Sample records for acrosin
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Effects of reduced glutathione on acrosin activity in frozen-thawed boar spermatozoa.
Estrada, Efrén; Rodríguez-Gil, Joan E; Rivera Del Álamo, Maria M; Peña, Alejandro; Yeste, Marc
2017-02-01
In pigs, acrosin activity in extended semen is correlated with reproductive performance and has recently been identified as a freezability marker. Reduced glutathione (GSH) is known to decrease sperm cryodamage and increase the reproductive performance of frozen-thawed boar spermatozoa. However, the effects of GSH on the acrosin activity of good and poor freezability ejaculates (GFE and PFE, respectively) is yet to be examined. The present study investigated how supplementing cryopreservation media with GSH affected acrosin activity in GFE and PFE, as well as the relationship between acrosin activity and reproductive performance in frozen-thawed boar spermatozoa. In addition, we examined whether the increase in fertility rates and litter sizes observed after the addition of 2mM GSH to cryopreservation extenders was related to acrosin activity. Supplementing freezing media with 2mM GSH partially counteracted the cryopreservation-related decrease in acrosin activity in GFE but not PFE. Acrosin activity was found to be significantly correlated with in vivo reproductive performance of frozen-thawed boar semen. In conclusion, the effects of adding GSH to freezing extenders on the acrosin activity of frozen-thawed boar spermatozoa rely on the intrinsic freezability of the ejaculate. Furthermore, the maintenance of proper acrosin activity could contribute to the increase in reproductive performance mediated by GSH.
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Glogowski, J; Demianowicz, W; Piros, B; Ciereszko, A
1998-10-15
A clinical assay to evaluate total acrosin activity developed for human semen has been optimized for use in boar spermatozoa. The main modifications included a decrease of sperm number per assay from 1.0 to 10.0 x 10(6) to 12.5 to 75.0 x 10(3) spermatozoa, and the time of incubation from 180 to 60 min. Linearity of response for differing quantities of spermatozoa was maintained. Extensive washing of spermatozoa was necessary to eliminate seminal plasma, the source of acrosin inhibitors. Seminal plasma that was diluted 1000 times inhibited acrosin activity by about 50%. To abolish the inhibitory effect of seminal plasma it was necessary to use 25,000-fold dilution. Total acrosin activity of boar spermatozoa was about 100 times higher than that of human spermatozoa. Acrosin activity of boar spermatozoa in extended semen decreased during 7 d of storage. These results indicate that the clinical assay of acrosin activity can be used for boar spermatozoa to evaluate the quality of boar semen.
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Discovery of novel human acrosin inhibitors by virtual screening
NASA Astrophysics Data System (ADS)
Liu, Xuefei; Dong, Guoqiang; Zhang, Jue; Qi, Jingjing; Zheng, Canhui; Zhou, Youjun; Zhu, Ju; Sheng, Chunquan; Lü, Jiaguo
2011-10-01
Human acrosin is an attractive target for the discovery of male contraceptive drugs. For the first time, structure-based drug design was applied to discover structurally diverse human acrosin inhibitors. A parallel virtual screening strategy in combination with pharmacophore-based and docking-based techniques was used to screen the SPECS database. From 16 compounds selected by virtual screening, a total of 10 compounds were found to be human acrosin inhibitors. Compound 2 was found to be the most potent hit (IC50 = 14 μM) and its binding mode was investigated by molecular dynamics simulations. The hit interacted with human acrosin mainly through hydrophobic and hydrogen-bonding interactions, which provided a good starting structure for further optimization studies.
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Słowińska, M; Liszewska, E; Dietrich, G J; Ciereszko, A
2012-09-15
This study was designed to identify the effect of liquid storage at 4 °C for 48 h and cryopreservation on the proacrosin/acrosin system of turkey spermatozoa. Anti-acrosin I antibodies were produced and used to demonstrate Western blot analysis profile of the proacrosin/acrosin system of sperm and seminal plasma and possible changes in the proacrosin/acrosin system of turkey sperm stored for 2.5, 24, and 48 h or cryopreserved. At the same time acrosin-like activity was examined by the measurement of amidase activity of sperm extracts, sperm suspension, and seminal plasma of turkey semen. A computer-assisted sperm analysis system was used to monitor the sperm motility characteristics of turkey sperm stored for 48 h or cryopreserved. Different profiles of the sperm proacrosin/acrosin system were observed regarding the presence or absence of inhibitors (p-nitrophenyl-p'-guanidine benzoate [NPGB] and Kazal family inhibitor) during the extraction process. When NPGB was present three main bands were observed with the molecular weight ranging from 66 to 35 kDa. Bands corresponding to acrosin I and II were not observed. In sperm extract without NPGB, three or four bands were observed with the molecular weight ranging from 41 to 30 kDa. The bands corresponding to acrosin I and II were observed. During liquid storage a decrease in sperm motility and an increase in sperm-extracted amidase activity were observed. After 24 and 48 h of storage, extracted amidase activity was higher than at 2.5 h by 24% and 31%, respectively. However, no changes in the Western blot analysis profiles of sperm extract and seminal plasma were visible during liquid storage. After cryopreservation a decrease in sperm motility and all sperm motility parameters were observed. In contrast to liquid storage, cryopreservation did not increase extracted amidase activity. However, changes in Western blot analysis profiles were visible in sperm extract and seminal plasma after cryopreservation. After freezing-thawing, additional bands appeared in sperm extract and seminal plasma. These bands were of different molecular weight regarding the presence or absence of NPGB. These data suggest that the mechanism of damage to the proacrosin/acrosin system is different for liquid storage and cryopreservation. Liquid storage seems to increase in the susceptibility of the proacrosin/acrosin system to be activated during extraction. Kazal inhibitors of turkey seminal plasma are involved in the control of proacrosin activation. The disturbances of the proacrosin/acrosin system of turkey spermatozoa can be related to a disturbance in the induction of the acrosome reaction. Our results may be important for a better understanding of the proacrosin/acrosin system of turkey spermatozoa and disturbance to this system during liquid storage and cryopreservation. Copyright © 2012 Elsevier Inc. All rights reserved.
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Stasiak, K; Janicki, B; Glogowski, J
2012-01-01
The aim of the study was to adapt a method to determine acrosin activity of human spermatozoa to arctic fox (Alopex lagopus L.) spermatozoa. We modified this method by reducing sperm count per sample from 1 divided by 10 x 10(6) to 25 divided by 200 x 10(3), incubation time from 180 minutes to 60 minutes, and Triton X-100 concentration in the reaction mixture from 0.01% to 0.005% per 100 cm3. It has also confirmed that arctic fox seminal plasma is rich in proteinases and their inhibitors. To completely abolish the inhibitory effect of seminal plasma on acrosin activity it is recommended to wash the spermatozoa four times. Benzamidine served an inhibitor of acrosin activity.
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Glogowski, J; Jankowski, J; Faruga, A; Ottobre, J S; Ciereszko, A
2001-09-15
We optimized a clinical assay developed for measuring total acrosin activity for mammalian and fish semen for use in turkey spermatozoa. The main modifications included dilution of semen to a final concentration of 25 to 1000 x 10(3) spermatozoa, an increase of Triton X-100 concentration to 0.05% and 1 hr preincubation without substrate, Acrosin activity in turkey spermatozoa was much higher than in human spermatozoa (about 100-times) but similar to that of boar sperm. To optimize this assay for turkey spermatozoa, it was necessary to use higher Triton X-100 concentrations in the reaction mixture. There was a better catalytic efficiency at higher temperatures and a special requirement for a preincubation period for proacrosin activation. We observed high inhibition of acrosin activity by zinc added during preincubation (90% at 0.01 mM of zinc chloride). Benzamidine also inhibited turkey acrosin, and the extent of inhibition was similar for the incubation or preincubation period. When zinc ions were added during incubation, this inhibition was lower (24%). The results suggest that zinc influences proacrosin activation of turkey spermatozoa. This influence may be important for successful long-term storage of spermatozoa in the hen's oviduct.
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Pinart, E; Yeste, M; Puigmulé, M; Barrera, X; Bonet, S
2013-08-01
The effect of increasing environmental temperature and radiation on the sperm quality and the field fertility of refrigerated seminal doses from AI boars (N = 30) was analyzed throughout four experimental months (from March through June). In each experimental month, analyses of sperm quality were performed at days 0, 1, 3, 5, 7, and 9 of refrigeration of seminal doses; pregnancy rate and litter size were evaluated using double monospermic inseminations of multiparous female animals using seminal doses at Days 1 to 2 and Days 3 to 4 of refrigeration. Sperm quality was assessed from the evaluation of conventional parameters of sperm concentration, sperm motility, sperm morphology, and sperm viability, and capacitation parameters of membrane lipid disorder, intracellular calcium content, and acrosin activity. Results showed that sperm quality of boar seminal doses was negatively affected by increasing temperature and radiation, which resulted in significantly decreased sperm motility and viability, acrosin activity, pregnancy rate, and litter size, and significantly increased intracellular calcium levels in the trials performed in June. In any experimental month, aging of refrigerated doses was associated with the progressive increase of intracellular calcium levels and inactivation of acrosin, that began from Day 5 of storage in the trials performed in March and April, from Day 3 in those of May, and from Day 0 in those of June. Among the sperm parameters analyzed, only acrosin activity exhibited a clearly differentiated pattern in association with increasing temperature and radiation, and a significant correlation with pregnancy rate and litter size. These results highlighted the potential role of acrosin activity as an indicator of boar sperm preservation at 17 °C in boars. Copyright © 2013 Elsevier Inc. All rights reserved.
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Effect of age and breeding season on sperm acrosin activity in the arctic fox (Alopex lagopus L.).
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.
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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.
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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.
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Zalata, Adel; El-Samanoudy, Ayman Z; Shaalan, Dalia; El-Baiomy, Youssef; Mostafa, Taymour
2015-01-01
Use of cellular phones emitting radiofrequency electromagnetic field (RF-EMF) has been increased exponentially and become a part of everyday life. This study aimed to investigate the effects of in vitro RF-EMF exposure emitted from cellular phones on sperm motility index, sperm DNA fragmentation and seminal clusterin (CLU) gene expression. In this prospective study, a total of 124 semen samples were grouped into the following main categories: i. normozoospermia (N, n=26), ii. asthenozoospermia (A, n=32), iii. asthenoteratozoospermia (AT, n=31) and iv. oligoasthenoteratozoospermia (OAT, n=35). The same semen samples were then divided into two portions non-exposed and exposed samples to cell phone radiation for 1 hour. Before and immediately after exposure, both aliquots were subjected to different assessments for sperm motility, acrosin activity, sperm DNA fragmentation and CLU gene expression. Statistical differences were analyzed using paired t student test for comparisons between two sub-groups where p<0.05 was set as significant. There was a significant decrease in sperm motility, sperm linear velocity, sperm linearity index, and sperm acrosin activity, whereas there was a significant increase in sperm DNA fragmentation percent, CLU gene expression and CLU protein levels in the exposed semen samples to RF-EMF compared with non-exposed samples in OAT>AT>A>N groups, respectively (p<0.05). Cell phone emissions have a negative impact on exposed sperm motility index, sperm acrosin activity, sperm DNA fragmentation and seminal CLU gene expression, especially in OAT cases.
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Nagdas, Subir K; Smith, Linda; Medina-Ortiz, Ilza; Hernandez-Encarnacion, Luisa; Raychoudhury, Samir
2016-03-01
Mammalian fertilization is accomplished by the interaction between sperm and egg. Previous studies from this laboratory have identified a stable acrosomal matrix assembly from the bovine sperm acrosome termed the outer acrosomal membrane-matrix complex (OMC). This stable matrix assembly exhibits precise binding activity for acrosin and N-acetylglucosaminidase. A highly purified OMC fraction comprises three major (54, 50, and 45 kDa) and several minor (38-19 kDa) polypeptides. The set of minor polypeptides (38-19 kDa) termed "OMCrpf polypeptides" is selectively solubilized by high-pH extraction (pH 10.5), while the three major polypeptides (55, 50, and 45 kDa) remain insoluble. Proteomic identification of the OMC32 polypeptide (32 kDa polypeptide isolated from high-pH soluble fraction of OMC) yielded two peptides that matched the NCBI database sequence of acrosin-binding protein. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38-19-kDa range with isoelectric points ranging between 4.0 and 5.1. Other than glycohydrolases, OMC32 may also be complexed to other acrosomal proteins. The present study was undertaken to identify and localize the OMC32 binding polypeptides and to elucidate the potential role of the acrosomal protein complex in sperm function. OMC32 affinity chromatography of a detergent-soluble fraction of bovine cauda sperm acrosome followed by mass spectrometry-based identification of bound proteins identified acrosin, lactadherin, SPACA3, and IZUMO1. Co-immunoprecipitation analysis also demonstrated the interaction of OMC32 with acrosin, lactadherin, SPACA3, and IZUMO1. Our immunofluorescence studies revealed the presence of SPACA3 and lactadherin over the apical segment, whereas IZUMO1 is localized over the equatorial segment of Triton X-100 permeabilized cauda sperm. Immunoblot analysis showed that a significant portion of SPACA3 was released after the lysophosphatidylcholine (LPC)-induced acrosome reaction, whereas the IZUMO1 and lactadherin polypeptides remain associated to the particulate fraction. Almost entire population of bovine sperm IZUMO1 relocates to the equatorial segment during the LPC-induced acrosome reaction. We propose that the interaction of OMC32 matrix polypeptide with detergent-soluble acrosomal proteins regulates the release of hydrolases/other acrosomal protein(s) during the acrosome reaction.
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Computer-assisted motility analysis (CASA) was used to evaluate the effect of cryopreservation and theophylline treatment on sperm motility of lake sturgeon (Acipenser fulvescens).Motility was recorded at 0 and 5 min postactivation.The effect of cryopreservation on sperm acrosin-...
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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 with either ascorbic acid or catalase, improvement in semen quality was noticed. It was inferred that incorporation of ascorbic acid, catalase and ascorbic acid + chlorpromazine in semen extender improved the post-thaw semen quality in crossbred bulls.
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Detection of sperm-reactive antibodies in wild sika deer and identification of the sperm antigens.
Kawase, Osamu; Jimbo, Mitsuru
2018-03-16
Antisperm antibodies potentially inhibit sperm functions causing the sterility in humans and experimentally treated animals. However, there is no information about antisperm antibodies emerging spontaneously in wildlife. In this study, we searched for the sperm-reactive antibodies, spontaneously produced in wild sika deer (Cervus nippon), and identified the sperm antigens. We collected 529 fecal masses of sika deer in Japanese cities, from which we extracted the mucosal antibodies to test them for reactivities to deer sperm proteins by ELISA. Two of the extracts contained IgAs that were highly reactive to the sperm proteins. The molecular weights of the active IgAs, partially purified by DEAE-sephadex A-50, were estimated at more than 100 kDa, suggesting that the IgAs evaded drastic digestion in the gastrointestinal tract. Two-dimensional electrophoresis and immunoblotting detected three major antigens, and the following LC-MS/MS analysis identified them as alpha-enolase, phosphoglycerate kinase 2 and acrosin-binding protein. The antibodies were cross-reactive to a recombinant human acrosin-binding protein. To our knowledge, this is the first research to find that the sperm-reactive antibodies are produced spontaneously in wildlife and they recognize a common antigen found in humans.
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Studies on the membrane integrity of human sperm treated with a new injectable male contraceptive.
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
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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
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Stasiak, K; Glogowski, J; Demianowicz, W; Kowalski, R; Nowak-Tkaczyk, A; Janicki, B
2014-01-01
The aim of this study was to use biochemical markers to evaluate the quality of fresh and cryopreserved semen from the arctic fox (Vulpes lagopus). Twenty-three manually collected ejaculates were analysed for the main indicators of semen quality (sperm concentration and ejaculate volume). Sperm motility and percentage of morphologically normal and abnormal spermatozoa were determined according to the stage of cryopreservation (fresh--measurement A; equilibrated--measurement B; frozen/thawed--measurement C). Furthermore, the seminal plasma and supernatants were analysed after equilibration and freeze/thawing for the activity of the enzymes alkaline phosphatase (ALP), acid phosphatase (AcP), lactate dehydrogenase (LDH) and aspartate aminotransferase (AspAT), and for the activity of acrosin inhibitors (AP). The mean concentration of sperm was 625.1 million/cm3, and ejaculate volume averaged 1.6 cm3. Seminal plasma was characterized by the highest activity of alkaline phosphatase (3.43 x 10(3) U/l) and lowest activity of acrosin inhibitors (4.55 x 10(3) U/l). After equilibration, the supernatants showed the highest activity of acid phosphatase (94.9 U/l) and after freeze-thawing, they showed a high activity of lactate dehydrogenase (535.8 U/l) and aspartate aminotransferase (577.1 U/l), which indicates that these proteins had leaked from spermatozoa into the extracellular medium during the biotechnique of semen cryopreservation. In addition, several significant relationships were found between some indicators of semen quality and plasma and/or supernatant enzyme activity.
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Hu, Jingmei; Zhao, Shidou; Xu, Chengyan; Zhang, Lin; Lu, Shaoming; Cui, Linlin; Ma, Jinlong; Chen, Zi-Jiang
2015-11-01
To compare the differences in the characteristics of post-thaw human sperm after storage in either liquid nitrogen (LN2; -196 °C) or LN2 vapor (-167 °C). Experimental study. University hospital. Thirty healthy volunteers who agreed to donate their normal semen samples for infertility or research were included in the study. Semen samples (n = 30) were divided into eight aliquots and frozen. Four aliquots of each human semen sample were stored in LN2 (-196 °C), and the other four aliquots were stored in LN2 vapor (-167 °C). After 1, 3, 6, or 12 months, samples were thawed and analyzed. The motility was evaluated by the manual counting method. The viability was estimated by eosin staining. The morphology was analyzed by Diff-Quik staining. The sperm DNA integrity was determined with acridine orange fluorescent staining, and acrosin activity was assayed by the modified Kennedy method. The characteristics of post-thaw human sperm, including motility, viability, morphology, DNA integrity, and acrosin activity, showed no significant difference between LN2 and LN2 vapor storage for the different time periods. LN2 vapor was comparable to LN2 in post-thaw sperm characteristics, suggesting that LN2 vapor may be substituted for LN2 for the long-term storage of human sperm. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.
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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.
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Correlations among assays of porcine semen quality following cryopreservation.
Hammitt, D G; Martin, P A
1989-09-01
Correlations between in vitro tests of semen quality, used to predict the in vivo fertilizing potential of sperm, indicate that the tests may substitute for each other in predicting fertilizing potential. Lack of correlation between tests suggest that both tests should be used to estimate the fertilizing potential. The purpose of this study was to establish correlations between several in vitro tests of porcine semen quality following freezing. Tests of motility with and without caffeine, spermatozoa with normal apical ridges, sephadex filtration with and without caffeine and acrosin activity were all correlated with each other. Correlations among these tests ranged from 0.45 to 0.83 (P<0.05). Assays for glutamic oxalacetic transaninase (GOT) were not consistently correlated with other tests. None of these tests of semen quality were correlated with the sperm penetration assay except for the test of motility without caffeine, which was correlated with the number of penetrations per hamster oocyte (r = 0.71, P<0.05).
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He, Lin; Li, Qing; Liu, Lihua; Wang, Yuanli; Xie, Jing; Yang, Hongdan; Wang, Qun
2015-01-01
The accessory gland (AG) is an important component of the male reproductive system of arthropods, its secretions enhance fertility, some AG proteins bind to the spermatozoa and affect its function and properties. Here we report the first comprehensive catalog of the AG secreted fluid during the mature phase of the Chinese mitten crab (Eriocheir sinensis). AG proteins were separated by one-dimensional gel electrophoresis and analyzed by reverse phase high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Altogether, the mass spectra of 1173 peptides were detected (1067 without decoy and contaminants) which allowed for the identification of 486 different proteins annotated upon the NCBI database (http://www.ncbi.nlm.nih.gov/) and our transcritptome dataset. The mass spectrometry proteomics data have been deposited at the ProteomeXchange with identifier PXD000700. An extensive description of the AG proteome will help provide the basis for a better understanding of a number of reproductive mechanisms, including potentially spermatophore breakdown, dynamic functional and morphological changes in sperm cells and sperm acrosin enzyme vitality. Thus, the comprehensive catalog of proteins presented here can serve as a valuable reference for future studies of sperm maturation and regulatory mechanisms involved in crustacean reproduction. PMID:26305468
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Cryopreservation of adult cervid testes.
Pothana, Lavanya; Devi, Lalitha; Goel, Sandeep
2017-02-01
Several species of cervids are currently classified as threatened or endangered due to a rapid decline in their populations. Sperm cryopreservation, in association with assisted reproductive technologies, can find application for the conservation of endangered cervids. In cases of unsuccessful sperm retrieval through other means prior to the death of the animal, adult testis is the only source of sperm. Recovery of viable sperm from adult testes depends on the effective preservation of testicular tissues through optimization of cryopreservation protocols. The present study evaluated combinations of 10% dimethyl sulfoxide (DMSO) with 0% or 80% fetal bovine serum (FBS) and 20% DMSO with 0 or 20% FBS for the cryopreservation of testicular tissues of three adult cervids using uncontrolled slow freezing protocol. The cryopreserved testis was compared to chilled tissue without cryoprotectants. Results revealed that testicular tissues of barking deer cryopreserved in 20% DMSO (D20) had all the analyzed 7 parameters (number of TNP1-, PRM2 and acrosin-expressing cells/tubule and, the number of viable, morphologically normal, acrosome intact, Annexin V-negative sperm) comparable to the chilled testis. However, testicular tissues of sambhar and hog deer cryopreserved only in D20S20 had 5 of 7 parameters comparable to the chilled testis. In conclusion, D20 is acceptable for cryopreservation of barking deer and D20S20 for sambar and hog deer testes. Copyright © 2016 Elsevier Inc. All rights reserved.
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MnSOD expression inhibited by electromagnetic pulse radiation in the rat testis.
Zeng, LiHua; Ji, XiTuan; Zhang, YanJun; Miao, Xia; Zou, ChangXu; Lang, HaiYang; Zhang, Jie; Li, YuRong; Wang, XiaoWu; Qi, HongXing; Ren, DongQin; Guo, GuoZhen
2011-12-01
Male Sprague Dawley rats were exposed to EMP irradiation of 100 kV/m peak-to-peak e-field intensity and different numbers of pulses. Rat sperm samples were prepared for analysis of sperm qualities; Testes were assessed by transmission electron microscopy and serum hormone concentrations were examined by radioimmunoassay; Enzymatic activities of Total-superoxide dismutase(T-SOD) and manganese-superoxide dismutase (MnSOD), the mRNA levels of MnSOD and cuprozinc-superoxide dismutase (CuZnSOD), and the density of malondialdehyde (MDA) were also determined. EMP irradiation did not affect spermatozoon morphology, micronucleus formation rate, sperm number or viability, but the acrosin reaction rate decreased at 24 h and 48 h and recovered by 72 h after irradiation as compared to the controls. The ultrastructure of rat testis displayed more serious damage at 24 h than at other time points (6 h, 12 h, 48 h). Serum levels of luteotrophic hormone (LH) and testosterone (T) were elevated in irradiated rats as compared to controls. After irradiation, enzymatic activities of T-SOD and MnSOD were reduced by 24 h, consistent with the changes observed in MnSOD mRNA expression; MDA content increased at 6 h in turn. These studies have quantified the morphological damage and dysfunction in the rat reproductive system induced by EMP. The mechanism of EMP induced damage may be associated with the inhibition of MnSOD expression.
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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.
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Sperm function and assisted reproduction technology
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
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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.
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Aquila, Saveria; Santoro, Marta; De Amicis, Francesca; Guido, Carmela; Bonofiglio, Daniela; Lanzino, Marilena; Cesario, Maria Grazia; Perrotta, Ida; Sisci, Diego; Morelli, Catia
2013-02-01
Myricetin is a natural flavonoid, particularly enriched in red wines, whose occurrence is widespread among plants. Despite extensive research, the beneficial effects of Myricetin on human health are still controversial. Here, we tested the estrogen-like effect of the phytoestrogen Myricetin on human ejaculated sperm biology. To this aim, human normozoospermic samples were exposed to increasing concentrations (10 nM, 100 nM, and 1 µM) of Myricetin. Motility, viability, capacitation-associated biochemical changes (i.e., cholesterol efflux and tyrosine phosphorylation), acrosin activity, as well as glucose utilization and fatty-acid oxidation (i.e., glucose and lipid metabolism) were all significantly increased by low doses of Myricetin. Importantly, both estrogen receptors α and β (ERs) and phosphatidylinositol-3-OH kinase (PI3K)/AKT signaling are activated in the presence of Myricetin since these were both abrogated by specific inhibitors of each pathway. Our results show how Myricetin, through ERs and PI3K/AKT signalings, potentiates sperm function. This effect is dose-dependent at low concentrations of Myricetin (up to 100 nM), whereas higher amounts do not seem to improve any further sperm motility, viability, or other tested features, and, in some cases, they reduced or even abrogated the efficacy exerted by lower doses. Further studies are needed to elucidate if high levels of Myricetin, which could be attained even with moderate wine consumption, could synergize with endogenous estrogens in the female reproductive tract, interfering with the physiological sperm fertilization process. Copyright © 2012 Wiley Periodicals, Inc.
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Stage-specific expression of DDX4 and c-kit at different developmental stages of the porcine testis.
Lee, Ran; Lee, Won-Young; Park, Hyun-Jung; Ha, Woo-Tae; Woo, Jae-Seok; Chung, Hak-Jae; Lee, Ji-Heon; Hong, Kwonho; Song, Hyuk
2018-03-01
Spermatogenesis begins with spermatogonial stem cells (SSCs), which are located in the basement membrane of the adult testes. Previous studies have described specific biomarkers for undifferentiated porcine spermatogonia or SSCs; however, these markers are not sufficient to understand spermatogenesis at different developmental stages. The objective of this study was characterize the expression of DEAD-Box polypeptide 4 (DDX4, also known as VASA) and tyrosine-protein kinase kit (c-kit), as potential markers of male germ cells in the porcine testis. In porcine testis tissue at prepubertal stages (5, 30, and 60 days), DDX4 and c-kit protein expression was detected in the most undifferentiated spermatogonia, which also express protein gene product 9.5 (PGP9.5). However, in porcine testis tissues from pubertal and postpubertal stages (90, 120, and 150 days), DDX4 and c-kit were not detected in PGP9.5-positive undifferentiated spermatogonia. The DDX4 expression pattern was similar to that of c-kit in the porcine testis. In adult porcine testes, DDX4-expressing cells were located on the lumenal side, compared to synaptonemal complex protein 3-positive primary spermatocytes, but DDX-4 was not co-expressed with acrosin, a known acrosome marker. In addition, DDX4 was detected in PGP9.5-expressing porcine SSCs in culture. Based on our results, we suggest that DDX4 and c-kit are putative markers of undifferentiated spermatogonia in the prepubertal porcine testis. While in the postpubertal porcine testis, they are markers of differentiated spermatocytes. These findings may facilitate future studies of porcine spermatogenesis. Copyright © 2018 Elsevier B.V. All rights reserved.
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Shah, Syed M; Saini, Neha; Singh, Manoj K; Manik, Radheysham; Singla, Suresh K; Palta, Prabhat; Chauhan, Manmohan S
2016-08-01
Testicular cells are believed to secrete various growth factors that activate signaling pathways finally leading to gametogenesis. In vitro gametogenesis is an obscure but paramountly important task primarily because of paucity of the precursor cells and first trimester gonadal tissues. To overcome these limitations for development of in vitro gametes, the present study was designed to induce differentiation of buffalo embryonic stem (ES) cells into germ lineage cells on stimulation by testicular cell-conditioned medium (TCM), on the basis of the assumption that ES cells have the intrinsic property to differentiate into any cell type and TCM would provide the necessary growth factors for differentiation toward germ cell lineage. For this purpose, buffalo ES cells were differentiated as embryoid bodies (EB) in floating cultures and as monolayer adherent cultures in different doses (10%, 20%, and 40%) of TCM for different culture intervals (4, 8, and 14 days), to identify the optimum dose-and-time period. We observed that 40% TCM dose induces highest expression of primordial germ cell-specific (DAZL, VASA, and PLZF), meiotic (SYCP3, MLH1, TNP1/2, and PRM2), spermatocyte-specific (BOULE and TEKT1), and oocyte-specific genes (GDF9 and ZP2/3) for a culture period of 14 days under both floating and adherent differentiation. Immunocytochemical analysis of EBs and adherent cultures revealed presence of primordial germ cell markers (c-KIT, DAZL, and VASA), meiotic markers (SYCP3, MLH1 and PROTAMINE1), spermatocyte markers (ACROSIN and HAPRIN), and oocyte markers (GDF9 and ZP4), indicating progression into post-meiotic gametogenesis. The detection of germ cell-specific proteins in Day 14 EBs like VASA, GDF9, and ZP4 by Western blotting further confirmed germ lineage differentiation. The significantly lower (P < 0.05) concentration of 5-methyl-2-deoxycytidine in optimally differentiated EBs is suggestive of the process of methylation erasure. Oocyte-like structures obtained in monolayer differentiation had a big nucleus and a surrounding ZP4 coat, the unique attributes of a female gamete. These oocyte-like structures, in extended cultures, showed embryonic development and progressed through two-cell, four-cell, eight-cell, morula, and blastocyst-like structures, indicative of their developmental competence. This, as per our knowledge, is first such study in higher mammals, especially farm animals, and assumes significance for its potential use in transgenic animal production, elite animal conservation and propagation, augmentation of reproductive performance in poor breeding buffalo species, and as a model for understanding human germ cell formation. Copyright © 2016 Elsevier Inc. All rights reserved.
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In vitro differentiation of rat spermatogonia into round spermatids in tissue culture.
Reda, A; Hou, M; Winton, T R; Chapin, R E; Söder, O; Stukenborg, J-B
2016-09-01
Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in vitro or testosterone production was observed. The human testis is very different in physiology from the rat testis, further investigations are still needed to optimize the organ culture system for future use in humans. The successful differentiation of undifferentiated spermatogonia using the testis explant culture system might be employed in future to produce sperm from human spermatogonia as a clinical tool for fertility preservation in boys and men suffering infertility. None. This work was supported financially by the Frimurare Barnhuset in Stockholm, the Paediatric Research Foundation, Jeanssons Foundation, Sällskåpet Barnåvard in Stockholm, Swedish Research Council/Academy of Finland, Emil and Wera Cornells Foundation, Samariten Foundation, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. All authors declare no conflicts of interests. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.
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In vitro differentiation of rat spermatogonia into round spermatids in tissue culture
Reda, A.; Hou, M.; Winton, T.R.; Chapin, R.E.; Söder, O.; Stukenborg, J.-B.
2016-01-01
STUDY QUESTION Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? SUMMARY ANSWER We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. WHAT IS KNOWN ALREADY Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in vitro or testosterone production was observed. LIMITATIONS, REASONS FOR CAUTION The human testis is very different in physiology from the rat testis, further investigations are still needed to optimize the organ culture system for future use in humans. WIDER IMPLICATIONS OF THE FINDINGS The successful differentiation of undifferentiated spermatogonia using the testis explant culture system might be employed in future to produce sperm from human spermatogonia as a clinical tool for fertility preservation in boys and men suffering infertility. LARGE SCALE DATA None. STUDY FUNDING AND COMPETING INTEREST(S) This work was supported financially by the Frimurare Barnhuset in Stockholm, the Paediatric Research Foundation, Jeanssons Foundation, Sällskåpet Barnåvard in Stockholm, Swedish Research Council/Academy of Finland, Emil and Wera Cornells Foundation, Samariten Foundation, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. All authors declare no conflicts of interests. PMID:27430551