OBJECTIVE : The sperm acrosome reaction is a Ca2+ -dependent exocytotic event that is triggered by adhesion to the mammalian egg's zona pellucida. Previous studies suggested a role of Ca2+ channels in acrosome reactions. This study was conducted to investigate the T-type calcium channel is operated in acrosome reaction of human spermatozoa. METHOD : Human semen samples were obtained from healthy donors with nomal criteria. The spermatozoa were divided into five groups: Group 1 were non-treated as a control; Group 2 where spermatozoa were exposed to 5 micrometer Ca2+ A23187 (Ca2+i); Group 3 where spermatozoa were exposed 5 micrometer Ca2+i and mibefradil; Group 4 where spermatozoa were exposed 5 micrometer Ca2+i and nifedipine, and Group 5 where spermatozoa were treated with 5 micrometer Ca2+i and both of mibefradil and nifedipine. Spermatozoa in all groups were retrieved after incubation for 15 and 30 minutes at 37degrees C. After staining with PSA-FITC, fluorescence was observed under a fluorescence microscope, and AR was evaluated on a total >100 spermatozoa/side. RESULT AND CONCLUSION : We observed on acrosome reaction inhibition rate in human spermatozoa the various of concentration of mibefradil, nifedipine. Maximum response was noted with 1.0 micrometer mibefradil and the decrease of acrosome reaction inhibition rate 45%. Nifedipine in acrosome reaction inhibition rate was only about 25%. The Ca2+i-induced AR of spermatozoa was significantly suppressed by mibefradil. Incidence of the suppression was depending on concentration of mibefradil. Results from the present study suggest that the human spermatozoa possess T-type channel. The observation that reversible inhibitor of T channels in male germ cells provides a new mechanism of contraceptive action.
Acrosome Reaction* ; Acrosome* ; Calcimycin ; Calcium Channels, T-Type ; Fluorescence ; Germ Cells ; Humans* ; Incidence* ; Male ; Mibefradil* ; Nifedipine ; Semen ; Spermatozoa* ; Tissue Donors ; Zona Pellucida

Successful male germ cell transplantation requires depletion of the host germ cells to allow efficient colonization of the donor spermatogonial stem cells. Although a sterilizing drug, busulfan, is commonly used for the preparation of recipient models before transplantation, the optimal dose of this drug has not yet been defined in dogs. In this study, 1-year-old mongrel dogs were intravenously injected with three different concentrations of busulfan (10, 15, or 17.5 mg/kg). Four weeks after busulfan treatment, no fully matured spermatozoa were detected in any of the busulfan-treated groups. However, small numbers of PGP9.5-positive spermatogonia were detected in all treatment groups, although no synaptonemal complex protein-3-positive spermatocytes were detected. Of note, acrosin-positive spermatids were not detected in the dogs treated with 15 or 17.5 mg/kg busulfan, but were detected in the other group. Eight weeks after busulfan treatment, the dogs treated with 10 mg/kg busulfan fully recovered, but those in the other groups did not. PGP9.5-positive spermatogonia were detected in the 10 mg/kg group, and at a similar level as in the control group, but these cells were rarely detected in the 15 and 17.5 mg/kg groups. These results suggest that a dose of 15-17.5 mg/kg is optimal for ablative treatment with busulfan to prepare the recipient dogs for male germ cell transplantation. At least eight weeks should be allowed for recovery. The results of this study might facilitate the production of recipient dogs for male germ cell transplantation and can also contribute to studies on chemotherapy.
Animals ; Busulfan* ; Colon ; Dogs* ; Drug Therapy ; Germ Cells ; Humans ; Male ; Spermatids ; Spermatocytes ; Spermatogonia ; Spermatozoa ; Stem Cell Transplantation* ; Stem Cells* ; Synaptonemal Complex ; Testis* ; Tissue Donors

OBJECTIVE: Lectins are cell-agglutinating and sugar specific proteins or glycoproteins of non-immune origin that precipitate glycoconjugates having saccharides of appropriate complementarity. Because of these properties, plant lectins have been used to help characterize the carbohydrate moieties of glycoproteins in the zona pellucida (ZP) of several mammalian species including pigs. Treatment of oocytes with various lectins blocks sperm binding to the ZP in various mammalian species. This study was undertaken to examine the distribution of sugar residues in the ZP of pig oocytes matured in vitro and the ability of spermatozoa to bind to ZP and in vitro penetration in oocytes treated with fluorescein isothiocyanate (FITC)-labelled lectins. MATERIALS AND METHODS: The lectins of Banderiaea simplicifolia (BS-II, bind to beta-D-Nacetylglucosamine), Canavalin ensiformis (Con A, bind to alpha-D-Mannose), Lens culinaris (LCA, bind to alpha-D-Mannose), Ricinus communis (RCA-I, bind to beta-D-Galactose) and Ulex europaeus (UEA-I, bind to alpha-L-Fucose) were examined for spermatozoa penetration, binding capacity to ZP and distribution of lectins. RESULTS: The penetration rates were significantry (p<0.05) higher in control oocytes (63%) than those treated with all lectins, but penetration rates (40~49%) were simililar in group treated with lectins. The incidence of monospermy was similar in oocytes untreated and UEA-I, but it was higher in oocytes treated with BS-II, Con A, RCA-I and LCA. The porcine oocytes cultured for 48 h in TC-199 medium were freed from cumulus cells and treated for 30 min with fluorescein isothiocyanate-labelled lectins. When examined under fluorescein illumination, higher (p<0.001) proportions of oocytes showed fluorescein of zona pellucida after treatment with Con A (93%), LCA (93%) and RCA-I (100%) than BS-II (37%) and UEA-I (50%). All of the oocytes treated with RCA-I exhibited strong fluorescein in the outer region of the zona pellucida while those treated with LCA exhibited strong fluorescein throughout the zona pellucida. BS-II bounded mainly to the outer region and UEA-I bounded mainly to the inner region of the zona pellucida, with either strong or weak fluorescein. At 120 min after insemination in vitro, fewer spermatozoa were bound to the zona pellucida of the oocytes treated with BS-II, Con-A and RCA-I. Of the lectins, Con A most inhibited sperm binding. CONCLUSIONS: These results suggest that beta-D-Galactose residues in the porcine zona pellucida may act as primary sperm receptors and inducers of the sperm acrosome reaction and these sugar residues may be involved in the block to polyspermy.
Acrosome Reaction ; Cumulus Cells ; Fluorescein ; Glycoconjugates ; Glycoproteins ; Herpes Zoster* ; Incidence ; Insemination ; Lectins* ; Lens Plant ; Lighting ; Oocytes ; Plant Lectins ; Ricinus ; Sperm-Ovum Interactions ; Spermatozoa ; Swine ; Ulex ; Zona Pellucida*

PURPOSE: The purpose of the present study was to investigate the effect of sesame oil on the reproductive parameters of diabetic male Wistar rats. MATERIALS AND METHODS: The adult male rats in a split plot design were divided into normal (n=10), normal 5% (n=5; 5% sesame oil enriched diet), diabetic (Streptozocin induced diabetes; n=9), diabetic 5% (n=9; 5% sesame oil enriched diet), and diabetic 10% (n=9; 10% sesame oil enriched diet) groups. Diet supplementation continued for 56 days. RESULTS: Sesame oil supplementation did not reduce the plasma glucose concentration of rats in the diabetic groups (p>0.05). The total spermatogonia, spermatocytes, Leydig cells/tubule, and the germ cell to Sertoli cell ratio were lower in the diabetic rats than the normal ones (p<0.05), and with the exception of spermatogonia counts, these values improved by the addition of sesame oil to the diet (p<0.05). The sperm progressive motility and viability were lower in the diabetic rats (p<0.05) and sesame oil supplementation did not improve them. Incorporation of sesame oil into the diet improved the plasma testosterone concentration of the diabetic rats in a dose-dependent manner (p<0.05). CONCLUSIONS: In summary, sesame oil supplementation improved the reproductive parameters of diabetic rats at the levels of the testicular microstructure and function, but was not effective in protecting the epididymal sperm.
Adult ; Animals ; Diabetes Mellitus ; Diet ; Germ Cells ; Male ; Rats ; Sesame Oil ; Sesamum ; Spermatocytes ; Spermatogonia ; Spermatozoa ; Testis ; Testosterone

Gossypol is found to induce an inhibition of fertility in various animals such as rat, dog and monkey. This study was undertaken to determine the exact site of the action of gossypol acetic acid at the testicular level concomitant with antifertility activity at various dose and time intervals in male hamsters. This study consisted of three groups of hamsters according to the dose of GAA with control group. After 12 weeks of treatment at the dose of 5mg/kg/day and 10mg/kg/day a rate of increase in body weight were statistically lower than that of control group. In contrary at the dose of 20mg/kg/day there was a tendency to decrease in body weight. Male hamsters became completely sterile after 8 weeks of treatment with 20mg/kg/day. A regaining of fertility was observed after the cessation of treatment in 5 male hamsters that lost fertility. In the group treated with 10mg/kg/day of GAA, the fertility rate of female at 4,8 and 12 weeks of treatment were reduced 54.5%, 37.5% and 35.7 % respectively whereas those of female in 5 mg/kg/day group were 91.7%, 69.2% and 71.4% respectively. The number of delivered hamsters also decreased with the increase in dosage and duration of treatment. A decrease of motility was observed and followed by reduced sperm population. It was not until 4 weeks after administration with 20mg/kg/day of GAA that a reduction of sperm population was statistically significant. Azoospermia was found as early as 8 weeks after treatment with 20mg/ke/day of GAA. ultrastructurally initial detectable damages were observed in mitochondrial sheath of epididymal sperm and the damage was progressed in order of early spermatid, primary and secondary spermatocyte. spermatogonia. Distinct ultrastructural changes were seen in mitochondria of germ cell. In Sertoli cell cytoplasmic vacuolization was noted and mitochondrial damage was observed in late phase. An aggregation of mitochondria in spermatogonia was observed after treatment with 20mg/kg/day of GAA for 12 weeks. Leydig cell was not altered significantly in this study. The results of this study confirmed antifertility effects of GAA in male hamster and suggested that a spermatogonia may be damaged by chronic effects of gossypol treatment.
Acetic Acid* ; Animals ; Azoospermia ; Birth Rate ; Body Weight ; Cricetinae* ; Cytoplasm ; Dogs ; Female ; Fertility* ; Germ Cells ; Gossypol* ; Haplorhini ; Humans ; Infertility ; Male* ; Mitochondria ; Rats ; Spermatids ; Spermatocytes ; Spermatogonia ; Spermatozoa ; Testis* ; Withholding Treatment

Histomorphologic study was performed on bilaterally testicular biopsies of 30 preadolescent patient with unilateral cryptorchidism so as to understand pathophysiology of the increased incidence or infertility, seen in unilateral cryptorchidism. The results demonstrated delayed and defective transformation of gonocytes to spermatogonia, delayed or failed transformation of spermatogonia to primary spermatocyte and decreased numbers of Leydig cells. These abnormalities were present in the unilaterally cryptorchid testis and their contralateral descended partners but they were more serious and earlier onset in the cryptorchid testis. That is to say, blunted surge in gonadotropins triggers atrophy of Leydig cells leading to delayed and defective maturation of germ cells leading to decreased numbers of germ cells. These findings are compatible with the hypothesis that hypo gonadotropic hypogonadism is the cause of cryptorchidism.
Atrophy ; Biopsy ; Cryptorchidism* ; Germ Cells ; Gonadotropins ; Humans ; Hypogonadism ; Incidence ; Infertility ; Leydig Cells ; Male ; Spermatocytes ; Spermatogonia ; Testis*

In human reproduction, fertilization is the first step for successful pregnancy. From the perspective of sperm physiology, the progressive motility and capacitation, including hyperactivation and acrosome reaction, are the most important factors in the fertilization of oocytes. Numerous studies have demonstrated the roles of calcium ions, cyclic nucleotides, and bicarbonate in the acquisition of progressive motility and capacitation. Among these factors, calcium ion plays the most important role. Sperm possess several calcium channels, including voltage-gated calcium channel, cyclic nucleotide-gated calcium channel, transient receptor potential channel, and channels of the CatSper family The CatSper family is a newly-identified group of four sperm-specific cation channels. CatSper1 and CatSper2 proteins localize on the sperm tail and play a critical role in sperm motility and fertilization. In contrast, CatSper3 and CatSper4 proteinsare expressed only in the acrosomal region of sperm head, which implies that they may have a role in the acrosome reaction. Taken together, the CatSper family is the most important group of calcium channels for regulating sperm physiology and appear to be an attractive target for non-hormonal male contraceptives.
Acrosome Reaction ; Calcium ; Calcium Channels ; Contraceptive Agents, Male ; Fertilization ; Humans ; Ions ; Nucleotides, Cyclic ; Oocytes ; Physiology ; Pregnancy ; Reproduction ; Sperm Head ; Sperm Motility ; Sperm Tail ; Spermatozoa

OBJECTIVE: Recapitulation of the spermatogenesis process in vitro is a tool for studying the biology of germ cells, and may lead to promising therapeutic strategies in the future. In this study, we attempted to transdifferentiate Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) into male germ cells using all-trans retinoic acid and Sertoli cell-conditioned medium. METHODS: Human WJ-MSCs were propagated by the explant culture method, and cells at the second passage were induced with differentiation medium containing all-trans retinoic acid for 2 weeks. Putative germ cells were cultured with Sertoli cell-conditioned medium at 36℃ for 3 more weeks. RESULTS: The gene expression profile was consistent with the stage-specific development of germ cells. The expression of Oct4 and Plzf (early germ cell markers) was diminished, while Stra8 (a premeiotic marker), Scp3 (a meiotic marker), and Acr and Prm1 (postmeiotic markers) were upregulated during the induction period. In morphological studies, approximately 5% of the cells were secondary spermatocytes that had completed two stages of acrosome formation (the Golgi phase and the cap phase). A few spermatid-like cells that had undergone the initial stage of tail formation were also noted. CONCLUSION: Human WJ-MSCs can be transdifferentiated into more advanced stages of germ cells by a simple two-step induction protocol using retinoic acid and Sertoli cell-conditioned medium.
Acrosome ; Biology ; Germ Cells* ; Humans* ; In Vitro Techniques ; Male* ; Mesenchymal Stromal Cells* ; Methods ; Spermatocytes ; Spermatogenesis ; Tail ; Transcriptome ; Tretinoin

OBJECTIVE: To determine the sperm-zona pellucida (ZP) binding and ZP-induced acrosome reaction in patients with unexplained infertility, and to discuss the relationship between ZP-induced acrosome reaction and fertilization rate. METHODS: We compared the fertilization rate and good embryo rate in patients with unexplained infertility after fertilization in 2 ways. Based on the causes of infertility, patients were divided into an unexplained infertility group (Group A) and a pure female tubal factor group (Group B). Oocytes which were obtained by super ovulation from 25 patients with unexplained infertility were randomly divided into 2 groups with conventional in vitro fertilization (IVF) (Group A1) and intracytoplasmic sperm injection (ICSI) fertilization (Group A2). The pure female tubal factor group (Group B) had conventional IVF. We conducted sperm-ZP binding and ZP-induced acrosome reaction experiments with 2 groups of men's sperms separately. We compared the number of sperm-egg binding and ZP-induced acrosome reaction rate and discussed the relationship between the ZP-induced acrosome reaction and fertilization rate, and also the fertilization rate, good embryo rate and pregnancy rate in patients with unexplained infertility after fertilization in 2 ways. RESULTS: The average number of sperm-egg binding (78.29 ± 16.31) and the ZP-induced acrosome reaction rate (55.87 ± 27.69) % in Group A were lower than those of Group B [94.63 ± 6.72, (82.53 ± 17.99)%]. The difference between the average number of sperm-egg binding and the ZP-induced acrosome reaction was significant (P <0.01). The fertilization rate of Group A1 was significantly lower than that of Group B and Group A2 (P <0.01). But there was no significant difference in the good embryo rate among the 3 groups. There was no significant difference between Group A2 and B in fertilization rate and good embryo rate (P <0.05). There was no significant difference in pregnancy rate between Group A and B (P <0.05). Fertilization rate and the rate of acrosome reaction had marked positive correlation with statistical significance (r =0.932, P <0.01). CONCLUSION ZP binding and ZP-induced acrosome reaction are very important experiments in sperm function test for patients with unexplained infertility. It can not only effectively avoid no embryo transferring due to complete failure of fertilization but also get a desirable outcome of pregnancy using half-ICSI fertilization in patients with unexplained infertility.
Acrosome Reaction ; Embryo Transfer ; Female ; Fertilization in Vitro ; Humans ; Infertility ; etiology ; therapy ; Male ; Oocytes ; physiology ; Ovulation Induction ; Sperm Injections, Intracytoplasmic ; Sperm-Ovum Interactions ; physiology ; Treatment Outcome ; Zona Pellucida ; physiology

The generation of artificial gametes is a real challenge for the scientific community today. In vitro development of human eggs and sperm will pave the way for the understanding of the complex process of human gametogenesis and will provide with human gametes for the study of infertility and the onset of some inherited disorders. However, the great promise of artificial gametes resides in their future application on reproductive treatments for all these people wishing to have genetically related children and for which gamete donation is now their unique option of parenthood. This is the case of infertile patients devoid of suitable gametes, same sex couples, singles and those fertile couples in a high risk of transmitting serious diseases to their progeny. In the search of the best method to obtain artificial gametes, many researchers have successfully obtained human germ cell-like cells from stem cells at different stages of differentiation. In the near future, this field will evolve to new methods providing not only viable but also functional and safe artificial germ cells. These artificial sperm and eggs should be able to recapitulate all the genetic and epigenetic processes needed for the correct gametogenesis, fertilization and embryogenesis leading to the birth of a healthy and fertile newborn.
Child ; Eggs ; Embryonic Development ; Epigenesis, Genetic ; Family Characteristics ; Female ; Fertilization ; Gametogenesis ; Germ Cells* ; Humans ; Infant, Newborn ; Infertility ; Ovum ; Parturition ; Pluripotent Stem Cells ; Pregnancy ; Spermatozoa ; Stem Cells*