followed over two spermatogenic cycles.

PubMed

Clément, C; Kneubühler, J; Urwyler, A; Witschi, U; Kreuzer, M

2010-07-15

Maca (Lepidium meyenii Walpers), is an Andean crop that grows between 3,800 and 4,500 m a.s.l. The persistent interest in this plant is based on its assumed effects on fertility of male mammals due to the prevalence of certain, partially specific, secondary compounds. The present study aimed at evaluating the effect of maca supplementation on quality and quantity of semen, mating behavior, and clinical status of peripubertal breeding bulls. The experiment followed a cross-over design lasting for 23 wk with 3 wk of adaptation and baseline measurements, and 2 x 10 wk of treatment feeding thus covering two times the complete 8-wk spermatogenic cycle. Seventy-eight 55 wk to 84 wk old breeding bulls received either no maca (control) or maca (233 mg dried hypocotyls/kg body weight/day) for 10 wk followed by 10 wk without maca (maca early) or maca only in the last 10 wk (maca late). Measurements were always made in the last 2 wk of each period. Apart from standard analyses, ejaculates were analyzed by flow cytometry. Data was evaluated by analysis of variance considering the repeated measurement structure of the data. Significant treatment by measurement period indicated direct or carry-over effects of maca. Maca supplementation had no direct effect on body weight, testes circumference, rectal temperature, mating behavior, and ejaculate volume. However, supplementing maca in the first 10 wk period increased the number of sperms in the second 10 wk period, i.e., when the animals no longer received maca. The DNA fragmentation index and the visually assessed motility of the sperms of bulls, that initially showed a borderline sperm quality, were significantly improved with early maca supplementation, while no such effect was observed in the two other groups. No effects occurred in the proportion of intact sperm plasma membranes or acrosomes or both. In conclusion, maca supplementation seems to improve sperm quantity and quality of bulls to a certain degree, while mating

Rediscovering sperm ion channels with the patch-clamp technique

PubMed Central

Kirichok, Yuriy; Lishko, Polina V.

2011-01-01

Upon ejaculation, mammalian spermatozoa have to undergo a sequence of physiological transformations within the female reproductive tract that will allow them to reach and fertilize the egg. These include initiation of motility, hyperactivation of motility and perhaps chemotaxis toward the egg, and culminate in the acrosome reaction that permits sperm to penetrate the protective vestments of the egg. These physiological responses are triggered through the activation of sperm ion channels that cause elevations of sperm intracellular pH and Ca2+ in response to certain cues within the female reproductive tract. Despite their key role in sperm physiology and their absolute requirement for the process of fertilization, sperm ion channels remain poorly understood due to the extreme difficulty in application of the patch-clamp technique to spermatozoa. This review covers the topic of sperm ion channels in the following order: first, we discuss how the intracellular Ca2+ and pH signaling mediated by sperm ion channels controls sperm behavior during the process of fertilization. Then, we briefly cover the history of the methodology to study sperm ion channels, which culminated in the recent development of a reproducible whole-cell patch-clamp technique for mouse and human cells. We further discuss the main approaches used to patch-clamp mature mouse and human spermatozoa. Finally, we focus on the newly discovered sperm ion channels CatSper, KSper (Slo3) and HSper (Hv1), identified by the sperm patch-clamp technique. We conclude that the patch-clamp technique has markedly improved and shifted our understanding of the sperm ion channels, in addition to revealing significant species-specific differences in these channels. This method is critical for identification of the molecular mechanisms that control sperm behavior within the female reproductive tract and make fertilization possible. PMID:21642646

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 �

Elevated aminopeptidase N affects sperm motility and early embryo development

PubMed Central

Ryu, Do-Yeal; Kwon, Woo-Sung

2017-01-01

Aminopeptidase N (APN) is a naturally occurring ectopeptidase present in mammalian semen. Previous studies have demonstrated that APN adversely affects male fertility through the alteration of sperm motility. This enzyme constitutes 0.5 to 1% of the seminal plasma proteins, which can be transferred from the prostasomes to sperms by a fusion process. In the present study, we investigated the molecular mechanism of action of APN and its role in regulating sperm functions and male fertility. In this in vitro study, epididymal mouse spermatozoa were incubated in a capacitating media (pH 7) containing 20 ng/mL of recombinant mouse APN for 90 min. Our results demonstrated that the supplementation of recombinant APN in sperm culture medium significantly increased APN activity, and subsequently altered motility, hyperactivated motility, rapid and medium swimming speeds, viability, and the acrosome reaction of mouse spermatozoa. These effects were potentially caused by increased toxicity in the spermatozoa. Further, altered APN activity in sperm culture medium affected early embryonic development. Interestingly, the effect of elevated APN activity in sperm culture medium was independent of protein tyrosine phosphorylation and protein kinase A activity. On the basis of these results, we concluded that APN plays a significant role in the regulation of several sperm functions and early embryonic development. In addition, increased APN activity could potentially lead to several adverse consequences related to male fertility. PMID:28859152

Sperm viability in the black-footed ferret (Mustela nigripes) is influenced by seminal and medium osmolality.

PubMed

Santymire, Rachel M; Marinari, Paul E; Kreeger, Julie S; Wildt, David E; Howard, JoGayle

2006-08-01

Fundamental knowledge of spermatozoa cryobiology can assist with optimizing cryopreservation protocols needed for genetic management of the endangered black-footed ferret. Objectives were to characterize semen osmolality and assess the influence of two media at various osmolalities on sperm viability. We examined the influence of Ham's F10 +Hepes medium (H) at 270, 400, 500 or 700 mOsm (adjusted with sucrose, a nonpermeating cryoprotectant) and TEST Yolk Buffer (TYB) with 0% (300 mOsm) versus 4% (900 mOsm) glycerol (a permeating cryoprotectant). Electroejaculates (n=16) were assessed for osmolality using a vapor pressure osmometer. For media comparison, semen (n=5) was collected in TYB 0%, split into six aliquots, and diluted in H270, H400, H500, H700, and TYB 0% or TYB 4%. Each sample was centrifuged (300 g, 8 min), resuspended in respective medium, and maintained at 37 degrees C for 3h. Sperm motility and forward progression were monitored every 30 min for 3h post-washing. Acrosomal integrity was monitored at 0 and 60 min post-washing. Results demonstrated that black-footed ferret semen has a comparatively high osmolality (mean+/-SEM, 513.1+/-32.6 mOsm; range, 366-791 mOsm). Ferret spermatozoa were sensitive to hyperosmotic stress. Specifically, sperm motility was more susceptible (P<0.01) to hyperosmotic conditions than acrosomal integrity, and neither were influenced (P>0.05) by hypotonic solutions. Exposure to TYB 4% glycerol retained more (P<0.01) sperm motility than a hyperosmotic Ham's (700 mOsm). These findings will guide the eventual development of assisted breeding with cryopreserved sperm contributing to genetic management of this rare species.

Influences of dietary supplementation with Lepidium meyenii (Maca) on stallion sperm production and on preservation of sperm quality during storage at 5 °C.

PubMed

Del Prete, C; Tafuri, S; Ciani, F; Pasolini, M P; Ciotola, F; Albarella, S; Carotenuto, D; Peretti, V; Cocchia, N

2018-03-01

Stallion semen is damaged by oxidative stress during cooling and transport. Semen processing and extenders have been tested to improve the fertilizing capacity of semen and to preserve semen during transport. Dietary supplementation with natural antioxidants has been proposed to prevent oxidative damages. In this study, for the first time, the effect of dietary supplementation with Lepidium meyenii (Maca) on the characteristics of fresh and chilled stallion semen was evaluated. Maca is a traditional Andean crop used as a nutraceutical for the fertility-enhancing properties that are linked with antioxidant activity. The diet of five stallions was supplemented with 20 g of Maca powder daily for a total of 60 days. A control group of five stallions received the same diet without Maca. Semen was collected once before the administration of Maca (D0), twice during the administration at 30 and 60 days (D30 and D60), and finally twice at 30 and 60 days after the end of the administration (D90 and D120). Ejaculates were processed for cooled shipping at 5 °C and evaluated in the laboratory for total and progressive motility, acrosome integrity, and lipid peroxidation after collection and after 24, 48, and 72 h of storage. Dietary supplementation with Maca improved sperm concentration (from 213 ± 80.4 to 447 ± 73.1 × 10 6 spz/mL) and total sperm count (from 10,880 ± 4377 to 24,783 ± 4419 × 10 6 spz). The beneficial effects of Maca supplementation on motility and acrosome integrity in the raw semen were detected from the end of treatment with Maca (D60) until the end of the study (D120). Furthermore, during cooling storage, total motility, progressive motility, and acrosome integrity declined more slowly in the Maca-treated group than in the control group. Lipid peroxidation did not change during cooling storage in either group and did not show a significant difference between the two groups. In this study, the dietary supplementation with Maca

Flying-fox (Pteropus spp.) sperm membrane fatty acid composition, its relationship to cold shock injury and implications for cryopreservation success.

PubMed

Melville, D F; Johnston, S D; Miller, R R

2012-12-01

The very large acrosome of Pteropus species spermatozoa is prone to damage during cooling procedures. Cryogenic succuss has been linked to membrane composition, therefore the lipid composition of five Pteropus species sperm acrosomal and plasma membranes were investigated to provide insight into reasons for cold shock susceptibility. Rapid chilling and re-warming of spermatozoa from three Pteropus species resulted in a decrease (P<0.05) in acrosomal integrity. Biochemical analysis of lipids revealed that stearic acid (18:0) was the predominant saturated fatty acid and oleic acid (18:1, n-9) the predominant unsaturated fatty acid in both acrosomal and plasma membranes. Linolenic acid (18:3, n-3) was only detected in plasma membranes of Pteropus hypomelanus and was detected in acrosomal membranes of all Pteropus spp. studied (except Pteropus giganteus). Although detected in both plasma and acrosomal membranes of Pteropus vampyrus, docosahexaenoic acid (22:6) was not detected at all in Pteropus poliocephalus, only in trace levels in the acrosomal and plasma membranes of P. giganteus and P. hypomelanus and not in acrosomal membranes of Pteropus rodricensis. No difference was seen in the levels of polyunsaturated fatty acids (PUFAs) within plasma membranes, however PUFAs were lower (P<0.05) in acrosomal membranes of P. giganteus compared with P. vampyrus. Pteropus spp. spermatozoa have a very low ratio of unsaturated/saturated membrane fatty acids (<0.5). Membranes containing more PUFAs are more fluid, so the use of cryogenic media which improves membrane fluidity should improve Pteropus spp. spermatozoal viability post-thaw. Copyright © 2012 Elsevier Inc. All rights reserved.

The benefits of cooling boar semen in long-term extenders prior to cryopreservation on sperm quality characteristics.

PubMed

Wasilewska, K; Zasiadczyk, Ł; Fraser, L; Mogielnicka-Brzozowska, M; Kordan, W

2016-10-01

This study investigated the effects of long-term extenders on post-thaw sperm quality characteristics following different holding times (HT) of boar semen at 17 and 10°C. Sperm-rich fractions, collected from five boars, were diluted in Androhep(®) Plus (AHP), Androstar(®) Plus (ASP), Safecell(®) Plus and TRIXcell(®) Plus (TCP) extenders. The extended semen samples were held for 2 hr at 17°C (HT 1) and additionally for 24 hr at 10°C (HT 2), after they were evaluated and frozen. CASA sperm motility and motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome integrity were assessed in the pre-freeze and frozen-thawed semen. The Vybrant Apoptosis Assay Kit was used to analyse the proportions of viable and plasma membrane apoptotic-like changes in spermatozoa. Results indicated that boar variability, extender and HT significantly affected the sperm quality characteristics, particularly after freezing-thawing. Differences in the pre-freeze semen were more marked in the sperm motion patterns between the HTs. Pre-freeze semen in HT 2 showed significantly higher VCL and VAP, whereas no marked effects were observed in the sperm membrane integrity and viability (YO-PRO-1(-) /PI(-) ) among the extenders. Post-thaw sperm TMOT and PMOT were significantly higher in the AHP and ASP extenders of HT 2 group, whereas VSL, VCL and VAP were markedly lower in the TCP extender. Furthermore, spermatozoa from the AHP- and ASP-extended semen of HT 2 group were characterized by higher MMP, PMI and NAR acrosome integrity following freezing-thawing. In most of the extenders, the incidence of frozen-thawed spermatozoa with apoptotic-like changes was greater in HT 1. The findings of this study indicate that holding of boar semen at 10°C for 24 hr in long-term preservation extenders modulates post-thaw sperm quality characteristics in an extender-dependent manner. These results will further contribute to the

Cryopreservation of bull spermatozoa alters the perinuclear theca.

PubMed

Martínez, Carmen Omega; Juárez-Mosqueda, María de Lourdes; Hernández, Jorge; Valencia, Javier

2006-11-01

The perinuclear theca (PT) is involved in several important sperm functions leading to fertilization. The objective of this study was to investigate the effect of cryopreservation of bull spermatozoa on the integrity of the PT and the relationship between PT integrity and semen characteristics. Semen from seven bulls was evaluated before and after cryopreservation, comparing the integrity of the plasma membrane (hypo-osmotic test), percentage of live and dead spermatozoa (triple stain), acrosome integrity (triple stain) and the integrity of the PT (negative stain by electron microscopy). Cryopreservation of bull semen caused substantial damage to the PT; the proportion of spermatozoa with a damaged PT was 15.2% versus 52.5% (P<0.05) in fresh versus frozen-thawed spermatozoa, respectively. Furthermore, on average, 67.4% (range, 64-72%) of fresh spermatozoa were live, compared to 53.1% (range, 49-58%) for frozen-thawed spermatozoa; there was an inverse correlation between the percentage of live spermatozoa and the percentage with damage to the PT. Although 59.1% of frozen-thawed spermatozoa had an intact acrosome, only 43.7% of them still remained alive. In frozen-thawed semen, there was a high correlation (r=0.69) between live spermatozoa with an intact acrosome and spermatozoa that maintained an intact PT. In conclusion, freezing/thawing of bull spermatozoa altered the PT and maintaining PT integrity may be necessary to maintain acrosome integrity.

Sperm structure and motility in the eusocial naked mole-rat, Heterocephalus glaber: a case of degenerative orthogenesis in the absence of sperm competition?

PubMed Central

2011-01-01

Background We have studied sperm structure and motility in a eusocial rodent where reproduction is typically restricted to a single male and behaviourally dominant queen. Males rarely compete for access to the queen during her estrus cycle, suggesting little or no role for sperm competition. Results Our results revealed an atypical mammalian sperm structure with spermatozoa from breeding, subordinate and disperser males being degenerate and almost completely lacking a "mammalian phylogenetic stamp". Sperm structure is characterized by extreme polymorphism with most spermatozoa classified as abnormal. Sperm head shapes include round, oval, elongated, lobed, asymmetrical and amorphous. At the ultrastructural level, the sperm head contains condensed to granular chromatin with large open spaces between the chromatin. Nuclear chromatin seems disorganized since chromatin condensation is irregular and extremely inconsistent. The acrosome forms a cap (ca 35%) over the anterior part of the head. A well defined nuclear fossa and neck with five minor sets of banded protein structures are present. The midpiece is poorly organized and contains only 5 to 7 round to oval mitochondria. The flagellar pattern is 9+9+2. A distinct degenerative feature of the tail principal piece is the absence of the fibrous sheath. Only 7% motile spermatozoa were observed which had exceptionally slow swimming speeds. Conclusion In this species, sperm form has simplified and degenerated in many aspects and represents a specialised form of degenerative orthogenesis at the cellular level. PMID:22142177

Trehalose improves rabbit sperm quality during cryopreservation.

PubMed

Zhu, Zhendong; Fan, Xiaoteng; Pan, Yang; Lu, Yinghua; Zeng, Wenxian

2017-04-01

High levels of reactive oxygen species are associated with spermatozoa cryopreservation, which bring damage to functional spermatozoa. The aim of the present study was to investigate whether and how the freezing extenders supplemented with trehalose was beneficial for the survival of rabbit spermatozoa. semen was diluted with Tris-citrate-glucose extender addition of different concentrations of trehalose. Addition of 100 mM trehaose significantly improved post-thaw rabbit sperm parameters, such as motility, acrosome integriy, membrane integrity and mitochondrial membrane potential. Moreover, when freezing extenders supplemented with trehalose, activities of catalase (CAT), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) of post-thaw spermatozoa were enhanced, meanwhile, reactive oxygen species (ROS) level and Malondialdehyde (MDA) content were decreased. The results suggest that freezing extenders supplemented with 100 mM trehalose resulted in less ROS level and MDA content, higher motility and mitochondrial membrane potential as well as the integrity of acrosome and plasma membrane. Supplementation of trehalose with freezing extenders is beneficial to the rabbit breeding industry. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of phospholipase activity in Rhinella arenarum sperm extract capable of inducing oocyte activation.

PubMed

Bonilla, Federico; Minahk, Carlos; Ajmat, María Teresa; Toranzo, Graciela Sánchez; Bühler, Marta Inés

2014-11-01

Egg activation, which includes cortical granule exocytosis, resumption and completion of meiosis and pronuclear formation culminates in the first mitotic cleavage. However, the mechanism through which the fertilizing sperm induces this phenomenon is still controversial. We investigated the effect of the microinjection of homologous sperm soluble fractions obtained by fast protein liquid chromatography (FPLC) from reacted sperm (without acrosome) and non-reacted sperm on the activation of Rhinella arenarum oocytes matured in vitro. The FPLC-purified sperm fraction obtained from reacted or non-reacted sperm is able to induce oocyte activation when it is microinjected. This fraction has a 24 kDa protein and showed phospholipase C (PLC) activity in vitro, which was inhibited by D-609 but not by n-butanol or neomycin, suggesting that it is a PLC that is specific for phosphatidylcholine (PC-PLC). The assays conducted using inhibitors of inositol triphosphate (IP3) and ryanodine receptors (RyRs) indicate that the fraction with biological activity would act mainly through the cADPr (cyclic ADP ribose) pathway. Moreover, protein kinase C (PKC) inhibition blocks the activation produced by the same fraction. Immunocytochemical studies indicate that this PC-PLC can be found throughout the sperm head.

Albumin is synthesized in epididymis and aggregates in a high molecular mass glycoprotein complex involved in sperm-egg fertilization.

PubMed

Arroteia, Kélen Fabíola; Barbieri, Mainara Ferreira; Souza, Gustavo Henrique Martins Ferreira; Tanaka, Hiromitsu; Eberlin, Marcos Nogueira; Hyslop, Stephen; Alvares, Lúcia Elvira; Pereira, Luís Antonio Violin Dias

2014-01-01

The epididymis has an important role in the maturation of sperm for fertilization, but little is known about the epididymal molecules involved in sperm modifications during this process. We have previously described the expression pattern for an antigen in epididymal epithelial cells that reacts with the monoclonal antibody (mAb) TRA 54. Immunohistochemical and immunoblotting analyses suggest that the epitope of the epididymal antigen probably involves a sugar moiety that is released into the epididymal lumen in an androgen-dependent manner and subsequently binds to luminal sperm. Using column chromatography, SDS-PAGE with in situ digestion and mass spectrometry, we have identified the protein recognized by mAb TRA 54 in mouse epididymal epithelial cells. The ∼65 kDa protein is part of a high molecular mass complex (∼260 kDa) that is also present in the sperm acrosomal vesicle and is completely released after the acrosomal reaction. The amino acid sequence of the protein corresponded to that of albumin. Immunoprecipitates with anti-albumin antibody contained the antigen recognized by mAb TRA 54, indicating that the epididymal molecule recognized by mAb TRA 54 is albumin. RT-PCR detected albumin mRNA in the epididymis and fertilization assays in vitro showed that the glycoprotein complex containing albumin was involved in the ability of sperm to recognize and penetrate the egg zona pellucida. Together, these results indicate that epididymal-derived albumin participates in the formation of a high molecular mass glycoprotein complex that has an important role in egg fertilization.

The ultrastructure of the spermatozoon of the lizard Iguana iguana (Reptilia, Squamata, Iguanidae) and the variability of sperm morphology among iguanian lizards

PubMed Central

Vieira, Gustavo H C; Colli, Guarino R; Báo, Sônia N

2004-01-01

The spermatozoon of Iguana iguana is filiform and resembles that of other iguanian lizards, being most similar to Tropidurus. All sperm synapomorphies of Tetrapoda, Amniota and Squamata are present in the sperm of Iguana iguana. By reconstructing the evolution of 30 sperm characters we identified a novel synapomorphy of Iguania: the presence of a well-developed acrosomal ridge at the level of the epinuclear lucent zone. Because of the poor topological resolution among iguanian clades we could not discount the possibility of convergence or neutral selection as determinant of the variability in characteristics of the sperm cell. In agreement with previous studies, we identified heterogeneous rates of evolution among the three main regions of the sperm cell, namely the head, midpiece and tail. PMID:15198687

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

Lack of voltage-dependent calcium channel opening during the calcium influx induced by progesterone in human sperm. Effect of calcium channel deactivation and inactivation.

PubMed

Guzmán-Grenfell, Alberto Martín; González-Martínez, Marco T

2004-01-01

Progesterone induces calcium influx and acrosomal exocytosis in human sperm. Pharmacologic evidence suggests that voltage-dependent calcium channels (VDCCs) are involved. In this study, membrane potential (Vm) and intracellular calcium concentration ([Ca(2+)](i)) were monitored simultaneously to assess the effect of VDCC gating on the calcium influx triggered by progesterone. Holding the Vm to values that maintained VDCCs in a deactivated (-71 mV) closed state inhibited the calcium influx induced by progesterone by approximately 40%. At this Vm, the acrosomal reaction induced by progesterone, but not by A23187, was inhibited. However, when the Vm was held at -15 mV (which maintains VDCCs in an inactivated closed state), the progesterone-induced calcium influx was stimulated. Furthermore, the progesterone and voltage-dependent calcium influxes were additive. These findings indicate that progesterone does not produce VDCC gating in human sperm.

Sperm-cell ultrastructure of North American sturgeons. IV. The pallid sturgeon (Scaphirhynchus albus Forbes and Richardson, 1905)

USGS Publications Warehouse

DiLauro, M.N.; Walsh, R.A.; Peiffer, M.; Bennett, R.M.

2001-01-01

Sperm-cell morphology and ultrastructure in the pallid sturgeon (Scaphirhynchus albus) were examined using transmission and scanning electron microscopy. Metrics and structure were compared with similar metrics obtained from other published descriptions of sturgeon sperm cells. General morphology was found to be similar to that of sperm cells of the white (Acipenser transmontanus), lake (A. fulvescens), stellate (A. stellatus), Chinese (A. sinensis), Russian (A. gueldenstaedti colchicus), and shortnose (A. brevirostrum) sturgeons, which all shared a gradual tapering of the nuclear diameter from posterior to anterior, unlike that of the Atlantic sturgeon (A. oxyrhynchus). The sperm cell of the pallid sturgeon was similar in size to that of the Atlantic sturgeon, being only slightly larger. The sperm cell of the pallid sturgeon differed from those of other sturgeons chiefly in the acrosomal region, where the posterolateral projections (PLP) have the shape of an acute triangle and are arranged in a spiral about the longitudinal axis of the cell. The PLP were longer than those of other sturgeons, being twice the length of those of the Atlantic sturgeon and 58% longer than those of the lake sturgeon. Also, in cross section the acrosome had the shape of a hollow cone rather than the cap of an oak tree acorn, as was found in ultrastructural studies of other sturgeons. In addition, we were able to confirm that the structural arrangement of the distal centriole of the midpiece is identical with that of the proximal centriole: nine sets of microtubular triplets around the periphery of the centriole. This information is of potential use to fishery biologists, forensic biologists, zoologists, reproductive physiologists, taxonomists, evolutionary biologists, and aquaculturists.

Group X secreted phospholipase A₂ specifically decreases sperm motility in mice.

PubMed

Escoffier, Jessica; Pierre, Virginie J; Jemel, Ikram; Munch, Léa; Boudhraa, Zied; Ray, Pierre F; De Waard, Michel; Lambeau, Gérard; Arnoult, Christophe

2011-10-01

Different mammalian secreted phospholipases A(2) (sPLA(2) s) are expressed in male reproductive organs and/or in sperm cells but their cellular functions are still not fully characterized. Because several reports indicate a link between cellular lipids and sperm motility, we have investigated the effect of mouse group IIA, IID, IIE, V, and X sPLA(2) s on sperm motility. Among these enzymes, only mouse group X sPLA(2) (mGX sPLA(2) ) acts as a potent inhibitor of sperm motility that decreases track speed (VCL) and lateral displacement of the head (ALH) of both noncapacitated and capacitated sperm. The inhibitory effect of mGX sPLA(2) is dependent on its enzymatic activity because (i) both the proenzyme form of mGX sPLA(2) (pro-mGX) and the H48Q mutant of mGX sPLA(2) have very weak enzymatic activity and are unable to modulate sperm motility and (ii) LY329722, a specific inhibitor of sPLA(2) s, blocks the inhibitory effect of mGX sPLA(2) . Moreover, mGX sPLA(2) exerts a gradual potency on sperm subpopulations with different velocities, an effect which may be linked to the heterogeneity of lipid composition in these sperm subpopulations. Finally, we found that endogenous mGX sPLA(2) released during spontaneous acrosome reaction modulates sperm motility of capacitated sperm. Together, our results suggest a new role of sPLA(2) in sperm physiology where the sPLA2 selects a sperm subpopulation for fertilization based on its effect on sperm motility. Copyright © 2010 Wiley-Liss, Inc.

Effect of age and breeding season on sperm acrosin activity in the arctic fox (Alopex lagopus L.).

PubMed

Stasiak, K; Janicki, B

2014-01-01

The objective of this study was to determine the effect of age and reproductive season on selected properties of semen from the arctic fox, Aloper lagopus L. The experiment used 40 ejaculates collected manually from 6 animals (3 foxes aged one year and 3 foxes older than three years). Statistically less semen (0.39 cm3) was collected from the young compared to the older animals, and the ejaculates obtained were characterized by higher concentration of spermatozoa (195.04 x 106/cm3). In turn, sperm acrosomal extracts from the older animals contained statistically more acrosin (6,4 mU/106 spermatozoa). In the sperm acrosomal extracts prepared during the first semen sampling, the mean acrosin activity did not exceed 2.3 mU/million spermatozoa. At subsequent semen sampling dates, the activity of the analysed enzyme increased to reach 7.72 mU/million spermatozoa. In the extracts obtained from the semen collected at the end of the breeding season of arctic foxes, the acrosin activity again reached a value obtained at the beginning of the season.

The pattern of tyrosine phosphorylation in human sperm in response to binding to zona pellucida or hyaluronic acid.

PubMed

Sati, Leyla; Cayli, Sevil; Delpiano, Elena; Sakkas, Denny; Huszar, Gabor

2014-05-01

In mammalian species, acquisition of sperm fertilization competence is dependent on the phenomenon of sperm capacitation. One of the key elements of capacitation is protein tyrosine phosphorylation (TP) in various sperm membrane regions. In previous studies performed, the pattern of TP was examined in human sperm bound to zona pellucida of oocytes. In the present comparative study, TP patterns upon sperm binding to the zona pellucida or hyaluronic acid (HA) were investigated in spermatozoa arising from the same semen samples. Tyrosine phosphorylation, visualized by immunofluorescence, was localized within the acrosomal cap, equatorial head region, neck, and the principal piece. Tyrosine phosphorylation has increased in a time-related manner as capacitation progressed, and the phosphorylation pattern was identical within the principal piece and neck, regardless of the sperm bound to the zona pellucida or HA. Thus, the data demonstrated that the patterns of sperm activation-related TP were similar regardless of the spermatozoa bound to zona pellucida or HA. Further, sperm with incomplete development, as detected by excess cytoplasmic retention, failed to exhibit TP.

The Pattern of Tyrosine Phosphorylation in Human Sperm in Response to Binding to Zona Pellucida or Hyaluronic Acid

PubMed Central

Sati, Leyla; Cayli, Sevil; Delpiano, Elena; Sakkas, Denny

2014-01-01

In mammalian species, acquisition of sperm fertilization competence is dependent on the phenomenon of sperm capacitation. One of the key elements of capacitation is protein tyrosine phosphorylation (TP) in various sperm membrane regions. In previous studies performed, the pattern of TP was examined in human sperm bound to zona pellucida of oocytes. In the present comparative study, TP patterns upon sperm binding to the zona pellucida or hyaluronic acid (HA) were investigated in spermatozoa arising from the same semen samples. Tyrosine phosphorylation, visualized by immunofluorescence, was localized within the acrosomal cap, equatorial head region, neck, and the principal piece. Tyrosine phosphorylation has increased in a time-related manner as capacitation progressed, and the phosphorylation pattern was identical within the principal piece and neck, regardless of the sperm bound to the zona pellucida or HA. Thus, the data demonstrated that the patterns of sperm activation-related TP were similar regardless of the spermatozoa bound to zona pellucida or HA. Further, sperm with incomplete development, as detected by excess cytoplasmic retention, failed to exhibit TP. PMID:24077441

Antioxidant Effect of Xanthan Gum on Ram Sperm after Freezing and Thawing.

PubMed

Gastal, G DA; Silva, E F; Mion, B; Varela Junior, A S; Rosa, C E; Corcini, C D; Mondadori, R G; Vieira, A D; Bianchi, I; Lucia, T

Xanthan gum is used as thickener in media to preserve food products, having cryoprotectant and antioxidant properties that may be relevant for sperm cryopreservation. To evaluate the effects of adding xanthan gum to freezing extenders on post-thawing quality and oxidant activity of ram sperm. Ejaculates from seven rams extended TRIS-egg yolk-glycerol were split in three treatments including xanthan gum (0.15%; 0.20%; and 0.25%) and a control with no xanthan gum. After thawing, motility and production of reactive oxygen species (ROS) with 0.20% and 0.25% xanthan gum were lower than for the control (P < 0.05), but mitochondrial functionality and integrity of membrane, acrosome and DNA did not differ (P > 0.05). Xanthan gum at 0.20% and 0.25% may be an efficient antioxidant for frozen-thawed ram sperm, due to the reduction in ROS production.

Boar variability in sperm cryo-tolerance after cooling of semen in different long-term extenders at various temperatures.

PubMed

Wasilewska, K; Fraser, L

2017-10-01

This study investigated individual boar variability in the quality of pre-freeze (PF) and post-thaw (PT) semen cooled in different long-term (LT) extenders and for different holding times (HT). Sperm rich fractions were diluted with Androhep ® Plus (AHP), Androstar ® Plus (ASP), Safecell ® Plus (SCP) and TRIXcell ® Plus (TCP) extenders, stored for 2h at 17°C (HT 1) and additionally for 24h at 10°C (HT 2) and the samples were subsequently evaluated and frozen. Besides the analysis of CASA sperm variables, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI), normal apical ridge (NAR) acrosome integrity, and viability (YO-PRO-1 - /PI - ) of sperm were assessed in the PF and PT semen. Results indicated that boar, extender and HT group affected the sperm quality characteristics. There were great variations in PMOT and the sperm motion patterns of the PF semen among the boars. Differences in the HT groups of the PF semen, with respect to the sperm membrane integrity, were less marked among the boars. Consistent variations in TMOT and PMOT in the PT semen were observed among the boars, being greater in the HT 2 group. Most of the CASA-analyzed sperm motion patterns were greater in the HT 2 group of the PT semen. Furthermore, sperm MMP, PMI and viability were greater in the HT 2 group of the PT semen in most of the boars, while consistent differences were observed among the boars for sperm NAR acrosome integrity in either HT group. The significant effect of the cryopreservation process on the sperm membrane proteome was evident from the number of protein bands, detected in the electrophoretic profiles of sperm of the HT 1 and HT 2 groups. The electrophoretic profiles of the PF and PT semen among boars with poor and good semen freezability, however, differed with respect to the abundance and types of sperm membrane-associated proteins. The overall results of this study provided evidence that there are differences among boars in response to the

Calcium/calmodulin and cAMP/protein kinase-A pathways regulate sperm motility in the stallion.

PubMed

Lasko, Jodi; Schlingmann, Karen; Klocke, Ann; Mengel, Grace Ann; Turner, Regina

2012-06-01

In spite of the importance of sperm motility to fertility in the stallion, little is known about the signaling pathways that regulate motility in this species. In other mammals, calcium/calmodulin signaling and the cyclic AMP/protein kinase-A pathway are involved in sperm motility regulation. We hypothesized that these pathways also were involved in the regulation of sperm motility in the stallion. Using immunoblotting, calmodulin and the calmodulin-dependent protein kinase II β were shown to be present in stallion sperm and with indirect immunofluorescence calmodulin was localized to the acrosome and flagellar principal piece. Additionally, inhibition of either calmodulin or protein kinase-A significantly reduced sperm motility without affecting viability. Following inhibition of calmodulin, motility was not restored with agonists of the cyclic AMP/protein kinase-A pathway. These data suggest that calcium/calmodulin and cyclic AMP/protein kinase-A pathways are involved in the regulation of stallion sperm motility. The failure of cyclic AMP/protein kinase-A agonists to restore motility of calmodulin inhibited sperm suggests that both pathways may be required to support normal motility. Copyright © 2012 Elsevier B.V. All rights reserved.

Dilution of boar ejaculates with BTS containing HEPES in place of bicarbonate immediately after ejaculation can reduce the increased inducibility of the acrosome reaction by treatment with calcium and calcium ionophore A23187, which is potentially associated with boar subfertility.

PubMed

Murase, Tetsuma; Imaeda, Noriaki; Yamada, Hiroto; Takasu, Masaki; Taguchi, Kazuo; Katoh, Tsutomu

2010-06-01

The present study investigated whether substitution of HEPES for bicarbonate in BTS (BTS-H) used to dilute boar ejaculates immediately after ejaculation could reduce the increased inducibility of the acrosome reaction by calcium and calcium ionophore A23187. When an ejaculate was split, diluted 5-fold with regular BTS (BTS-B) and BTS-H and stored at 17 C for 12 h or 60 h, the extender or storage time had no significant influence on sperm motility or viability measured by the eosin-nigrosin method. When spermatozoa diluted serially with BTS-B and stored (36 h) were stimulated with Ca2+ (3 mM) and A23187 (0.3 microM), the proportion of spermatozoa that underwent the acrosome reaction (% acrosome reactions) significantly increased as the magnifications of dilution increased (bicarbonate content almost unchanged by dilution). By contrast, the % acrosome reactions in spermatozoa similarly diluted and stored with BTS-H decreased with the increasing magnifications of dilution (bicarbonate decreased). Sperm motility immediately after the end of incubation without A23178 tended to be lower for BTS-H than BTS-B, and the ejaculates for BTS-H had a tendency to have a lower total protein in seminal plasma than those for BTS-B. These results implied that the samples for BTS-H could be used as a model for ejaculates possibly collected during summer and showing subfertility. When an ejaculate was split, diluted serially with BTS-B and BTS-H and stored, viability measured by staining with propidium iodide was extremely similar between the 2 extenders and among the different dilution magnifications, regardless of whether spermatozoa were washed (stored for 36-66 h) or not (stored for 66-72 h). These results suggest that boar ejaculate can be stored with BTS-H at least according to the results for sperm motility and viability and that hypersensitivity of spermatozoa to Ca2+ and A23187 potentially associated with boar subfertility could be lessened by diluting ejaculates with BTS-H.

A membrane-associated adenylate cyclase modulates lactate dehydrogenase and creatine kinase activities required for bull sperm capacitation induced by hyaluronic acid.

PubMed

Fernández, Silvina; Córdoba, Mariana

2017-04-01

Hyaluronic acid, as well as heparin, is a glycosaminoglycan present in the female genital tract of cattle. The aim of this study was to evaluate oxidative metabolism and intracellular signals mediated by a membrane-associated adenylate cyclase (mAC), in sperm capacitation with hyaluronic acid and heparin, in cryopreserved bull sperm. The mAC inhibitor, 2',5'-dideoxyadenosine, was used in the present study. Lactate dehydrogenase (LDH) and creatine kinase (CK) activities and lactate concentration were determined spectrophotometrically in the incubation medium. Capacitation and acrosome reaction were evaluated by chlortetracycline technique, while plasma membrane and acrosome integrity were determined by trypan blue stain/differential interference contrast microscopy. Heparin capacitated samples had a significant decrease in LDH and CK activities, while in hyaluronic acid capacitated samples LDH and CK activities both increased compared to control samples, in heparin and hyaluronic acid capacitation conditions, respectively. A significant increase in lactate concentration in the incubation medium occurred in hyaluronic acid-treated sperm samples compared to heparin treatment, indicating this energetic metabolite is produced during capacitation. The LDH and CK enzyme activities and lactate concentrations in the incubation medium were decreased with 2',5'-dideoxyadenosine treatment in hyaluronic acid samples. The mAC inhibitor significantly inhibited heparin-induced capacitation of sperm cells, but did not completely inhibit hyaluronic acid capacitation. Therefore, hyaluronic acid and heparin are physiological glycosaminoglycans capable of inducing in vitro capacitation in cryopreserved bull sperm, stimulating different enzymatic pathways and intracellular signals modulated by a mAC. Hyaluronic acid induces sperm capacitation involving LDH and CK activities, thereby reducing oxidative metabolism, and this process is mediated by mAC. Copyright © 2017 Elsevier B.V. All

The impact of bacteriospermia on boar sperm storage and reproductive performance.

PubMed

Kuster, C E; Althouse, G C

2016-01-01

Bacteriospermia is a documented risk to reproductive performance when using extended boar semen for artificial insemination. A substantial list of bacteria have been recovered from boar semen attributed to fecal, preputial, skin, and hair microorganisms, with these and other environmental bacteria from processing areas identified in doses prepared for artificial insemination. Gram-negative bacteria are most commonly recovered from extended doses, including both Enterobacteriaceae and environmental contaminants, such as those that inhabit water purification systems. The method of processing, distributing, and storing fresh liquid boar semen before insemination plays a role in bacterial growth dynamics and the degree to which the bacteria may damage the sperm or affect the sow. Not all bacterial isolates or contamination levels have the same impact on sperm, with multiple factors governing if and when storage longevity will be reduced through sperm-to-sperm agglutination, impaired motility, acrosome disruption, or loss of membrane viability. Suboptimal reproductive performance can occur because of reduced fertilizing capacity of the sperm or induction of a uterine environment hostile to sperm and/or embryonic survival. Effective bacterial control strategies are necessary to minimize the risk of bacteria contaminating extended semen doses, including monitoring programs designed for quick detection and intervention, should the need arise. Copyright © 2016 Elsevier Inc. All rights reserved.

Human spermatozoa possess a calcium-dependent chloride channel that may participate in the acrosomal reaction

PubMed Central

Orta, Gerardo; Ferreira, Gonzalo; José, Omar; Treviño, Claudia L; Beltrán, Carmen; Darszon, Alberto

2012-01-01

Motility, maturation and the acrosome reaction (AR) are fundamental functions of mammalian spermatozoa. While travelling through the female reproductive tract, spermatozoa must mature through a process named capacitation, so that they can reach the egg and undergo the AR, an exocytotic event necessary to fertilize the egg. Though Cl− is important for sperm capacitation and for the AR, not much is known about the molecular identity of the Cl− transporters involved in these processes. We implemented a modified perforated patch-clamp strategy to obtain whole cell recordings sealing on the head of mature human spermatozoa. Our whole cell recordings revealed the presence of a Ca2+-dependent Cl− current. The biophysical characteristics of this current and its sensitivity to niflumic acid (NFA) and 4,4′-diisothiocyano-2,2′-stilbene disulphonic acid (DIDIS) are consistent with those displayed by the Ca2+-dependent Cl− channel from the anoctamin family (TMEM16). Whole cell patch clamp recordings in the cytoplasmic droplet of human spermatozoa corroborated the presence of these currents, which were sensitive to NFA and to a small molecule TMEM16A inhibitor (TMEM16Ainh, an aminophenylthiazole). Importantly, the human sperm AR induced by a recombinant human glycoprotein from the zona pellucida, rhZP3, displayed a similar sensitivity to NFA, DIDS and TMEM16Ainh as the sperm Ca2+-dependent Cl− currents. Our findings indicate the presence of Ca2+-dependent Cl− currents in human spermatozoa, that TMEM16A may contribute to these currents and also that sperm Ca2+-dependent Cl− currents may participate in the rhZP3-induced AR. PMID:22473777

Characteristics and fate of the spermatozoa of Inachus phalangium (Decapoda, Majidae): description of novel sperm structures and evidence for an additional mechanism of sperm competition in Brachyura.

PubMed

Rorandelli, Rocco; Paoli, Francesco; Cannicci, Stefano; Mercati, David; Giusti, Fabiola

2008-03-01

Various aspects of the reproductive anatomy of the spider crab Inachus phalangium are investigated utilizing light and electron microscopy. Spermatozoal ultrastructure reveals the presence of a glycocalyx in the peripheral region of the periopercular rim, never recorded before in crustacean sperm cells. Sperm cell morphological traits such as semi-lunar acrosome shape, centrally perforate and flat operculum, and absence of a thickened ring, are shared only with Macropodia longirostris, confirming a close phylogenetic relationship of these species and their separation from the other members of the family Majidae. Spermatozoa are transferred to females inside spermatophores of different sizes, but during ejaculate transfer, larger spermatophores might be ruptured by tooth-like structures present on the ejaculatory canal of the male first gonopod, releasing free sperm cells. Such a mechanism could represent the first evidence of a second form of sperm competition in conflict with sperm displacement, the only mechanism of sperm competition known among Brachyura, enabling paternity for both dominant and smaller, non-dominant, males. In addition, we propose several hypotheses concerning the remote and proximal causes of the existence of large seminal receptacles in females of I. phalangium. Among these, genetically diverse progeny, reduction of sexual harassment and phylogenetic retention seem the most plausible, while acquisition of nutrients from seminal fluids, demonstrated in other arthropods, and suggested by previous studies, could be discarded on the basis of the presented data.

Vitelline envelope of Bufo arenarum: biochemical and biological characterization.

PubMed

Barisone, Gustavo A; Hedrick, Jerry L; Cabada, Marcelo O

2002-04-01

Vitelline envelopes (VEs) of Bufo arenarum were isolated in order to study their composition and their role in fertilization. VEs are composed of four glycoproteins, with molecular masses of 120, 75, 41, and 38 kDa. To characterize its biological properties, we quantitatively determined sperm-VE binding and the induction of the acrosome reaction. Heterologous binding of B. arenarum sperm to Xenopus laevis VE components was observed with about one-third the efficiency of homologous binding. Equivalent binding of X. laevis sperm to the B. arenarum VE was observed. When B. arenarum sperm were incubated with fluorescein isothiocyanate-labeled VE, the labeled glycoproteins bound to the anterior end of the sperm head, showing a lateral distribution. Induction of the acrosome reaction was evaluated by incubating sperm in hypotonic saline media with VE glycoproteins. VEs induced the acrosome reaction in a time- and concentration-dependent manner. The acrosome reaction was maximal after 10 min. The half-maximal effect was obtained at a glycoprotein concentration of 1 microg/ml. Specificity was determined using fertilization envelope glycoproteins, which failed to induce the acrosome reaction. The B. arenarum VE is biochemically similar to other egg envelopes. It also seems that its biological properties are similar to other species in regard to sperm binding and induction of the acrosome reaction. However, as far as we are aware, this is the first observation of the VE inducing the sperm acrosome reaction in amphibians. The relatively small differences observed in heterologous sperm-VE binding in X. laevis and B. arenarum are inconsistent with the current paradigm that species specificity in fertilization is regulated at the sperm-VE binding step.

Use of testicular sperm for intracytoplasmic sperm injection in men with high sperm DNA fragmentation: a SWOT analysis

PubMed Central

Esteves, Sandro C; Roque, Matheus; Garrido, Nicolás

2018-01-01

Spermatozoa retrieved from the testis of men with high levels of sperm DNA fragmentation (SDF) in the neat semen tend to have better DNA quality. Given the negative impact of SDF on the outcomes of Assisted Reproductive Technology (ART), an increased interest has emerged about the use of testicular sperm for intracytoplasmic sperm injection (Testi-ICSI). In this article, we used a SWOT (strengths, weaknesses, opportunities, and threats) analysis to summarize the advantages and drawbacks of this intervention. The rationale of Testi-ICSI is bypass posttesticular DNA fragmentation caused by oxidative stress during sperm transit through the epididymis. Hence, oocyte fertilization by genomically intact testicular spermatozoa may be optimized, thus increasing the chances of creating a normal embryonic genome and the likelihood of achieving a live birth, as recently demonstrated in men with high SDF. However, there is still limited evidence as regards the clinical efficacy of Testi-ICSI, thus creating opportunities for further confirmatory clinical research as well as investigation of Testi-ICSI in clinical scenarios other than high SDF. Furthermore, Testi-ICSI can be compared to other laboratory preparation methods for deselecting sperm with damaged DNA. At present, the available literature supports the use of testicular sperm when performing ICSI in infertile couples whose male partners have posttesticular SDF. Due to inherent risks of sperm retrieval, Testi-ICSI should be offered when less invasive treatments for alleviating DNA damage have failed. A call for continuous monitoring is nonetheless required concerning the health of generated offspring and the potential complications of sperm retrieval. PMID:28440264

Use of testicular sperm for intracytoplasmic sperm injection in men with high sperm DNA fragmentation: a SWOT analysis.

PubMed

Esteves, Sandro C; Roque, Matheus; Garrido, Nicolás

2018-01-01

Spermatozoa retrieved from the testis of men with high levels of sperm DNA fragmentation (SDF) in the neat semen tend to have better DNA quality. Given the negative impact of SDF on the outcomes of Assisted Reproductive Technology (ART), an increased interest has emerged about the use of testicular sperm for intracytoplasmic sperm injection (Testi-ICSI). In this article, we used a SWOT (strengths, weaknesses, opportunities, and threats) analysis to summarize the advantages and drawbacks of this intervention. The rationale of Testi-ICSI is bypass posttesticular DNA fragmentation caused by oxidative stress during sperm transit through the epididymis. Hence, oocyte fertilization by genomically intact testicular spermatozoa may be optimized, thus increasing the chances of creating a normal embryonic genome and the likelihood of achieving a live birth, as recently demonstrated in men with high SDF. However, there is still limited evidence as regards the clinical efficacy of Testi-ICSI, thus creating opportunities for further confirmatory clinical research as well as investigation of Testi-ICSI in clinical scenarios other than high SDF. Furthermore, Testi-ICSI can be compared to other laboratory preparation methods for deselecting sperm with damaged DNA. At present, the available literature supports the use of testicular sperm when performing ICSI in infertile couples whose male partners have posttesticular SDF. Due to inherent risks of sperm retrieval, Testi-ICSI should be offered when less invasive treatments for alleviating DNA damage have failed. A call for continuous monitoring is nonetheless required concerning the health of generated offspring and the potential complications of sperm retrieval.

Dynamin Regulates Specific Membrane Fusion Events Necessary for Acrosomal Exocytosis in Mouse Spermatozoa

PubMed Central

Reid, Andrew T.; Lord, Tessa; Stanger, Simone J.; Roman, Shaun D.; McCluskey, Adam; Robinson, Phillip J.; Aitken, R. John; Nixon, Brett

2012-01-01

Mammalian spermatozoa must complete an acrosome reaction prior to fertilizing an oocyte. The acrosome reaction is a unique exocytotic event involving a series of prolonged membrane fusions that ultimately result in the production of membrane vesicles and release of the acrosomal contents. This event requires the concerted action of a large number of fusion-competent signaling and scaffolding proteins. Here we show that two different members of the dynamin GTPase family localize to the developing acrosome of maturing mouse germ cells. Both dynamin 1 and 2 also remain within the periacrosomal region of mature mouse spermatozoa and are thus well positioned to regulate the acrosome reaction. Two pharmacological inhibitors of dynamin, dynasore and Dyngo-4a, blocked the in vitro induction of acrosomal exocytosis by progesterone, but not by the calcium ionophore A23187, and elicited a concomitant reduction of in vitro fertilization. In vivo treatment with these inhibitors also resulted in spermatozoa displaying reduced acrosome reaction potential. Dynamin 1 and 2 phosphorylation increased on progesterone treatment, and this was also selectively blocked by dynasore. On the basis of our collective data, we propose that dynamin could regulate specific membrane fusion events necessary for acrosomal exocytosis in mouse spermatozoa. PMID:22977254

Sperm collection by transrectal ultrasound-guided massage of the accessory sex glands is less stressful than electroejaculation without altering sperm characteristics in conscious goat bucks.

PubMed

Abril-Sánchez, Silvia; Freitas-de-Melo, Aline; Beracochea, Florencia; Damián, Juan Pablo; Giriboni, Julia; Santiago-Moreno, Julián; Ungerfeld, Rodolfo

2017-08-01

In anesthetized non-domestic ruminants transrectal ultrasound-guided massage of the accessory sex glands (TUMASG) is an alternative method to collect semen slightly less stressful than electroejaculation (EE). However, some sperm characteristics are better when semen is collected with EE than with TUMASG. As anesthesia reduces the response to stressors, the advantages of TUMASG may be reduced in anesthetized animals, and thus, TUMASG may be even more advantageous in conscious animals. Therefore, the aim of the present study was to compare the stress response and the characteristics of the sperm collected with TUMASG and EE in conscious goat bucks. Semen was collected in 10 bucks with both procedures. During each procedure, the time required for ejaculation, the number of electric pulses applied and the number of vocalizations were recorded. Rectal temperature, heart rate, serum cortisol concentration, biochemical and hematological parameters were measured before and after each procedure. Sperm characteristics [ejaculated volume, sperm concentration, sperm mass motility (scale 0-5), sperm vigor (scale 0-5), the percentages of motile and progressive motile sperm, of sperm vitality, of sperm with plasma membrane integrity, and with acrosome damage and morphological abnormalities] were also determined. Electroejaculation required more electric pulses than TUMASG (P < 0.0001), but TUMASG took more time than EE (P < 0.0001). The EE provoked more vocalizations (P = 0.02) and a greater increase of cortisol concentrations than TUMASG (P = 0.04). Heart rate also tended to be greater with EE than with TUMASG (P = 0.07). The sperm characteristics did not differ between TUMASG and EE. In conclusion, TUMASG was less stressful and probably less painful than EE without affecting the semen quality. Thus, although it required more time, TUMASG is an alternative procedure to decrease the welfare concerns raised by EE in conscious goat bucks. Copyright © 2017 Elsevier Inc

Involvement of a Na+/HCO-3 cotransporter in mouse sperm capacitation.

PubMed

Demarco, Ignacio A; Espinosa, Felipe; Edwards, Jennifer; Sosnik, Julian; De La Vega-Beltran, Jose Luis; Hockensmith, Joel W; Kopf, Gregory S; Darszon, Alberto; Visconti, Pablo E

2003-02-28

Mammalian sperm are incapable of fertilizing eggs immediately after ejaculation; they acquire fertilization capacity after residing in the female tract for a finite period of time. The physiological changes sperm undergo in the female reproductive tract that render sperm able to fertilize constitute the phenomenon of "sperm capacitation." We have demonstrated that capacitation is associated with an increase in the tyrosine phosphorylation of a subset of proteins and that these events are regulated by an HCO(3)(-)/cAMP-dependent pathway involving protein kinase A. Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. Here we present evidence that, in addition to its role in the regulation of adenylyl cyclase, HCO(3)(-) has a role in the regulation of plasma membrane potential in mouse sperm. Addition of HCO(3)(-) but not Cl(-) induces a hyperpolarizing current in mouse sperm plasma membranes. This HCO(3)(-)-dependent hyperpolarization was not observed when Na(+) was replaced by the non-permeant cation choline(+). Replacement of Na(+) by choline(+) also inhibited the capacitation-associated increase in protein tyrosine phosphorylation as well as the zona pellucida-induced acrosome reaction. The lack of an increase in protein tyrosine phosphorylation was overcome by the presence of cAMP agonists in the incubation medium. The lack of a hyperpolarizing HCO(3)(-) current and the inhibition of the capacitation-dependent increase in protein tyrosine phosphorylation in the absence of Na(+) suggest that a Na(+)/HCO(3)(-) cotransporter is present in mouse sperm and is coupled to events regulating capacitation.

The influence of benign prostatic hyperplasia on sperm morphological features and sperm DNA integrity in dogs.

PubMed

Flores, R B; Angrimani, Dsr; Rui, B R; Brito, M M; Abreu, R A; Vannucchi, C I

2017-04-01

Benign prostatic hyperplasia (BPH) has a high incidence in older intact dogs. Due to the increased prostatic oxidative stress and hormonal imbalance of BPH, sperm damage can arise, such as sperm morphological alterations and DNA fragmentation. This study aimed to compare the reproductive potential of healthy dogs and those affected by benign prostatic hyperplasia. Ten dogs were assigned to two experimental groups: dogs without BPH (control; n = 5) and dogs diagnosed with BPH (n = 5), based on clinical signs and ultrasonographic findings. Three semen collections were performed from each dog within one month and analysed using computer-assisted sperm analysis (CASA) and functional tests. Control group showed higher percentage of sperm DNA integrity (95 ± 1.8%) compared to the BPH group (79.2 ± 6.4%). On the other hand, the percentage of minor sperm defects, amplitude of lateral sperm head displacement of the spermatozoa and medium sperm mitochondrial activity were higher in the BPH group. In conclusion, BPH decreases sperm DNA integrity, increases mitochondrial activity, as well as modifies sperm movement pattern. Therefore, a careful sperm analysis of aged dogs with BPH is required before a reproductive programme can be established for such patients. © 2016 Blackwell Verlag GmbH.

Loss of heat shock protein 70 from apical region of buffalo (Bubalus bubalis) sperm head after freezing and thawing.

PubMed

Varghese, Tincy; Divyashree, Bannur C; Roy, Sudhir C; Roy, Kajal S

2016-03-15

revealed that HSP70 proteins were distributed over the apical region of sperm head and/or acrosome, post-acrosomal, and middle piece regions of NC buffalo spermatozoa. However, the fluorescence signal of apical region of sperm head was lost significantly (P < 0.05) after a cycle of freezing and thawing. Thus, the present study confirmed that there was loss of HSP70 from buffalo sperm head after freezing and thawing of buffalo spermatozoa, and this may be one of the causes of the reduced post-thaw fertility of sperm in this species. Copyright © 2016 Elsevier Inc. All rights reserved.

Fibroblast Growth Factor Receptors (FGFRs) in Human Sperm: Expression, Functionality and Involvement in Motility Regulation.

PubMed

Saucedo, Lucía; Buffa, Gabriela N; Rosso, Marina; Guillardoy, Tomás; Góngora, Adrian; Munuce, María J; Vazquez-Levin, Mónica H; Marín-Briggiler, Clara

2015-01-01

Fibroblast growth factors receptors (FGFRs) have been widely characterized in somatic cells, but there is scarce evidence of their expression and function in mammalian gametes. The objective of the present study was to evaluate the expression of FGFRs in human male germ cells, to determine sperm FGFR activation by the FGF2 ligand and their participation in the regulation of sperm motility. The expression of FGFR1, 2, 3 and 4 mRNAs and proteins in human testis and localization of these receptors in germ cells of the seminiferous epithelium was demonstrated. In ejaculated sperm, FGFRs were localized to the acrosomal region and flagellum. Sperm exposure to FGF2 caused an increase in flagellar FGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB or Akt) signaling pathways. Incubation with FGF2 led to a significant increase in the percentage of total and progressive sperm motility, as well as in sperm kinematics. All responses were prevented by sperm preincubation with BGJ398, a specific inhibitor of FGFR tyrosine kinase activity. In addition to confirming the expression of FGFRs in germ cells of the human testis, our study describes for the first time the presence, localization and functionality of human sperm FGFRs, and provides evidence of the beneficial effect of FGF2 upon sperm motility.

Fibroblast Growth Factor Receptors (FGFRs) in Human Sperm: Expression, Functionality and Involvement in Motility Regulation

PubMed Central

Saucedo, Lucía; Buffa, Gabriela N.; Rosso, Marina; Guillardoy, Tomás; Góngora, Adrian; Munuce, María J.

2015-01-01

Fibroblast growth factors receptors (FGFRs) have been widely characterized in somatic cells, but there is scarce evidence of their expression and function in mammalian gametes. The objective of the present study was to evaluate the expression of FGFRs in human male germ cells, to determine sperm FGFR activation by the FGF2 ligand and their participation in the regulation of sperm motility. The expression of FGFR1, 2, 3 and 4 mRNAs and proteins in human testis and localization of these receptors in germ cells of the seminiferous epithelium was demonstrated. In ejaculated sperm, FGFRs were localized to the acrosomal region and flagellum. Sperm exposure to FGF2 caused an increase in flagellar FGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB or Akt) signaling pathways. Incubation with FGF2 led to a significant increase in the percentage of total and progressive sperm motility, as well as in sperm kinematics. All responses were prevented by sperm preincubation with BGJ398, a specific inhibitor of FGFR tyrosine kinase activity. In addition to confirming the expression of FGFRs in germ cells of the human testis, our study describes for the first time the presence, localization and functionality of human sperm FGFRs, and provides evidence of the beneficial effect of FGF2 upon sperm motility. PMID:25970615

Effects of addition of sodium lauryl sulfate on frozen-thawed canine spermatozoa.

PubMed

Hori, Tatsuya; Kaseki, Hanae; Fukuhara, Youko; Oba, Hiromichi; Mizutani, Tatsuji; Kawakami, Eiichi; Tsutsui, Toshihiko

2006-10-01

The addition of Orvus ES paste (OEP) to extender may be essential for preparing frozen dog semen. The major ingredient of OEP is sodium lauryl sulfate (SLS). In this study, we compared the effect of SLS on frozen dog semen with that of OEP. There were no significant differences between the 2-mg/ml SLS group and OEP group concerning sperm motility, viability and the percentage of viable sperm with intact acrosomes after freeze-thawing. These results suggest that the effectiveness of frozen dog semen extender containing 2 mg/ml of SLS is similar effective to that demonstrated for OEP.

Heat shock proteins on the human sperm surface.

PubMed

Naaby-Hansen, Soren; Herr, John C

2010-01-01

The sperm plasma membrane is known to be critical to fertilization and to be highly regionalized into domains of head, mid- and principal pieces. However, the molecular composition of the sperm plasma membrane and its alterations during genital tract passage, capacitation and the acrosome reaction remains to be fully dissected. A two-dimensional gel-based proteomic study previously identified 98 human sperm proteins which were accessible for surface labelling with both biotin and radioiodine. In this report twelve dually labelled protein spots were excised from stained gels or PDVF membranes and analysed by mass spectrometry (MS) and Edman degradation. Seven members from four different heat shock protein (HSP) families were identified including HYOU1 (ORP150), HSPC1 (HSP86), HSPA5 (Bip), HSPD1 (HSP60), and several isoforms of the two testis-specific HSP70 chaperones HSPA2 and HSPA1L. An antiserum raised against the testis-specific HSPA2 chaperone reacted with three 65kDa HSPA2 isoforms and three high molecular weight surface proteins (78-79kDa, 84kDa and 90-93kDa). These proteins, together with seven 65kDa HSP70 forms, reacted with human anti-sperm IgG antibodies that blocked in vitro fertilization in humans. Three of these surface biotinylated human sperm antigens were immunoprecipitated with a rabbit antiserum raised against a linear peptide epitope in Chlamydia trachomatis HSP70. The results indicate diverse HSP chaperones are accessible for surface labelling on human sperm. Some of these share epitopes with C. trachomatis HSP70, suggesting an association between genital tract infection, immunity to HSP70 and reproductive failure. 2009 Elsevier Ireland Ltd. All rights reserved.

Hyaluronic acid improves frozen-thawed sperm quality and fertility potential in rooster.

PubMed

Lotfi, Saied; Mehri, Morteza; Sharafi, Mohsen; Masoudi, Reza

2017-09-01

Beneficial effects of Hyaluronic acid (HA) has not been yet assessed for cryopreservation of rooster sperm. This study was conducted to evaluate the effects of different concentrations of HA (0, 1, 2, 4 and 8mM) in Beltsville extender on the cryopreservation of rooster sperm. Semen samples were collected from six Ross broiler breeders (24-week) using abdominal massage, then divided into five equal aliquots and cryopreserved in Beltsville extender that contained different concentrations of HA. Motion characteristics, morphology, membrane functionality, viability, acrosome integrity, lipid peroxidation and fertility potential of sperm were assessed after thawing. HA at concentration of 2mM (HA2) resulted in the highest (P<0.05) total motility (55.3±1.1%) and progressive motility (25.2±0.8%) compared to the other groups. HA8 produced the lowest significant (P<0.05) percentage of total (38.6±1.1%) and progressive (14.7±0.8%) motility. High significant percentage of membrane functionality were observed in HA1 and HA2 (43.2±1.0 and 46.1±1.0%, respectively) compared to HA4 (40.1±1.0%) and HA8 (32.5±1.0%). Moreover, HA1 and HA2 produced the higher percentage of acrosome integrity (54.8±1.2 and 57.5±1.2, respectively) compared to other groups. HA1 and HA2 reduced (P<0.05) malondialdehyde formation (3.66±0.08 and 3.75±0.08 nmol/ml) compared to other groups. Fertility rate and hatching rate obtained from artificial insemination were significantly higher in HA1 (63.7 and 54.7%) and HA2 (67.5 and 57.7%) compared to control group (40 and 37%). Our results showed that supplementation of Beltsville extender with 1 and 2mM HA significantly improved the quality of rooster sperm after freeze thawing. Copyright © 2017 Elsevier B.V. All rights reserved.

Adverse effects of members of the Enterobacteriaceae family on boar sperm quality.

PubMed

Ubeda, Juan Luis; Ausejo, Raquel; Dahmani, Yahya; Falceto, Maria V; Usan, Adan; Malo, Clara; Perez-Martinez, Francisco C

2013-10-01

Semen samples collected in 2012 from 1785 boars belonging to five different breeds were recruited from the quality control laboratory of Magapor SL, Spain. These samples came from 43 boar studs and resulted from diluting the ejaculates in commercial semen extenders. Evaluation of the semen sample characteristics (color, smell, pH, osmolality, concentration, motility of sperm cells, agglutination, acrosome integrity, short hypoosmotic swelling test, and abnormal forms) revealed that they met the international standards. The samples were also tested for the presence of aerobic bacterial contamination. In the present study, 14.73% (n = 263) of the semen samples were contaminated above 3 × 10(2) colony-forming units/mL with at least one type of bacteria. The Enterobacteriaceae family was by far the major contaminant, being present in 40.68% of the contaminated samples (n = 107). Bacterial strains of the Enterobacteriaceae family isolated from boar semen samples were in order of incidence (percentage of the contaminated samples): Serratia marcescens (12.55%), Klebsiella oxytoca (11.79%), Providencia stuartii (9.12%), Morganella morganii (3.80%), Proteus mirabilis (1.90%), and Escherichia coli (1.52%). We have seen that the presence in semen samples of S. marcescens, K. oxytoca, M. morganii, or P. mirabilis, but not P. stuartii or E. coli, was negatively associated with sperm motility (P < 0.05). The mean sperm concentration (P < 0.05), the mean percentage of spermatozoa with curled tails after the short hypoosmotic swelling test (P < 0.01), and the incidence of morphologically normal acrosomes (P < 0.05) were also lower in semen samples infected with M. morganii compared with uninfected ones. Moreover, P. mirabilis was negatively associated with the presence of abnormal forms. Thus, on the basis of the pathological effects that some of these strains may have on boar sperm quality, bacterial contamination should always be examined in semen samples prepared for

The "soluble" adenylyl cyclase in sperm mediates multiple signaling events required for fertilization.

PubMed

Hess, Kenneth C; Jones, Brian H; Marquez, Becky; Chen, Yanqiu; Ord, Teri S; Kamenetsky, Margarita; Miyamoto, Catarina; Zippin, Jonathan H; Kopf, Gregory S; Suarez, Susan S; Levin, Lonny R; Williams, Carmen J; Buck, Jochen; Moss, Stuart B

2005-08-01

Mammalian fertilization is dependent upon a series of bicarbonate-induced, cAMP-dependent processes sperm undergo as they "capacitate," i.e., acquire the ability to fertilize eggs. Male mice lacking the bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC), the predominant source of cAMP in male germ cells, are infertile, as the sperm are immotile. Membrane-permeable cAMP analogs are reported to rescue the motility defect, but we now show that these "rescued" null sperm were not hyperactive, displayed flagellar angulation, and remained unable to fertilize eggs in vitro. These deficits uncover a requirement for sAC during spermatogenesis and/or epididymal maturation and reveal limitations inherent in studying sAC function using knockout mice. To circumvent this restriction, we identified a specific sAC inhibitor that allowed temporal control over sAC activity. This inhibitor revealed that capacitation is defined by separable events: induction of protein tyrosine phosphorylation and motility are sAC dependent while acrosomal exocytosis is not dependent on sAC.

Comparison of Cryopreservation Protocols (Single and Two-steps) and Thawing (Fast and Slow) for Canine Sperm.

PubMed

Brito, Maíra M; Lúcio, Cristina F; Angrimani, Daniel S R; Losano, João Diego A; Dalmazzo, Andressa; Nichi, Marcílio; Vannucchi, Camila I

2017-01-02

In addition to the existence of several cryopreservation protocols, no systematic research has been carried out in order to confirm the suitable protocol for canine sperm. This study aims to assess the effect of adding 5% glycerol during cryopreservation at 37°C (one-step) and 5°C (two-steps), in addition of testing two thawing protocols (37°C for 30 seconds, and 70°C for 8 seconds). We used 12 sperm samples divided into four experimental groups: Single-Step - Slow Thawing Group; Two-Step - Slow Thawing Group; Single-Step - Fast Thawing Group; and Two-Step - Fast Thawing Group. Frozen-thawed samples were submitted to automated analysis of sperm motility, evaluation of plasmatic membrane integrity, acrosomal integrity, mitochondrial activity, sperm morphology, sperm susceptibility to oxidative stress, and sperm binding assay to perivitellinic membrane of chicken egg yolk. Considering the comparison between freezing protocols, no statistical differences were verified for any of the response variables. When comparison between thawing protocols was performed, slow thawing protocol presented higher sperm count bound to perivitelline membrane of chicken egg yolk, compared to fast thawing protocol. Regardless of the freezing process, the slow thawing protocol can be recommended for the large scale cryopreservation of canine semen, since it shows a consistent better functional result.

Blastocyst production after intracytoplasmic sperm injection with semen from a stallion with testicular degeneration.

PubMed

Roels, K; Smits, K; Ververs, C; Govaere, J; D'Herde, K; Van Soom, A

2018-06-01

In horse breeding, intracytoplasmic sperm injection (ICSI) has gained interest to obtain offspring from subfertile individuals. This paper presents a case report of a stallion with severe testicular degeneration. Semen analysis showed very low motility and 83.5% of detached heads. Histology of a testicular biopsy showed severely decreased spermatogenesis, while transmission electron microscopy of the sperm cells revealed no significant abnormalities. A total of 39 oocytes were fertilized by ICSI with frozen-thawed spermatozoa of this stallion: 25 oocytes with intact spermatozoa and 24 with detached heads. When using intact sperm cells, 8 out of the 25 oocytes cleaved, and 1 developed to the blastocyst stage 9 days after ICSI. None of the oocytes injected with a detached sperm head cleaved. Studies on the paternal influence on ICSI outcome are limited in the horse and further research is needed to define which stallion factors may influence ICSI results. Here, we report the possibility to produce a blastocyst by ICSI of a stallion suffering from testicular degeneration with a poor spermiogram, as long as an intact sperm cell containing a centriole is selected. © 2018 Blackwell Verlag GmbH.

Influence of cooling rates and addition of Equex pasta on cooled and frozen-thawed semen of generic gray (Canis lupus) and Mexican gray wolves (C. l. baileyi).

PubMed

Zindl, C; Asa, C S; Günzel-Apel, A-R

2006-10-01

A current priority for the preservation of the endangered Mexican gray wolf (Canis lupus baileyi) is the development of a sperm-based genome resource bank for subsequent use in artificial insemination. To optimize the quality of cryopreserved sperm, the procedures involved in processing semen before and during freezing need to be improved. The aim of this study were to examine the effects of: (i) different cooling periods before freezing and (ii) addition of Equex pasta (Minitüb, Tübingen, Germany) on the characteristics of sperm from the generic gray wolf and the Mexican gray wolf after cooling and cryopreservation. For Mexican wolf sperm, cooling for 0.5 and 1.0 h had a less detrimental effect on cell morphology than cooling for 2.5 h, whereas the slower cooling rate (2.5 h) had a less detrimental effect on functional parameters and seemed to cause less damage to plasma membrane and acrosome integrity than 0.5 and 1.0 h. For the generic gray wolf, cooling semen for 2.5 h had less detrimental effect on plasma membrane integrity and viability; together with the 0.5 h cooling time, it yielded the highest percentages of intact acrosomes. As previously shown in the domestic dog, Equex pasta had no beneficial effect on sperm characteristics in either wolf species.

Cysteine protects rabbit spermatozoa against reactive oxygen species-induced damages

PubMed Central

Fan, Xiaoteng; Pan, Yang; Lv, Shan; Pan, Chuanying; Lei, Anmin

2017-01-01

The process of cryopreservation results in over-production of reactive oxygen species, which is extremely detrimental to spermatozoa. The aim of this study was to investigate whether addition of cysteine to freezing extender would facilitate the cryosurvival of rabbit spermatozoa, and if so, how cysteine protects spermatozoa from cryodamages. Freshly ejaculated semen was diluted with Tris-citrate-glucose extender supplemented with different concentrations of cysteine. The motility, intact acrosomes, membrane integrity, mitochondrial potentials, 8-hydroxyguanosine level and sperm-zona pellucida binding capacity were examined. Furthermore, glutathione peroxidase (GPx) activity, glutathione content (GSH), and level of reactive oxygen species (ROS) and hydrogen peroxide of spermatozoa were analyzed. The values of motility, intact acrosomes, membrane integrity, mitochondrial potentials and sperm-zona pellucida binding capacity of the frozen-thawed spermatozoa in the treatment of cysteine were significantly higher than those of the control. Addition of cysteine to extenders improved the GPx activity and GSH content of spermatozoa, while lowered the ROS, DNA oxidative alterations and lipid peroxidation level, which makes spermatozoa avoid ROS to attack DNA, the plasma membrane and mitochondria. In conclusion, cysteine protects spermatozoa against ROS-induced damages during cryopreservation and post-thaw incubation. Addition of cysteine is recommended to facilitate the improvement of semen preservation for the rabbit breeding industry. PMID:28700739

Body mass index is not associated with sperm-zona pellucida binding ability in subfertile males.

PubMed

Sermondade, Nathalie; Dupont, Charlotte; Faure, Céline; Boubaya, Marouane; Cédrin-Durnerin, Isabelle; Chavatte-Palmer, Pascale; Sifer, Christophe; Lévy, Rachel

2013-09-01

Lifestyle factors, such as weight and nutritional status may affect male fertility, including sperm fertilization ability. The objective of this retrospective study was to evaluate the association between body mass index (BMI) and sperm-zona pellucida binding ability assessed according to the zona binding (ZB) test, which has been described to be a relevant diagnostic tool for the prediction of in vitro fertilization (IVF) ability. Three hundred and six male patients from couples diagnosed with primary idiopathic or mild male factor infertility were included. Correlations between BMI and semen parameters according to ZB test indices were assessed, together with frequencies of positive and negative tests across the BMI categories. In this selected population, BMI was not related to conventional semen parameters or sperm quality assessed according to the ability of spermatozoa to bind to the zona pellucida. The previously described poor outcomes of IVF procedures in cases of male obesity could be due to other sperm defects, such as alterations of sperm capacitation or acrosome reaction. The link between male BMI and biological outcomes during IVF procedures, such as fertilization rates, should be further evaluated.

Effect of feeding fescue seed containing ergot alkaloid toxins on stallion spermatogenesis and sperm cells.

PubMed

Fayrer-Hosken, R; Stanley, A; Hill, N; Heusner, G; Christian, M; De La Fuente, R; Baumann, C; Jones, L

2012-12-01

The cellular effects of tall fescue grass-associated toxic ergot alkaloids on stallion sperm and colt testicular tissue were evaluated. This was a continuation of an initial experiment where the effects of toxic ergot alkaloids on the stallion spermiogram were investigated. The only spermiogram parameter in exposed stallions that was affected by the toxic ergot alkaloids was a decreased gel-free volume of the ejaculate. This study examined the effect of toxic ergot alkaloids on chilling and freezing of the stallion sperm cells. The effect of toxic ergot alkaloids on chilled extended sperm cells for 48 h at 5°C was to make the sperm cells less likely to undergo a calcium ionophore-induced acrosome reaction. The toxic ergot alkaloids had no effect on the freezability of sperm cells. However, if yearling colts were fed toxic ergot alkaloids, then the cytological analysis of meiotic chromosome synapsis revealed a significant increase in the proportion of pachytene spermatocytes showing unpaired sex chromosomes compared to control spermatocytes. There was little effect of ergot alkaloids on adult stallions, but there might be a significant effect on yearling colts. © 2012 Blackwell Verlag GmbH.

Effect of Feeding Fescue Seed Containing Ergot Alkaloid Toxins on Stallion Spermatogenesis and Sperm Cells

PubMed Central

Fayrer-Hosken, R; Stanley, A; Hill, N; Heusner, G; Christian, M; Fuente, R De La; Baumann, C; Jones, L

2012-01-01

Contents The cellular effects of tall fescue grass–associated toxic ergot alkaloids on stallion sperm and colt testicular tissue were evaluated. This was a continuation of an initial experiment where the effects of toxic ergot alkaloids on the stallion spermiogram were investigated. The only spermiogram parameter in exposed stallions that was affected by the toxic ergot alkaloids was a decreased gel-free volume of the ejaculate. This study examined the effect of toxic ergot alkaloids on chilling and freezing of the stallion sperm cells. The effect of toxic ergot alkaloids on chilled extended sperm cells for 48 h at 5 °C was to make the sperm cells less likely to undergo a calcium ionophore–induced acrosome reaction. The toxic ergot alkaloids had no effect on the freezability of sperm cells. However, if yearling colts were fed toxic ergot alkaloids, then the cytological analysis of meiotic chromosome synapsis revealed a significant increase in the proportion of pachytene spermatocytes showing unpaired sex chromosomes compared to control spermatocytes. There was little effect of ergot alkaloids on adult stallions, but there might be a significant effect on yearling colts. PMID:22524585

Epididymosomes: transfer of fertility-modulating proteins to the sperm surface

PubMed Central

Martin-DeLeon, Patricia A

2015-01-01

A variety of glycosylphosphatidylinositol (GPI)-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane (TM) proteins, including plasma membrane Ca2+-ATPase 4 (PMCA4). Synthesized in the testis, PMCA4 is an essential protein and the major Ca2+ efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion–competent sites on membranes. On the basis of knowledge of PMCA4's interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca2+-dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach. PMID:26112481

Epididymosomes: transfer of fertility-modulating proteins to the sperm surface.

PubMed

Martin-DeLeon, Patricia A

2015-01-01

A variety of glycosylphosphatidylinositol (GPI)-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane (TM) proteins, including plasma membrane Ca 2 + -ATPase 4 (PMCA4). Synthesized in the testis, PMCA4 is an essential protein and the major Ca 2 + efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion-competent sites on membranes. On the basis of knowledge of PMCA4's interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca 2 + -dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach.

Correlation between sperm DNA fragmentation index and CMA3 positive spermatozoa in globozoospermic patients.

PubMed

Hosseinifar, H; Yazdanikhah, S; Modarresi, T; Totonchi, M; Sadighi Gilani, M A; Sabbaghian, M

2015-05-01

The absence of the acrosome causes the situation which is called globozoospermia. There are a few studies, mostly as case reports, about correlation between levels of sperm DNA damage in patients with total round-headed spermatozoa. We investigated this correlation as well as CMA3 positive spermatozoa in 20 globozoospermic men (with more than 90% round-headed spermatozoa) attending to Royan Institute. Semen samples divided into three parts to semen analysis, to measure DNA fragmentation index (DFI) using sperm chromatin structure assay (SCSA) and to detect CMA3(+) sperm cells by chromomycin A3 staining and fluorescent microscopy. Our results showed that there were significant differences in sperm concentration, total sperm motility, and normal morphology between patients and controls group (p < 0.001). Moreover, the average of DFI and CMA3 positive spermatozoa in patients group significantly increases compared with control group (p < 0.001). A significant correlation between DFI and CMA3(+) in total population was also detected in patients group (r = 0.45, p = 0.046). To our knowledge, this is the largest study about correlation between DNA damage levels and CMA3 positive spermatozoa with round head sperm cells in total globozoospermic men. It seems that the increase in DNA damage may be because of defective sperm DNA compaction, as we detected CMA3 positive sperm cells in these patients. © 2015 American Society of Andrology and European Academy of Andrology.

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.

Antioxidant effect of rosemary (Rosmarinus officinalis L.) extract in soybean lecithin-based semen extender following freeze-thawing process of ram sperm.

PubMed

Motlagh, Mahdi Khodaei; Sharafi, Mohsen; Zhandi, Mahdi; Mohammadi-Sangcheshmeh, Abdollah; Shakeri, Malak; Soleimani, Masoud; Zeinoaldini, Saeed

2014-10-01

The aim of current study was to evaluate effect of rosemary aqueous extract on post-thawed ram sperm quality in a soybean lecithin-based (SL) extender. Ram semen samples were obtained, extended with SL extender and supplemented with 0% (SL-R0), 2% (SL-R2), 4% (SL-R4), 6% (SL-R6), and 8% (SL-R8) rosemary aqueous extract. Following equilibration, the straws were frozen, and then plunged into the liquid nitrogen. After thawing, sperm motility and velocity parameters, plasma membrane functionality, viability, acrosomal and capacitation status were evaluated. Membrane lipid peroxidation was also analyzed through the malondialdehyde (MDA) concentration. Our results showed that SL-R4 and SL-R6 groups resulted in higher (p < 0.05) percentages of total motility, progressive motility, and plasma membrane functionality, as compared with other groups. Highest (p < 0.05) viable and lowest (p < 0.05) dead spermatozoa were observed in SL-R6 group compared to the other groups. The acrosomal and capacitation status were not affected (p > 0.05) by different levels of rosemary aqueous extract. Lower (p < 0.05) MDA concentration has been observed in SL-R4 and SL-R6 groups. The results of this study demonstrate that supplementation of SL extender with rosemary aqueous extract influences post-thawed ram sperm quality in a dose dependent manner. Copyright © 2014 Elsevier Inc. All rights reserved.

The presence of seminal plasma, especially derived from stallion semen, helps preserve chilled Asian elephant (Elephas maximus) sperm motility.

PubMed

Pinyopummin, A; Mahasawangkul, S; Kornkaewrat, K; Rattanapirom, S; Leartsang, W; Kitkha, S

2017-08-01

The effects of seminal plasma (SP), derived from autologous, homologous and heterologous species (stallion, boar and dog) on chilled Asian elephant sperm quality, were determined. Semen was collected from eight males and samples with ≥30% motile spermatozoa were used in the study. Semen was diluted with Tris-glucose-egg yolk extender, supplemented with different SP types and preserved at 4°C for 48 hr. Experiment 1 (n = 31), showed that the presence of SP (autologous) helped to preserve sperm quality in terms of sperm motility and acrosome integrity (p < .05). Homologous SP did not result in better sperm quality than autologous SP. Heterologous SP from stallion provided higher sperm motility and velocities compared to autologous SP (p < .05). Experiment 2 (n = 14) determined the effect of different SP from four stallions. All stallion SP gave higher (p < .05) results for motile spermatozoa and sperm velocities than autologous SP. In conclusion, the presence of SP helps preserve Asian elephant sperm quality and stallion SP supports the motility of Asian elephant spermatozoa during cold storage. © 2016 Blackwell Verlag GmbH.

Potential Changes in Rat Spermatogenesis and Sperm Parameters after Inhalation of Boswellia papyrifera and Boswellia carterii Incense

PubMed Central

Ahmed, Mukhtar; Al-Daghri, Nasser; Alokail, Majed S.; Hussain, Tajamul

2013-01-01

In this study the effect of Boswellia papyrifera (B. papyrifera) and Boswellia carterii (B. carterii) smoke exposure on spermatogenesis and sperm parameters in male albino rats was investigated. Rats (n = 11) were exposed daily in smoking chambers to smoke emanated by burning 4 g each of either B. papyrifera or B. carterii for 48 days. At the end of exposure duration rats were killed, and the testes were excised and analysed for histopathological and ultrastructural changes. Sperm analysis including total sperm count, motility, velocity and relative percentage of abnormal sperms were recorded. Rats exposed to B. papyrifera and B. carterii showed significant disturbances in spermatogenetic patterns and changes in sperm kinetics compared to unexposed rats. Atrophied seminiferous tubules with dynamic changes were also noticed. The boundaries of intercellular and intracellular vacuoles were seen in the Sertoli cells. Furthermore, in spermatids acrosomal vesicles were not fully formed. Degenerating spermatids were devoid of their nuclear membrane with electron dense matrix and vacuolization. Structural changes in Leydig cells were observed. Sperm analysis in exposed rats exhibited significant decrease in the sperm count, motility, speed and an increase in sperm anomalies when compare to controls. These findings demonstrate that the B. papyrifera and B. carterii smoke affects the process of spermatogenesis and sperm parameters and indicate the detrimental effects of these incense materials on human reproductive system. PMID:23449005

Membrane status and in vitro capacitation of porcine sperm preserved in long-term extender at 16 degrees C.

PubMed

Conejo-Nava, J; Fierro, R; Gutierrez, C G; Betancourt, M

2003-01-01

Preservation of porcine semen in long-term extenders at 15-18 degrees C for more than 5 days results in decreased farrowing rates and reduced litter size after artificial insemination, despite the high progressive motility rates of sperm. To improve this preservation system it is necessary to understand sperm physiology under storage conditions. The purpose of this study was to determine the effect of storing diluted porcine semen (during 0, 2, 4, 6, and 8 days) on the sperm membranes status and the ability of sperm to respond to in vitro capacitation treatment. Ten semen samples from 5 adult boars were analyzed. Two aliquots were obtained from the sperm-rich fraction: one was used to assess fresh semen and the other was diluted in Reading extender and stored at 16 degrees C. Both semen samples were stained with chlortetracycline to assess the status of sperm membranes and with Hoechst 33258 to determine viability. Semen storage for 4-8 days increased the proportion of prematurely capacitated sperm. After 4 days of storage, in vitro capacitation treatment did not increase the percentage of capacitated sperm, but increased the percentage of acrosome reacted sperm. This phenomenon could explain the reduced fertilizing ability of porcine semen stored at 16 degrees C for over 4 days, in spite of the acceptable sperm viability and progressive motility.

Effect of semen extenders on frozen-thawed boar sperm characteristics and distribution in the female genital tract after deep intrauterine insemination in sows.

PubMed

Noguchi, Michiko; Yoshioka, Koji; Hikono, Hirokazu; Suzuki, Chie; Kikuchi, Kazuhiro

2015-12-01

We compared the effects of extenders of frozen-thawed semen on post-thaw sperm characteristics and the distribution of frozen-thawed spermatozoa in the female genital tract after fixed-timed deep intrauterine insemination (DIUI) in sows. Frozen semen samples were thawed and diluted in either modified Modena solution (mMS) or porcine fertilization medium (PFM) containing theophylline, adenosine and cysteine. Sperm quality, assessed in vitro based on motility using a computer-assisted sperm analyzer and the integrity of the plasma and acrosomal membranes using flow cytometry, was evaluated at 0.5, 1.5, 3 and 6h after thawing. Progressive motility and the percentage of spermatozoa with damaged acrosomal membranes in PFM were significantly better than in mMS throughout the 6h. Sows with estrus synchronized using prostaglandin F2 alpha, equine chorionic gonadotropin and human chorionic gonadotropin (hCG) were inseminated once with mMS- or PFM-diluted 5 × 10(8) frozen-thawed spermatozoa by DIUI at 34 h after the hCG injection. At 4h after DIUI, reproductive tracts were recovered from 30 sows. There were significantly fewer polymorphonuclear leukocytes (PMNs) and more spermatozoa outside PMNs in the uterine horn after PFM treatment than with mMS. When 22 sows were administered DIUI with 10 × 10(8) frozen-thawed spermatozoa at 36 h after hCG, the pregnancy rates did not differ significantly between the mMS- (36%) and PFM- (64%) treated groups. Thus, PFM enhanced progressive sperm motility but increased sperm membrane damage compared with mMS; it also suppressed the migration of PMNs into the uterine lumen. Copyright © 2015 Elsevier B.V. All rights reserved.

A two-step dilution tris-egg yolk extender containing Equex STM significantly improves sperm cryopreservation in the African wild dog (Lycaon pictus).

PubMed

Van den Berghe, Femke; Paris, Monique Christina Johanna; Briggs, Michael Brent; Farstad, Wenche Kristin; Paris, Damien Boyd Bertrand Paul

2018-02-01

Conservation management of endangered African wild dogs (AWD; Lycaon pictus) can benefit greatly from development of sperm freezing and artificial insemination. Previous freezing attempts yielded nearly 0% motile sperm within 2 h of thawing. In this study, two canine freezing protocols were tested: Protocol 1: a one-step dilution in TRIS-20% egg yolk containing 8% glycerol; and Protocol 2: a two-step dilution in TRIS-20% egg yolk containing a final extender concentration of 5% glycerol and 0.5% Equex STM, coupled with a TRIS-citrate-fructose thawing solution. Semen was collected by electroejaculation from n = 24 AWDs, of which eight ejaculates of sufficient quality (four good quality with initial sperm motility of 75.0 ± 4.4% and four poor quality; showing rapid decrease in sperm motility to 3.3 ± 3.3% prior to freezing) were frozen. For good quality samples, motility and sperm motility index persisted for up to 8 h for Protocol 2, and was higher between 2 and 6 h after thawing with a decrease from 4 h of incubation. Motility dropped to nearly 0% after 2 h incubation for Protocol 1. Viability was higher for Protocol 2 throughout the 8 h of incubation, with a decrease after 6 h, compared to 4 h for Protocol 1. Acrosome integrity was higher for Protocol 2 throughout post-thaw incubation, with a decrease after 2 h for both protocols. Protocols did not differ in normal sperm morphology or DNA integrity. Poor quality samples yielded similar results, except for acrosome integrity, which declined for Protocol 2. In conclusion, a two-step dilution in TRIS-egg yolk-glycerol extender containing Equex STM yields significantly improved post-thaw quality and longevity of AWD spermatozoa, making it suitable for sperm banking and artificial insemination initiatives. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Monogamy in a new species of wild guinea pigs (Galea sp.)

NASA Astrophysics Data System (ADS)

Hohoff, Christa; Solmsdorff, Katrin; Löttker, Petra; Kemme, Kristina; Epplen, Jörg; Cooper, Trevor; Sachser, Norbert

2002-08-01

A new species of wild guinea pigs from Bolivia was recently discovered: Galea sp. So far no data are available concerning its social and mating systems. We therefore investigated behavioural compatibility, sexual dimorphism, paternity in a male-competing situation, testicular and epididymal size, and sperm morphology and motility. The results showed a complete intrasexual and moderate intersexual behavioural incompatibility between unfamiliar animals, a reversed sexual dimorphism with females larger than males, and litters always fathered by single males. Furthermore, both the reproductive organ weights and the percentage of motile sperm and sperm with intact acrosomes were significantly lower compared with the promiscuous congeneric Galea musteloides. In summary, these data strongly suggest a monogamous social and mating system for the recently discovered Galea sp.

Anatase titanium dioxide nanoparticles in mice: evidence for induced structural and functional sperm defects after short-, but not long-, term exposure

PubMed Central

Smith, Michelle A; Michael, Rowan; Aravindan, Rolands G; Dash, Soma; Shah, Syed I; Galileo, Deni S; Martin-DeLeon, Patricia A

2015-01-01

Titanium dioxide (TiO2) nanoparticles (TNPs) are widely used commercially and exist in a variety of products. To determine if anatase TNPs (ATNPs) in doses smaller than previously used reach the scrotum after entry in the body at a distant location and induce sperm defects, 100% ATNP (2.5 or 5 mg kg−1 body weight) was administered intraperitoneally to adult males for three consecutive days, followed by sacrifice 1, 2, 3, or 5 weeks later (long-) or 24, 48 or 120 h (short-term exposure). Transmission electron microscopy revealed the presence of ANTP in scrotal adipose tissues collected 120 h postinjection when cytokine evaluation showed an inflammatory response in epididymal tissues and fluid. At 120 h and up to 3 weeks postinjection, testicular histology revealed enlarged interstitial spaces. Significantly increased numbers of terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling-positive (apoptotic) germ (P = 0.002) and interstitial space cells (P = 0.04) were detected in treated males. Caudal epididymal sperm from the short-term, but not a long-term, arm showed significantly (P < 0.001) increased frequencies of flagellar abnormalities, excess residual cytoplasm (ERC), and unreacted acrosomes in treated versus controls (dose-response relationship). A novel correlation between ERC and unreacted acrosomes was uncovered. At 120 h, there were significant decreases in hyperactivated motility (P < 0.001) and mitochondrial membrane potential (P < 0.05), and increased reactive oxygen species levels (P < 0.00001) in treated versus control sperm. These results indicate that at 4–8 days postinjection, ANTP induce structural and functional sperm defects associated with infertility, and DNA damage via oxidative stress. Sperm defects were transient as they were not detected 10 days to 5 weeks postinjection. PMID:25370207

Defective sperm head decondensation undermines the success of ICSI in the bovine.

PubMed

Águila, Luis; Felmer, Ricardo; Arias, María Elena; Navarrete, Felipe; Martin-Hidalgo, David; Lee, Hoi Chang; Visconti, Pablo; Fissore, Rafael

2017-09-01

The efficiency of intracytoplasmic sperm injection (ICSI) in the bovine is low compared to other species. It is unknown whether defective oocyte activation and/or sperm head decondensation limit the success of this technique in this species. To elucidate where the main obstacle lies, we used homologous and heterologous ICSI and parthenogenetic activation procedures. We also evaluated whether in vitro maturation negatively impacted the early stages of activation after ICSI. Here we showed that injected bovine sperm are resistant to nuclear decondensation by bovine oocytes and this is only partly overcome by exogenous activation. Remarkably, when we used heterologous ICSI, in vivo -matured mouse eggs were capable of mounting calcium oscillations and displaying normal PN formation following injection of bovine sperm, although in vitro -matured mouse oocytes were unable to do so. Together, our data demonstrate that bovine sperm are especially resistant to nuclear decondensation by in vitro -matured oocytes and this deficiency cannot be simply overcome by exogenous activation protocols, even by inducing physiological calcium oscillations. Therefore, the inability of a suboptimal ooplasmic environment to induce sperm head decondensation limits the success of ICSI in the bovine. Studies aimed to improve the cytoplasmic milieu of in vitro -matured oocytes and to replicate the molecular changes associated with in vivo capacitation and acrosome reaction will deepen our understanding of the mechanism of fertilization and improve the success of ICSI in this species. © 2017 Society for Reproduction and Fertility.

Hyper-activated motility in sperm capacitation is mediated by phospholipase D-dependent actin polymerization.

PubMed

Itach, Sarit Bar-Sheshet; Finklestein, Maya; Etkovitz, Nir; Breitbart, Haim

2012-02-15

In order to fertilize the oocyte, sperm must undergo a series of biochemical changes in the female reproductive tract, known as capacitation. Once capacitated, spermatozoon can bind to the zona pellucida of the egg and undergo the acrosome reaction (AR), a process that enables its penetration and fertilization of the oocyte. Important processes that characterize sperm capacitation are actin polymerization and the development of hyper-activated motility (HAM). Previously, we showed that Phospholipase D (PLD)-dependent actin polymerization occurs during sperm capacitation, however the role of this process in sperm capacitation is not yet known. In the present study, we showed for the first time the involvement of PLD-dependent actin polymerization in sperm motility during mouse and human capacitation. Sperm incubated under capacitation conditions revealed a time dependent increase in actin polymerization and HAM. Inhibition of Phosphatidic Acid (PA) formation by PLD using butan-1-ol, inhibited actin polymerization and motility, as well as in vitro fertilization (IVF) and the ability of the sperm to undergo the AR. The inhibition of sperm HAM by low concentration of butan-1-ol is completely restored by adding PA, further indicating the involvement of PLD in these processes. Furthermore, exogenous PA enhanced rapid actin polymerization that was followed by a rise in the HAM, as well as an increased in IVF rate. In conclusion, our results demonstrate that PLD-dependent actin polymerization is a critical step needed for the development of HAM during mouse and human sperm capacitation. Copyright © 2011 Elsevier Inc. All rights reserved.

[The incidence of agglutination and its influence on sperm quality and fertility of boar semen].

PubMed

Bollwein, Heinrich; Petschow, Karola; Weber, Frank; Leiding, Claus; Stolla, Rudolf

2004-01-01

The aim of this study was to examine the incidence of sperm agglutinations and their relationships with sperm quality and fertility. Semen samples of 40 boars of an AI-station were investigated. Nineteen of the 40 investigated boars showed a constantly low (< 10% agglutinated sperm), 3 an intermediate (10-20%) and 6 boars a high level (> 20%) of agglutination in raw semen. The degree of agglutination in sperm samples of 12 boars varied distinctly during the investigation period. During summer more (P < 0.05) agglutinated sperm were observed (11.0 +/- 11.6%) than during winter (6.2 +/- 7.3%). There was no association between bacterial contamination and incidence of agglutinations (P > 0.05). After dilution in extender the percentage of agglutinated sperm decreased from 6.2 +/- 7.3% to 1.1 +/- 1.4% (P < 0.0001). Twenty-four hours after dilution the percentage of progressively motile sperm was 7.4% lower (P < 0.05) in ejaculates with an initially high degree of agglutination (> 20% agglutinated sperm) compared to samples with an initially low degree of agglutinated sperm (< 10%). Plasma membrane integrity, mitochondrial membrane potential, acrosome reaction and chromatin structure were independent (P > 0.05) from the level of agglutination. Fertility data did not differ (P > 0.05) between boars with low and high numbers of agglutinated sperm in raw semen. The results show that there are individual, ejaculatory and seasonal variations in the incidence and degree of agglutination. Agglutinations have a negative effect on motility of sperm and disappear to a large extent after dilution in sperm extender. They have no negative consequences on fertility.

Incubation of boar spermatozoa in viscous media by addition of methylcellulose improves sperm quality and penetration rates during in vitro fertilization.

PubMed

González-Abreu, David; García-Martínez, Soledad; Fernández-Espín, Vanesa; Romar, Raquel; Gadea, Joaquín

2017-04-01

This work was designed to study whether viscous media can improve the in vitro sperm functionality in pigs by using methylcellulose as a thickener. Viscosity of porcine oviductal fluid (POF) was compared with culture medium (Tyrode's) supplemented with methylcellulose (MET 0, 0.5 and 1% w/v). Spermatozoa were incubated in the different media (0, 1 and 2 h) and sperm motion parameters, lipid membrane disorder, plasma membrane integrity and reactive oxygen species (ROS) formation were assessed. Fertilization results were assessed i) preincubating spermatozoa in the viscous media followed by gamete coculture in a non-viscous medium; and ii) gamete coculture in the viscous media. Viscosity of POF from early luteal phase was higher than late follicular phase. Medium without methylcellulose presented constant viscosity with increased shear rate, while viscosity of the POF and media with methylcellulose was reduced by increased shear rates. Methylcellulose improved sperm linearity, straightness and the proportion of fast-linear spermatozoa. Moreover, methylcellulose increased the rate of viable spermatozoa with intact acrosome and low lipid disorder, reducing the ROS generation. Preincubation in viscous media increased the penetration rate and the mean number of spermatozoa bound to the zona pellucida (both with 0.5 and 1% MET) and reduced monospermy with 1% MET. On the other hand fertilization in the viscous media reduced penetration rate and increased monospermy. The efficiency of the IVF system was not improved with the use of viscous media. The results show the relevance of increasing viscosity thus making the in vitro media more comparable to physiological conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of low dose of vinclozolin on reproductive tract development and sperm parameters in CD1 outbred mice.

PubMed

Elzeinova, Fatima; Novakova, Vendula; Buckiova, Daniela; Kubatova, Alena; Peknicova, Jana

2008-01-01

The effect of a low dose of vinclozolin within the development of the reproductive tract during gestation (VIN-GD 15-22) and puberty (VIN-PND 23-44) in CD1 mice was tested. We found a decrease in the anogenital distance, prostate weight and pathology of testes in both experimental groups. Sperm counts decreased to 46% (VIN-GD) and to 81% (VIN-PND), and also the acrosomal state (evaluated by antiacrosomal antibody) decreased in both groups to 89% in comparison to the control group (100%). Sperm head abnormalities increased by approximately 18% and 13%, respectively. In this connection, the expression of some genes was changed (arosome-related gene (Acr), apoptosis related genes (p53, p21)). In conclusion, a low dose of vinclozolin affected the reproductive tract, sperm parameters and expression of selected genes in both experimental groups.

Dietary polyunsaturated fatty acid supplementation of young post-pubertal dairy bulls alters the fatty acid composition of seminal plasma and spermatozoa but has no effect on semen volume or sperm quality.

PubMed

Byrne, C J; Fair, S; English, A M; Holden, S A; Dick, J R; Lonergan, P; Kenny, D A

2017-03-01

assessed either immediately following collection or post-thawing. Membrane fluidity and oxidative stress of sperm were also not affected by diet. The percentage of sperm with intact-acrosomes was lower in CTL bulls compared to those fed SO (P < 0.01). In conclusion, while the lipid composition of semen was altered following dietary supplementation with either n-6 or n-3 based PUFA, this did not lead to measurable improvements in the quantity or quality of semen produced by young post-pubertal dairy bulls. Copyright © 2017 Elsevier Inc. All rights reserved.

Hydrogen Peroxide Modifies Human Sperm Peroxiredoxins in a Dose-Dependent Manner1

PubMed Central

O'Flaherty, Cristian; Rico de Souza, Angela

2010-01-01

Low levels of reactive oxygen species (ROS) modulate signaling pathways required for human sperm activation, but high levels impair sperm function, leading to infertility. Peroxiredoxins (PRDXs) are enzymes with a dual role as ROS scavengers and modulators of ROS-dependent signaling. The present study aimed to characterize PRDXs in human spermatozoa and possible modifications resulting from hydrogen peroxide (H2O2). We found PRDX1, PRDX4, PRDX5, and PRDX6 in both seminal plasma and spermatozoa. Using immunocytochemistry, we demonstrated that these PRDXs are differentially localized in the head, acrosome, mitochondrial sheath, and flagellum. These observations were confirmed by immunoblotting using cytosolic, Triton-soluble and -insoluble, and head and flagella sperm fractions. PRDXs are dose-dependently modified by H2O2, as seen by the formation of disulfide bridges and high-molecular-mass complexes. This first study, to our knowledge, on PRDXs in human spermatozoa indicates that PRDX1, PRDX4, PRDX5, and PRDX6 are modified when spermatozoa are challenged with H2O2. This suggests that PRDXs may protect these cells at high levels of H2O2 but could also control H2O2 levels within different cell compartments so that normal sperm activation can occur. PMID:20864641

Plasma membrane Ca2+-ATPase 4: interaction with constitutive nitric oxide synthases in human sperm and prostasomes which carry Ca2+/CaM-dependent serine kinase

PubMed Central

Andrews, Rachel E.; Galileo, Deni S.; Martin-DeLeon, Patricia A.

2015-01-01

Deletion of the gene encoding the widely conserved plasma membrane calcium ATPase 4 (PMCA4), a major Ca2+ efflux pump, leads to loss of sperm motility and male infertility in mice. PMCA4's partners in sperm and how its absence exerts its effect on fertility are unknown. We hypothesize that in sperm PMCA4 interacts with endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) which are rapidly activated by Ca2+, and that these fertility-modulating proteins are present in prostasomes, which deliver them to sperm. We show that in human sperm PMCA4 is present on the acrosome, inner acrosomal membrane, posterior head, neck, midpiece and the proximal principal piece. PMCA4 localization showed inter- and intra-individual variation and was most abundant at the posterior head/neck junction, co-localizing with NOSs. Co-immunoprecipitations (Co-IP) revealed a close association of PMCA4 and the NOSs in Ca2+ ionophore-treated sperm but much less so in uncapacitated untreated sperm. Fluorescence resonance energy transfer (FRET) showed a similar Ca2+-related association: PMCA4 and the NOSs are within 10 nm apart, and preferentially so in capacitated, compared with uncapacitated, sperm. FRET efficiencies varied, being significantly (P < 0.001) higher at high cytosolic Ca2+ concentration ([Ca2+]c) in capacitated sperm than at low [Ca2+]c in uncapacitated sperm for the PMCA4-eNOS complex. These dynamic interactions were not seen for PMCA4-nNOS complexes, which had the highest FRET efficiencies. Further, along with Ca2+/CaM-dependent serine kinase (CASK), PMCA4 and the NOSs are present in the seminal plasma, specifically in prostasomes where Co-IP showed complexes similar to those in sperm. Finally, flow cytometry demonstrated that following co-incubation of sperm and seminal plasma, PMCA4 and the NOSs can be delivered in vitro to sperm via prostasomes. Our findings indicate that PMCA4 interacts simultaneously with the NOSs preferentially at high [Ca2+]c in

Evaluation of cryopreserved stallion semen from Tori and Estonian breeds using CASA and flow cytometry.

PubMed

Kavak, A; Johannisson, A; Lundeheim, N; Rodriguez-Martinez, H; Aidnik, M; Einarsson, S

2003-04-15

Methods to evaluate the quality of frozen-thawed stallion semen are still needed, particularly those considering the sperm function. The present study evaluated sperm motility, membrane and acrosome integrity and the capacitation status of frozen-thawed spermatozoa from seven Tori and six Estonian breed stallions by way of computer assisted sperm analysis (CASA), a triple fluorophore stain combination and Merocyanine 540, respectively, the latter ones using flow cytometry. Two ejaculates from each stallion were cryopreserved using the Hannover method in 0.5 ml plastic straws. Two straws per ejaculate per stallion were thawed at 37 degrees C for 30s. Motility was analysed with CASA immediately after thawing, while for flow cytometry spermatozoa were cleansed by 70:40% Percoll discontinuous density gradient separation before analysed for sperm viability, acrosome integrity (stained with SNARF, PI and FITC-PSA) and capacitation status (stained with Merocyanine 540/Yo-Pro-1). Results (as least square means) were as follows: the motility of frozen-thawed semen was 43.4% for Tori stallions and 42.3% for Estonian stallions (P>0.05). After Percoll separation 79.3% of the spermatozoa from Tori stallions had intact acrosomes and 1.7% of them showed early signs of capacitation. The same parameters for Estonian stallions were 84.5 and 2.3%, respectively. There were no statistically significant differences between breeds or ejaculates within breed for any evaluated parameter. We conclude that triple staining and flow cytometry are valuable techniques to evaluate frozen-thawed stallion spermatozoa, and that no differences in quality of frozen semen were registered between Tori and Estonian breed stallions, allowing implementation of this technology in the Estonian horse population.

Dimethyleacetamide improves the cryosurvivability of Indian red jungle fowl (Gallus gallus murghi) sperm.

PubMed

Rakha, B A; Ansari, M S; Akhter, S; Zafar, Z; Naseer, A; Hussain, I; Santiago-Moreno, J; Blesbois, E

2017-11-01

It was hypothesized that dimethyleacetamide (DMA) can be used as an alternate to glycerol for cryopreservation of Indian red jungle fowl semen. Four concentrations of DMA (4%, 6%, 8% and 10%) in extender were compared with previously optimized cryopreservation protocol based on 20% glycerol (control) for Indian red jungle fowl. Sperm motility, plasma membrane integrity, viability, and acrosome integrity were assessed at the stage of post-dilution, cooling, equilibration, and freeze-thawing. The whole experiment was repeated/replicated for five times independently. Sperm motility, plasma membrane integrity, viability and acrosome integrity were recorded highest (P < 0.05) at post-dilution, cooling, equilibration, and freeze-thawing in extender having 6% DMA compared to control and other experimental extenders. The highest (P < 0.05) recovery rates of all aforementioned parameters were also recorded in extender having 6% DMA; thus, 6% DMA was further compared with control (20% glycerol) for fertility after artificial insemination. Eggs were collected for five days after artificial insemination with semen cryopreserved in extender containing 6% DMA and control. The higher no. of fertilized eggs, fertility, no. of hatched eggs, hatch (%) and hatchability were recorded with semen cryopreserved in extender having 6% DMA compared to control. It is concluded that 6% DMA maintained higher post-thaw quality and fertility of Indian red jungle fowl semen and is a better replacement of glycerol. Copyright © 2017 Elsevier Inc. All rights reserved.

Recombinant mouse sperm ZP3-binding protein (ZP3R/sp56) forms a high order oligomer that binds eggs and inhibits mouse fertilization in vitro.

PubMed

Buffone, Mariano G; Zhuang, Tiangang; Ord, Teri S; Hui, Ling; Moss, Stuart B; Gerton, George L

2008-05-02

Many candidates have been proposed as zona pellucida-binding proteins. Without precluding a role for any of those candidates, we focused on mouse sperm protein ZP3R/sp56, which is localized in the acrosomal matrix. The objective of this study was to analyze the role of ZP3R/sp56 in mouse fertilization. We expressed recombinant ZP3R/sp56 as a secreted protein in HEK293 cells and purified it from serum-free, conditioned medium. In the presence of reducing agents, the recombinant ZP3R/sp56 exhibited a molecular weight similar to that observed for the native ZP3R/sp56. Reminiscent of the native protein, recombinant ZP3R/sp56 formed a high molecular weight, disulfide cross-linked oligomer consisting of six or more monomers under non-reducing conditions. Recombinant ZP3R/sp56 bound to the zona pellucida of unfertilized eggs but not to 2-cell embryos, indicating that the changes that take place in the zona pellucida at fertilization affected the interaction of this protein with the zona pellucida. The extent of in vitro fertilization was reduced in a dose-dependent manner when unfertilized eggs were preincubated with recombinant ZP3R/sp56 (74% drop at the maximum concentrations assayed). Eggs incubated with the recombinant protein showed an absence of or very few sperm in the perivitelline space, suggesting that the reduction in the fertilization rate is caused by the inhibition of sperm binding and/or penetration through the zona pellucida. These results indicate that sperm ZP3R/sp56 is important for sperm-zona interactions during fertilization and support the concept that the acrosomal matrix plays an essential role in mediating the binding of sperm to the zona pellucida.

Effect of Vitamin E and Polyunsaturated Fatty Acids on Cryopreserved Sperm Quality in Bos taurus Bulls Under Testicular Heat Stress.

PubMed

Losano, João D A; Angrimani, Daniel S R; Dalmazzo, Andressa; Rocha, Carolina C; Brito, Maíra M; Perez, Eduardo G A; Tsunoda, Roberta H; Góes, Paola A A; Mendes, Camilla M; Assumpção, Mayra E O A; Barnabe, Valquiria H; Nichi, Marcilio

2018-04-03

Taurine bulls are highly susceptible to heat stress, leading to increased oxidative stress (OS) and impaired sperm viability. Polyunsaturated fatty acids (PUFAs) supplementation can be an alternative to improve semen quality, which also results in more sperm susceptibility to lipid peroxidation. Moreover, this deleterious effect can be exacerbated in animals affected by heat stress. Vitamin E is a key antioxidant that counteracts lipid peroxidation of sperm membrane caused by OS. Thus, combining PUFAs with vitamin E may improve sperm quality. In this context, this study aimed to evaluate the effect of interaction between PUFAs and vitamin E on sperm quality in Bos taurus bulls under testicular heat stress. Sixteen taurine bulls under testicular heat stress were randomly assigned in four groups: Control, Vitamin E, PUFA, and PUFA + Vitamin E. All groups lasted for 60 days. Samples were cryopreserved/thawed and analyzed for motility variables (CASA), membrane and acrosome integrity, mitochondrial activity, susceptibility to oxidative stress, DNA integrity, and sperm-binding capacity. Results showed that vitamin E had a beneficial effect on some sperm characteristics, whereas PUFA supplementation had an adverse effect when the two treatments were evaluated separately. Finally, the association between PUFAs and vitamin E did not improve sperm quality.

The post-thaw quality of ram sperm held for 0 to 48 h at 5 degrees C prior to cryopreservation.

PubMed

Purdy, P H

2006-06-01

The effects of holding diluted ram semen at 5 degrees C for up to 48 h prior to cryopreservation were investigated. Semen from six rams was collected by electro-ejaculation in the autumn and again from six different rams in the spring. The sperm concentration and motility were determined using spectrophotometry and computerized automated semen analysis, respectively. Samples were diluted at 23 degrees C to 400 x 10(6)cells/ml in a one-step Tris-egg yolk-glycerol (5%, v/v) media, cooled to 5 degrees C over 2h and maintained at 5 degrees C for the duration of the experiments. Aliquots were loaded into 0.5 ml French straws at 0, 24 or 48 h after cooling, frozen in liquid nitrogen vapor for 12-13 min, 4.5 cm above the liquid nitrogen, and plunged into liquid nitrogen for storage. After thawing, autumn samples frozen after 0, 24, or 48 h of storage exhibited similar percentages of motility (29, 31, 36%, respectively), progressively motility (16, 15, 17%, respectively), plasma membrane integrity (28, 35, 29%, respectively) and live acrosome-reacted cells (0.4, 0.6, 0.8%, respectively; P>0.05). In addition, the quantity of sperm that bound to hen's egg perivitelline membranes after being held at 5 degrees C for 0, 24, or 48 h was not significantly different when the values were expressed as means of the quantity of sperm (155, 177, 106 sperm, respectively) or as the proportion of sperm inseminated (0.39, 0.49, 0.34, respectively; P>0.05). Likewise, ram sperm collected in the spring and frozen at 0, 24 and 48 h after cooling had similar (P>0.05) total motility (21, 25, 20%, respectively), progressive motility (14, 15, 11%, respectively), plasma membrane integrity (26, 33, 31%, respectively) and live acrosome-reacted cells (3.7, 3.5, 3.2%, respectively; P>0.05). The 0 h holding time had significantly less sperm bound to a hen's egg perivitelline membrane compared to the 48 h holding time (250 and 470 sperm, respectively) although the 24h holding time was not different from

Dynamics of Bovine Sperm Interaction with Epithelium Differ Between Oviductal Isthmus and Ampulla.

PubMed

Ardon, Florencia; Markello, Ross D; Hu, Lian; Deutsch, Zarah I; Tung, Chih-Kuan; Wu, Mingming; Suarez, Susan S

2016-10-01

In mammals, many sperm that reach the oviduct are held in a reservoir by binding to epithelium. To leave the reservoir, sperm detach from the epithelium; however, they may bind and detach again as they ascend into the ampulla toward oocytes. In order to elucidate the nature of binding interactions along the oviduct, we compared the effects of bursts of strong fluid flow (as would be caused by oviductal contractions), heparin, and hyperactivation on detachment of bovine sperm bound in vitro to epithelium on intact folds of isthmic and ampullar mucosa. Intact folds of oviductal mucosa were used to represent the strong attachments of epithelial cells to each other and to underlying connective tissue that exist in vivo. Effects of heparin on binding were tested because heparin binds to the Binder of SPerm (BSP) proteins that attach sperm to oviductal epithelium. Sperm bound by their heads to beating cilia on both isthmic and ampullar epithelia and could not be detached by strong bursts of fluid flow. Addition of heparin immediately detached sperm from isthmic epithelium but not ampullar epithelium. Addition of 4-aminopyridine immediately stimulated hyperactivation of sperm but did not detach them from isthmic or ampullar epithelium unless added with heparin. These observations indicate that the nature of binding of sperm to ampullar epithelium differs from that of binding to isthmic epithelium; specifically, sperm bound to isthmic epithelium can be detached by heparin alone, while sperm bound to ampullar epithelium requires both heparin and hyperactivation to detach from the epithelium. © 2016 by the Society for the Study of Reproduction, Inc.

Impaired hypothalamic-pituitary-testicular axis activity, spermatogenesis, and sperm function promote infertility in males with lead poisoning.

PubMed

Gandhi, Jason; Hernandez, Rafael J; Chen, Andrew; Smith, Noel L; Sheynkin, Yefim R; Joshi, Gargi; Khan, Sardar Ali

2017-04-01

Lead poisoning is a stealthy threat to human physiological systems as chronic exposure can remain asymptomatic for long periods of time before symptoms manifest. We presently review the biophysical mechanisms of lead poisoning that contribute to male infertility. Environmental and occupational exposure of lead may adversely affect the hypothalamic-pituitary-testicular axis, impairing the induction of spermatogenesis. Dysfunction at the reproductive axis, namely testosterone suppression, is most susceptible and irreversible during pubertal development. Lead poisoning also appears to directly impair the process of spermatogenesis itself as well as sperm function. Spermatogenesis issues may manifest as low sperm count and stem from reproductive axis dysfunction or testicular degeneration. Generation of excessive reactive oxygen species due to lead-associated oxidative stress can potentially affect sperm viability, motility, DNA fragmentation, membrane lipid peroxidation, capacitation, hyperactivation, acrosome reaction, and chemotaxis for sperm-oocyte fusion, all of which can contribute to deter fertilization. Reproductive toxicity has been tested through cross-sectional analysis studies in humans as well as in vivo and in vitro studies in animals.

Glutathione in combination with trehalose has supplementary beneficial effects on cryopreserved red deer (cervus elaphus) sperm.

PubMed

Wang, Yan; Dong, Shude

2017-01-01

In this study, we evaluated the effects of glutathione in combination with trehalose addition to semen extenders on the quality parameters of frozen-thawed red deer (cervus elaphus) spermatozoa. The semen samples collected from six mature red deer once a week were diluted with Tris-egg yolk-based extenders. The diluted semen samples were supplemented with glutathione (8 mmol L -1 ) and or trehalose (5%, w/v), cryopreserved, thawed and then subjected to sperm quality parameter evaluation. Both glutathione and trehalose addition to the extender significantly improved progressive motility, acrosome integrity, membrane integrity, superoxide dismutase and glutathione peroxidase activity and decreased percentage abnormality and sperm malondialdehyde level compared with the control group (P<.05). Moreover, glutathione in combination with trehalose addition to semen extenders had higher efficiency compared with the glutathione or trehalose addition alone (P<.05). Therefore, glutathione in combination with trehalose could be a promising cryoprotectant for red deer sperm. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Specific LED-based red light photo-stimulation procedures improve overall sperm function and reproductive performance of boar ejaculates

PubMed Central

Yeste, Marc; Codony, Francesc; Estrada, Efrén; Lleonart, Miquel; Balasch, Sam; Peña, Alejandro; Bonet, Sergi; Rodríguez-Gil, Joan E.

2016-01-01

The present study evaluated the effects of exposing liquid-stored boar semen to different red light LED regimens on sperm quality and reproductive performance. Of all of the tested photo-stimulation procedures, the best pattern consisted of 10 min light, 10 min rest and 10 min of further light (10-10-10 pattern). This pattern induced an intense and transient increase in the majority of motility parameters, without modifying sperm viability and acrosome integrity. While incubating non-photo-stimulated sperm at 37 °C for 90 min decreased all sperm quality parameters, this reduction was prevented when the previously-described light procedure was applied. This effect was concomitant with an increase in the percentage of sperm with high mitochondrial membrane potential. When sperm were subjected to ‘in vitro’ capacitation, photo-stimulation also increased the percentage of sperm with capacitation-like changes in membrane structure. On the other hand, treating commercial semen doses intended for artificial insemination with the 10-10-10 photo-stimulation pattern significantly increased farrowing rates and the number of both total and live-born piglets for parturition. Therefore, our results indicate that a precise photo-stimulation procedure is able to increase the fertilising ability of boar sperm via a mechanism that could be related to mitochondrial function. PMID:26931070

NAADP and the two-pore channel protein 1 participate in the acrosome reaction in mammalian spermatozoa

PubMed Central

Arndt, Lilli; Castonguay, Jan; Arlt, Elisabeth; Meyer, Dorke; Hassan, Sami; Borth, Heike; Zierler, Susanna; Wennemuth, Gunther; Breit, Andreas; Biel, Martin; Wahl-Schott, Christian; Gudermann, Thomas; Klugbauer, Norbert; Boekhoff, Ingrid

2014-01-01

The functional relationship between the formation of hundreds of fusion pores during the acrosome reaction in spermatozoa and the mobilization of calcium from the acrosome has been determined only partially. Hence, the second messenger NAADP, promoting efflux of calcium from lysosome-like compartments and one of its potential molecular targets, the two-pore channel 1 (TPC1), were analyzed for its involvement in triggering the acrosome reaction using a TPCN1 gene–deficient mouse strain. The present study documents that TPC1 and NAADP-binding sites showed a colocalization at the acrosomal region and that treatment of spermatozoa with NAADP resulted in a loss of the acrosomal vesicle that showed typical properties described for TPCs: Registered responses were not detectable for its chemical analogue NADP and were blocked by the NAADP antagonist trans-Ned-19. In addition, two narrow bell-shaped dose-response curves were identified with maxima in either the nanomolar or low micromolar NAADP concentration range, where TPC1 was found to be responsible for activating the low affinity pathway. Our finding that two convergent NAADP-dependent pathways are operative in driving acrosomal exocytosis supports the concept that both NAADP-gated cascades match local NAADP concentrations with the efflux of acrosomal calcium, thereby ensuring complete fusion of the large acrosomal vesicle. PMID:24451262

Human testicular protein TPX1/CRISP-2: localization in spermatozoa, fate after capacitation and relevance for gamete interaction.

PubMed

Busso, D; Cohen, D J; Hayashi, M; Kasahara, M; Cuasnicú, P S

2005-04-01

Testicular protein Tpx-1, also known as CRISP-2, is a cysteine-rich secretory protein specifically expressed in the male reproductive tract. Since the information available on the human protein is limited to the identification and expression of its gene, in this work we have studied the presence and localization of human Tpx-1 (TPX1) in sperm, its fate after capacitation and acrosome reaction (AR), and its possible involvement in gamete interaction. Indirect immunofluorescence studies revealed the absence of significant staining in live or fixed non-permeabilized sperm, in contrast to a clear labelling in the acrosomal region of permeabilized sperm. These results, together with complementary evidence from protein extraction procedures strongly support that TPX1 would be mainly an intra-acrosomal protein in fresh sperm. After in vitro capacitation and ionophore-induced AR, TPX1 remained associated with the equatorial segment of the acrosome. The lack of differences in the electrophoretic mobility of TPX1 before and after capacitation and AR indicates that the protein would not undergo proteolytical modifications during these processes. The possible involvement of TPX1 in gamete interaction was evaluated by the hamster oocyte penetration test. The presence of anti-TPX1 during gamete co-incubation produced a significant and dose-dependent inhibition in the percentage of penetrated zona-free hamster oocytes without affecting sperm motility, the AR or sperm binding to the oolema. Together, these results indicate that human TPX1 would be a component of the sperm acrosome that remains associated with sperm after capacitation and AR, and is relevant for sperm-oocyte interaction.

The effect of glycerol-related osmotic changes on post-thaw motility and acrosomal integrity of ram spermatozoa.

PubMed

Fiser, P S; Fairfull, R W

1989-02-01

Ram semen, collected by artificial vagina, was diluted and processed for long-term storage as described by P. S. Fiser, L. Ainsworth, and R. W. Fairfull (Canad. J. Anim. Sci. 62, 425-428, 1982). The concentration of the cryoprotectant, glycerol, was adjusted to 4% in the diluted semen prior to freezing by a one-step addition at 30 degrees C (Method 1), by cooling the semen to 5 degrees C and addition of the glycerol gradually over 30 min (Method 2), by one-step addition of glycerol prior to equilibration for 2 hr (Method 3), or by cooling to 5 degrees C, followed by a holding period of 2 hr at 5 degrees C, and the one-step addition of glycerol just prior to freezing (Method 4). After thawing, the glycerol concentration of the semen was reduced by stepwise dilution from 4 to 0.4% over 15 or 30 min or by a one-step ten-fold dilution. The average post-thaw percentage of motile spermatozoa was significantly lower after addition of glycerol by Method 1 (39.9%) than when the glycerol was added by the other three methods (range, 44.0-46.4% averaged over the glycerol dilution). The average post-thaw percentage of intact acrosomes (61.2%), highest in semen in which the glycerol was added by Method 2, was not significantly different from those in which glycerol was added to semen by Methods 3 and 4, but it was significantly higher than that found in semen in which the glycerol was added by Method 1 (54.4%). However, when averaged over the method of glycerolation, the post-thaw percentage of motile spermatozoa (range, 43.7-44.2%) and the percentage of intact acrosomes (range, 56.8-59.5%) did not differ significantly in semen subjected to gradual decrease in glycerol concentration and diluent osmolality (over 15 and 30 min) or by a one-step, 10-fold dilution. These data indicate that post-thaw survival of spermatozoa can be influenced by the way in which glycerol is added prior to freezing. However, post-thaw spermatozoa motility and acrosomal integrity can be maintained even

Unresolved issues in mammalian fertilization.

PubMed

Olds-Clarke, Patricia

2003-01-01

This review considers the role of the sperm in fertilization, addressing areas of misunderstanding and unfounded assumptions and taking particular advantage of the large body of data resulting from work with rodent species in vitro. Considerable attention is given to the appropriate use and interpretation of assays for capacitation, acrosomal exocytosis, hyperactivation, and sperm protein phosphorylation, as well as tests for sperm-zona and sperm-oocyte membrane interactions. The lack of general agreement on the means of sperm adhesion to and penetration of the zona pellucida is addressed, and the need for new approaches to this problem is pointed out. Some molecular advances in our understanding of specific steps in the process of fertilization are discussed in the context of intact cell-matrix and cell-cell interaction. This review should provide practical information for researchers just beginning the study of fertilization and interesting but not widely known observations to stimulate new ideas in experienced scientists.

Dynamics of Bovine Sperm Interaction with Epithelium Differ Between Oviductal Isthmus and Ampulla1

PubMed Central

Ardon, Florencia; Markello, Ross D.; Hu, Lian; Deutsch, Zarah I.; Tung, Chih-Kuan; Wu, Mingming; Suarez, Susan S.

2016-01-01

In mammals, many sperm that reach the oviduct are held in a reservoir by binding to epithelium. To leave the reservoir, sperm detach from the epithelium; however, they may bind and detach again as they ascend into the ampulla toward oocytes. In order to elucidate the nature of binding interactions along the oviduct, we compared the effects of bursts of strong fluid flow (as would be caused by oviductal contractions), heparin, and hyperactivation on detachment of bovine sperm bound in vitro to epithelium on intact folds of isthmic and ampullar mucosa. Intact folds of oviductal mucosa were used to represent the strong attachments of epithelial cells to each other and to underlying connective tissue that exist in vivo. Effects of heparin on binding were tested because heparin binds to the Binder of SPerm (BSP) proteins that attach sperm to oviductal epithelium. Sperm bound by their heads to beating cilia on both isthmic and ampullar epithelia and could not be detached by strong bursts of fluid flow. Addition of heparin immediately detached sperm from isthmic epithelium but not ampullar epithelium. Addition of 4-aminopyridine immediately stimulated hyperactivation of sperm but did not detach them from isthmic or ampullar epithelium unless added with heparin. These observations indicate that the nature of binding of sperm to ampullar epithelium differs from that of binding to isthmic epithelium; specifically, sperm bound to isthmic epithelium can be detached by heparin alone, while sperm bound to ampullar epithelium requires both heparin and hyperactivation to detach from the epithelium. PMID:27605344

Ejaculate traits in the Namibian cheetah (Acinonyx jubatus): influence of age, season and captivity.

PubMed

Crosier, Adrienne E; Marker, Laurie; Howard, JoGayle; Pukazhenthi, Budhan S; Henghali, Josephine N; Wildt, David E

2007-01-01

The objective was to examine the influence of animal age, season and captivity status on seminal quality in wild-born cheetahs (Acinonyx jubatus) in Namibia, Africa. Animals were divided into three age categories: juvenile (14-24 months; n = 16 males, 23 ejaculates); adult (25-120 months; n = 76 males, 172 ejaculates); and aged (>120 months; n = 5 males, 5 ejaculates). Seasons were categorised into hot-wet (January-April), cold-dry (May-August) and hot-dry (September-December). A comparison between freshly wild-caught (n = 29 males, 41 ejaculates) and captive-held cheetahs (n = 68 males, 159 ejaculates) was also conducted. Raw ejaculates contained 69.0 +/- 1.1% motile spermatozoa (mean +/- s.e.m.) with 73.6 +/- 1.5% of these cells containing an intact acrosome. Overall, 18.4 +/- 0.9% of spermatozoa were morphologically normal, with midpiece anomalies being the most prevalent (approximately 39%) defect. Juvenile cheetahs produced ejaculates with poorer sperm motility, forward progressive status, lower seminal volume and fewer total motile spermatozoa than adult and aged animals. Spermatogenesis continued unabated throughout the year and was minimally influenced by season. Proportions of sperm malformations were also not affected by season. Ejaculates from captive cheetahs had increased volume and intact acrosomes, but lower sperm density than wild-caught counterparts. In summary, Namibian cheetahs produce an extraordinarily high proportion of pleiomorphic spermatozoa regardless of age, season or living (captive versus free-ranging) status. Young males less than 2 years of age produce poorer ejaculate quality than adult and aged males. Because (1) all study animals were wild born and (2) there was little difference between freshly caught males and those maintained in captivity for protracted periods, our results affirm that teratospermia in the cheetah is mostly genetically derived. It also appears that an ex situ environment for the Namibian cheetah can ensure sperm

A Role for the Chemokine Receptor CCR6 in Mammalian Sperm Motility and Chemotaxis

PubMed Central

Caballero-Campo, Pedro; Buffone, Mariano G.; Benencia, Fabian; Conejo-García, José R.; Rinaudo, Paolo F.; Gerton, George L.

2013-01-01

Although recent evidence indicates that several chemokines and defensins, well-known as inflammatory mediators, are expressed in the male and female reproductive tracts, the location and functional significance of chemokine networks in sperm physiology and sperm reproductive tract interactions are poorly understood. To address this deficiency in our knowledge, we examined the expression and function in sperm of CCR6, a receptor common to several chemoattractant peptides, and screened several reproductive tract fluids for the presence of specific ligands. CCR6 protein is present in mouse and human sperm and mainly localized in the sperm tail with other minor patterns in sperm from mice (neck and acrosomal region) and men (neck and midpiece regions). As expected from the protein immunoblotting and immunofluorescence results, mouse Ccr6 mRNA is expressed in the testis. Furthermore, the Defb29 mRNA encoding the CCR6 ligand, β-defensin DEFB29, is expressed at high levels in the epididymis. As determined by protein chip analysis, several chemokines (including some that act through CCR6, such as CCL20/MIP-3α (formerly Macrophage Inflammatory Protein 3α) and protein hormones were present in human follicular fluid, endometrial secretions, and seminal plasma. In functional chemotaxis assays, capacitated human sperm exhibited a directional movement towards CCL20, and displayed modifications in motility parameters. Our data indicate that chemokine ligand/receptor interactions in the male and female genital tracts promote sperm motility and chemotaxis under non-inflammatory conditions. Therefore, some of the physiological reactions mediated by CCR6 ligands in male reproduction extend beyond a pro-inflammatory response and might find application in clinical reproduction and/or contraception. PMID:23765988

The sperm of Hylodinae species (Anura, Leptodactylidae): ultrastructural characteristics and their relevance to interspecific taxonomic relationships.

PubMed

Aguiar, O; Giaretta, A A; Recco-Pimentel, S M

2006-09-01

Hylodinae leptodactylids (sensu Lynch 1971) form a group of diurnal frogs, which is hypothesized on the basis of morphological traits to be the closest relatives of the dendrobatid frogs. Our study describes ultrastructural characteristics of sperm from three hylodine species (Hylodes phyllodes, Crossodactylus sp. n. and Megaelosia massarti) to reassess the intergeneric relationships within the Hylodinae, as well as the supposed relationship between the Hylodinae and Dendrobatidae. The ultrastructure of the sperm is very similar among the three species and is indicative of its conserved nature within the Hylodinae. The structure of the acrosomal complex was very similar to that of other leptodactylid species, to most of the remaining species included in the Bufonoidea lineage, and also to that observed in the dendrobatid species examined so far. Since such a structure has been considered a plesiomorphic trait, it contributes little to our understanding of the relationships between the Hylodinae and Dendrobatidae. The flagellar apparatus of Crossodactylus sp. n. is very similar to that of most leptodactylids. The sperm of Megaelosia massarti and Hylodes phyllodes display a distinctive condition in their axial and juxtaxonemal fibers. This distinctive flagellar condition expands the already known variability in sperm structure within the Leptodactylidae.

Creatine enhances the duration of sperm capacitation: a novel factor for improving in vitro fertilization with small numbers of sperm.

PubMed

Umehara, Takashi; Kawai, Tomoko; Goto, Masaaki; Richards, JoAnne S; Shimada, Masayuki

2018-06-01

Why are many sperm required for successful fertilization of oocytes in vitro, even though fertilization occurs in vivo when only a few sperm reach the oocyte? Creatine produced in the ovary promotes efficient fertilization in vivo; however, in vitro, creatine is not contained in the in vitro fertilization (IVF) medium. The IVF medium enables capacitation of sperm. However, the IVF medium does not fully mimic the in vivo environment during fertilization. Consequently, fertilization in vitro is more inefficient than in the oviduct. Follicular and oviductal fluids were collected and then analyzed for creatine and glucose levels. To determine the physiological functions of creatine, the creatine antagonist 3-guanidinopropionic acid (GPA) was injected into hormonally primed mice. Using conventional IVF protocols, sperm were pre-incubated in IVF medium with creatine and then co-cultured with 10 ovulated cumulus-oocyte complexes (1-1000 per oocyte) in 50 μl medium droplets. Glucose and creatine levels were measured using commercial enzymatic assay kits. The effect of creatine in vivo was assessed by mating experiments using mice treated with or without GPA just before ovulation. To assess the functions of sperm incubated in IVF medium containing creatine, we analyzed (1) the motility of sperm using computer-assisted sperm assay, (2) the capacitation level of sperm by western blot analyses, and (3) the condition of sperm acrosomes by peanut agglutinin lectin-FITC staining. Oviductal creatine levels were significantly increased following ovulation. Injecting mice with GPA just before ovulation significantly reduced the number of fertilized oocytes. The addition of creatine to IVF medium enhanced sperm capacitation by increasing ATP levels. Successful fertilization was achieved with as few as five sperm/oocyte in the creatine group, and the number of fertilized oocytes was significantly higher than in the control without creatine (P < 0.01). In the present study, a

Effect of dietary supplementation with amino acids on boar sperm quality and fertility.

PubMed

Dong, Hong-Jun; Wu, De; Xu, Sheng-Yu; Li, Qiang; Fang, Zheng-Feng; Che, Lian-Qiang; Wu, Cai-Mei; Xu, Xue-Yu; Lin, Yan

2016-09-01

The aim of this study was to evaluate the effects of dietary supplementation with amino acids on sperm quality and fertility rates after insemination with boar semen. Twelve Yorkshire boars were paired by age and allocated to one of two dietary treatments composed of total lysine levels of 0.64% (T1) and 0.96% (T2), with the lysine: methionine: threonine: tryptophan: valine ratio in the diets set to 100:27:73:19:69 through the addition of synthetic amino acids. Semen was collected twice weekly (phase 1, 1-12 wk); every other day (phase 2, 13-16 wk); twice weekly (phase 3, 17-26 wk); and daily (phase 4, 27-28 wk). Semen was collected from boars during phase 3 and used to inseminate 64 multiparous sows. Our results showed that sperm concentration and total sperm cells were greater in boars in T2 than in boars in T1 in phases 2 and 4 (P<0.05). Sperm motility parameters, morphologically normal sperm, and acrosome integrity in T2 boars were greater than those in T1 boars (P<0.05) during the experiment. Free amino acid concentrations in seminal plasma increased in T2 boars (P<0.05). Furthermore, sows inseminated with semen collected from T2 boars gave birth to more live piglets than those inseminated with semen collected from T1 boars (P=0.04). In conclusion, supplementation of boar diet with amino acids improves sperm quality, and subsequently increases fertilization capacity and the number of live piglets. Copyright © 2016. Published by Elsevier B.V.