The Effects of Endosulfan on the Testes of Bluegill Fish, Lepomis macrochirus: A Histopathological Study

The effect of endosulfan, an Organochlorine pesticide, on bluegill testes was studied. Endosulfan is aqua-toxic and has an immediate effect on fish and other aquatic life. In this experiment, we exposed the fish for 24-, 48-, 72-, 96-h, and 1- and 2-week periods. A second group of fish without exposure to endosulfan served as the control. The control testis appeared structurally normal. The seminiferous tubules were of round or oval shape and contained primary spermatogonia, primary spermatocytes, secondary spermatocytes, spermatozoa, spermatids, Sertoli cells, and interstitial cells of Leydig. Within the connective tissue that connected the seminiferous tubules were Leydig cells. After 24 h of exposure, there was evidence of slight signs of connective tissue splintering. The 48-h exposure resulted in breakage of primary spermatocyte walls and separation from the seminiferous tubules. The 72-h testis showed further connective tissue damage and migration of primary spermatogonia into the lumen. After 96 h, there was significant damage to connective tissue and the seminiferous tubules were less pronounced. After 1 and 2 weeks, the seminiferous tubule walls were disrupted and missing in places and the structure of the testis was very disorganized compared to the control testis. Biometric analysis indicated that the diameter of the primary spermatogonia decreased from 24 h to two weeks. There also appeared to be fewer Leydig cells, responsible for testosterone production, over the exposure period and damaged Sertoli cells, which support, protect, and nourish the spermatogonic cells, synthesize ABP, and assist in maintaining the necessary concentration of testosterone in the seminiferous tubules so that spermatogenesis can progress. These kinds of damage could affect the spermatids and spermatozoa and possibly have a negative impact on spermatogenesis and male fertility, affecting bluegill fish population.     

Role of ghrelin on testosterone secretion and the mRNA expression of androgen receptors in adult rat testis

The present study was designed to determine the effects of ghrelin on in vivo and in vitro secretion of testosterone (T) and the expression of androgen receptor (AR) mRNA in the adult rat testis. The distribution of growth hormone secretagogue receptors (GHS-R(1a)) in the testis was also investigated. GHS-R(1a) immunoreactivity presented mainly in Sertoli and Leydig cells, primary spermatocytes, and secondary spermatocytes. Adult rats that were intracerebroventricularly (i.c.v.) administrated different dosages (1 nmol and 3 nmol) of ghrelin could significantly inhibit the secretion of T. The experession of AR mRNA in the testis was also notably reduced with 3 nmol ghrelin. Additionaly, in vitro exposure of the Leydig cells to increasing concentrations of ghrelin resulted in no obvious changes of T secretion in the culture media and AR mRNA expression of Leydig cells. Overall, our data demonstrate that the i.c.v. injection of ghrelin plays a physiological role in T secretion and AR mRNA expression in the testis, further confirming the reproductive role of ghrelin.     

Role of ghrelin on testosterone secretion and the mRNA expression of androgen receptors in adult rat testis

The present study was designed to determine the effects of ghrelin on in vivo and in vitro secretion of testosterone (T) and the expression of androgen receptor (AR) mRNA in the adult rat testis. The distribution of growth hormone secretagogue receptors (GHS-R_1a) in the testis was also investigated. GHS-R_1a immunoreactivity presented mainly in Sertoli and Leydig cells, primary spermatocytes, and secondary spermatocytes. Adult rats that were intracerebroventricularly (i.c.v.) administrated different dosages (1 nmol and 3 nmol) of ghrelin could significantly inhibit the secretion of T. The experession of AR mRNA in the testis was also notably reduced with 3 nmol ghrelin. Additionaly, in vitro exposure of the Leydig cells to increasing concentrations of ghrelin resulted in no obvious changes of T secretion in the culture media and AR mRNA expression of Leydig cells. Overall, our data demonstrate that the i.c.v. injection of ghrelin plays a physiological role in T secretion and AR mRNA expression in the testis, further confirming the reproductive role of ghrelin.     

CONTROL OF SPERMATOGENESIS IN PRIMATE AND PROSPECT OF MALE CONTRACEPTION

The present review is a summary of mechanisms of spermatogenesis in primates with emphasis on anti-spermatogenesis of testosterone (T), gossypol, and “testicular heat stress” for development of male contraception. Both FSH and testosterone stimulate all phases of spermatogenesis. FSH is capable of amplifying the population of the differentiated spermatogonia (B1, B2, B3 and B4) and controls the spermatogonia production rate, and, in synergy with testosterone, regulating spermatogenesis in adult monkeys. Pituitary FSH beta gene expression is governed by a feedback of Beta inhibin, which is a major component of the testicular negative feedback signals. Beta inhibin secreted by Sertoli cells is in turn inhibited by testosterone from Leydig cells under the control of LH. Disturbance of the normal interaction of pituitary FSH with Sertoli cell Beta inhibin is responsible for azoospermia or oligozoospermia induced by exogenous T. Three possible regimens of T, gossypol and “heat stress” have been suggested for male contraception. They act on different sites and stages of spermatogenesis in testis or sperm activity in epididymis. Apoptosis induced by testosterone occurs mainly at stages VII–VIII of spermatogenesis while that by testicular “heat stress” mostly occurs at stages I–IV and X–XII. Low dose of gossypol mainly influences the sperm activity in the epididymis although it also acts on testicular spermatids.     

SET在小鼠睾丸组织中的表达

目的：检测不同年龄段小鼠睾丸组织中SET蛋白和mRNA的表达,讨论其在精子发生及睾酮生成方面的潜在功能。方法：实验中用1周龄的ICR雄性小鼠作为幼年期组（相当于人类幼年期,n=12）,4周龄的ICR雄性小鼠作为性发育期组（相当于人类的青春期前,n=12）,12周龄的ICR雄性小鼠作为成年期组（相当于人类成年期,n=12）,12个月龄的ICR雄性小鼠作为中老年组（相当于人类的中老年,n=12）。免疫组织化学法观察SET在各年龄段小鼠的定位表达;实时逆转录聚合酶链反应（real-time RT-PCR）和蛋白质印迹（Western Blot）分别检测睾丸中SET mRNA和蛋白水平表达。结果：SET蛋白表达定位于生精小管中的精原细胞、精母细胞,在性发育期组和成年期组的单倍体和四倍体生殖细胞中高表达;成年期组和中老年组的睾丸间质细胞中也有SET的表达;支持细胞中很少量表达。性发育期组SET mRNA与幼年期组相比表达量升高（P=0.020）,而成年期组小鼠睾丸中SET蛋白的表达水平最高。结论：SET主要表达于精原细胞和精母细胞,少量表达于支持细胞,表明SET可能与精子发生有关。SET还表达于睾丸间质细胞,与睾酮生成有关。     

Immunocytochemical expression of p75LNGFR and trkA in Leydig cells of the human testis

The authors demonstrate for the first time the immunocytochemical expression of the low-affinity neurotrophic growth factor (NGF) receptor p75LNGFR and the high-affinity NGF receptor trkA in the human testis. Employing the polyclonal anti-p75LNGFR antibody and the polyclonal anti-trkA antibody they report strong immunoreactivity for those antigens in the interstitial Leydig cells. Sertoli cells and some cellular elements of the germinative epithelium were also trkA-positive. These findings implicate the neurotrophic factor NGF and its receptors p75LNGFR and trkA in the autocrine and paracrine regulation of the steroidogenic activity of Leydig cells of the human testis.     

INSLF3-LGR8 ligand-receptor system in testes of mature rats after exposure to ethane dimethanesulphonate (short communication)

The cytotoxic agent ethane-1,2-dimethanesulphonate (EDS) specifically destroys the Leydig cells (LC) in the adult testis, followed by a complete regeneration. The process of LC renewal after exposure to EDS shows homology to the development of the adult-type LC population in prepubertal testis. INSL3, also known as Leydig insulin-like peptide or relaxin-like factor, is a peptide hormone, a novel member of the insulin/relaxin family, and seems to be localized predominantly in the gonadal tissues. INSL3 mRNA is expressed in the LC in a constitutive fashion and INSL3 thus seems to be a useful marker of LC differentiation status. The present study was aimed at establishing the chronology and dynamic of expression of INSL3 and its specific receptor LGR8 in the LC repopulation after exposure to mature rats to EDS. As material, testes of mature Wistar rats that received single intraperitoneal injection of EDS (75 mg/kg body weight) were used. The animals were killed 1, 7, 14, 21 and 35 days after the initial treatment. The pattern of INSL3-LGR8 expression in newly formed LC after EDS administration was established using a high sensitive immunohistochemical polymer detection kit. After treatment with EDS, the immunoreactivity for INSL3 and LGR8 disappeared from the testis and reappeared again at the time of regeneration of the first LC, 14 days after EDS. The INSL3-LGR8 positive cells grew in number concomitantly with the increase of the LC repopulation. Thirty-five days after EDS destruction a larger number of immunopositive LC were seen in form of clusters corresponding with the regeneration of adult type LC population. The present findings support the hypothesis that EDS-treated rats can serve as a model for studying the LC development in the prepubertal testis and indicate a specific role of hormonal factors like INSL3 in this process.     

Role of the Y-located putative gonadoblastoma gene in human spermatogenesis

The gonadoblastoma locus on the human Y chromosome (GBY) is postulated to serve normal functions in spermatogenesis, but could exert oncogenic properties in predisposing susceptible germ cells to tumorigenesis in incompatible niches such as streaked gonads in XY sex reversed patients or dysfunctional testis in males. The testis-specific protein Y-linked (TSPY) repeat gene has recently been demonstrated to be the putative gene for GBY, based on its location on the GBY critical region, expression patterns in early and late stages of gonadoblastoma and ability to induce gonadoblastoma-like structures in the ovaries of transgenic female mice. Over-expression of TSPY accelerates G(2)/M progression in the cell cycle by enhancing the mitotic cyclin B-CDK1 kinase activities. Currently the normal functions of TSPY in spermatogenesis are uncertain. Expression studies of TSPY, and its X-homologue, TSPX, in normal human testis suggest that TSPY is co-expressed with cyclin B1 in spermatogonia and various stages of spermatocytes while TSPX is principally expressed in Sertoli cells in the human testis. The co-expression pattern of TSPY and cyclin B1 in spermatogonia and spermatocytes suggest respectively that 1) TSPY is important for male spermatogonial cell replication and renewal in the testis; and 2) TSPY could be a catalyst/meiotic factor essential for augmenting the activities of cyclin B-cyclin dependent kinases, important for the differentiation of the spermatocytes in prophase I and in preparation for consecutive rounds of meiotic divisions without an intermediate interphase during spermatogenesis.     

EXPRESSION OF CLAUDIN-1 IN MOUSE TESTIS

In testis, tight junctions (TJs) between adjacent Sertoli cells are important for the formation of the blood-testis barrier (BTB) and crucial for spermatogenesis. The present study aimed to find postnatal changes in the expression of claudin-1, one of the TJ genes in mouse testis. By semiquantitative RT-PCR, it was found that claudin-1 expression in testis increased up to a peak at 10 days after birth and decreased thereafter. Western blot analysis showed abundant expression of 21-kDa protein in testis, lung, and brain from the adult mouse. The developmental change in the expression of claudin-1 protein in testis coincided with that from the RT-PCR. Testosterone treatment significantly increased claudin-1 expression in immature Sertoli cells, suggesting the possible regulation of claudin-1 expression by androgen in mouse Sertoli cells. Claudin-1 expression appears to be developmentally regulated in the mouse testis.     

RELATIONSHIP BETWEEN MAST CELL AND INOS EXPRESSION IN TESTICULAR TISSUE ASSOCIATED WITH INFERTILITY

The objective of this study was to investigate mast cells and iNOS expression in testis tissue, and to correlate these results with spermatogenetic disorders. A total of 136 testicular biopsies were obtained from the testes of 80 patients with infertility. Their age ranged from 21 to 45 years. The biopsy specimens were immunohistochemically stained with antihuman tryptase for mast cells. In each section, all interstitial fields were evaluated for the total number of mast cells as well as the total number of Leydig cells. The number of mast cells per Leydig cell was calculated and recorded as mast cell index. Immunohistochemical iNOS staining was evaluated semiquantitatively according to intensity and the proportion of the stained cells. There was a significant increase of the mast cell index in all groups with testicular disorder compared with normal spermatogenesis group (p < 0.05). Increase of the index was in the order of hypospermatogenesis, maturation arrest and SCO, and index of SCO group was especially higher, i.e., more than twice than other groups. iNOS score was significantly higher in the SCO group than in the men with normal spermatogenesis, hypospermatogenesis, and maturation arrest (p < 0.05). Finally, a significant statistical correlation was found between the iNOS score and mast cells index (r = 0,758, p = 0,001). Increase of mast cell index was observed in the groups of infertile testis, and high expression of iNOS in Leydig cells was associated with the highest mast cell index in SCO, the lesion with the most severe damage of the germ cell.     

Aromatase and 11beta-hydroxysteroid dehydrogenase 2 localisation in the testes of pigs from birth to puberty linked to changes of hormone pattern and testicular morphology

Oestrogens and glucocorticoids are important for spermatogenesis and are regulated via aromatase for oestradiol synthesis and 11beta-hydroxysteroid dehydrogenase 2 (11beta-HSD 2) as an inactivator of cortisol. In the present study postnatal changes of these two enzymes were monitored together with testicular development and hormone concentrations. Pigs were assigned to three periods: Weeks 0-5, Weeks 5-11 or Weeks 11-17. In Period 1, groups of four piglets were killed after each week. Blood plasma and testes were sampled immediately post mortem. For Periods 2 and 3, groups of six pigs were fitted with vein catheters for daily blood collection. Testes from all pigs were obtained after killing. Levels of testosterone, oestradiol, LH, FSH and cortisol were determined radioimmunologically. The 11beta-HSD 2- and aromatase-expressing cells were stained immunocytochemically. All hormones were maximal 2 weeks after birth. A rise of LH, testosterone and oestradiol occurred again at Week 17. FSH and cortisol remained basal. Parallel to the first postnatal rise, the presence of aromatase and 11beta-HSD 2 in Leydig cells increased, together with germ and Sertoli cell numbers. Expression was low from 3 to 5 weeks, was resumed after Week 5 and was maximal at Week 17. The amount of 11beta-HSD 2 in germ cells was greatest at birth, decreased thereafter and was absent after Week 3.     

Effect of fenugreek seeds extract on cyclophosphamide-induced histomorphometrical, ultrastructural and biochemical changes in testes of albino mice

Cyclophosphamide (CPA) is an anticancer drug used in the treatment of a variety of neoplastic lesions. On the other hand, treatment with CPA was accompanied by different toxic effects on different body organs. The present work was conducted to study the effect of fenugreek seed extract on histomorphometrical and ultrastructural changes induced by CPA in testes of albino mice. Twenty animals were given CPA (7.0 mg/kg body weight) three times/week orally for 8 weeks and were killed after 4 and 8 weeks. Testis of CPA-treated mice showed many histological alterations including appearance of irregular seminiferous tubules, reduction in the number of all spermatogenic cells, degeneration of Leydig cells and appearance of intertubular hemorrhage. Concerning the ultrastructural changes, abnormalities in spermatogonia (A and B), spermatocytes, round and elongated spermatids were observed. Degenerated Sertoli cells and degenerated interstitial tissue with abnormal Leydig cells were also seen. Moreover, administration of CPA to animals significantly increased malondialdehyde (MDA, lipid peroxidation marker) and decreased superoxide dismutase (SOD) and catalase (CAT). These changes were time-dependent. Treating animals with CPA and fenugreek seed extract (0.4 g/kg body weight) led to an improvement in the histological and ultrastructural pictures of the testis together with reduction in the level of serum MDA and increase in the activities of serum SOD and CAT. In conclusion, the results of the present work indicated that fenugreek had ameliorative effect against testis damage induced by CPA and this may be mediated by its potent antioxidant activities.     

Role of the Y-located putative gonadoblastoma gene in human spermatogenesis

The gonadoblastoma locus on the human Y chromosome (GBY) is postulated to serve normal functions in spermatogenesis, but could exert oncogenic properties in predisposing susceptible germ cells to tumorigenesis in incompatible niches such as streaked gonads in XY sex reversed patients or dysfunctional testis in males. The testis-specific protein Y-linked (TSPY) repeat gene has recently been demonstrated to be the putative gene for GBY, based on its location on the GBY critical region, expression patterns in early and late stages of gonadoblastoma and ability to induce gonadoblastoma-like structures in the ovaries of transgenic female mice. Over-expression of TSPY accelerates G₂/M progression in the cell cycle by enhancing the mitotic cyclin B-CDK1 kinase activities. Currently the normal functions of TSPY in spermatogenesis are uncertain. Expression studies of TSPY, and its X-homologue, TSPX, in normal human testis suggest that TSPY is co-expressed with cyclin B1 in spermatogonia and various stages of spermatocytes while TSPX is principally expressed in Sertoli cells in the human testis. The co-expression pattern of TSPY and cyclin B1 in spermatogonia and spermatocytes suggest respectively that 1) TSPY is important for male spermatogonial cell replication and renewal in the testis; and 2) TSPY could be a catalyst/meiotic factor essential for augmenting the activities of cyclin B-cyclin dependent kinases, important for the differentiation of the spermatocytes in prophase I and in preparation for consecutive rounds of meiotic divisions without an intermediate interphase during spermatogenesis.     

Ultrastructure of the Testis in Klinefelter's Syndrome

The ultrastructure of the testis in 5 cases of Klinefelter's syndrome was studied. In 3 cases there was complete hyalinization of the seminiferous tubules. In one case Sertoli cells were present, and in another case focal areas of spermatogenesis were seen in the tubules. Hyperplasia of the Leydig cells was found in every case. The Leydig cells had usually abundant smooth endoplasmic reticulum and lipofuscin pigment in the cytoplasm. This suggested that the function of the Leydig cells was normal. The Sertoli cells contained lipid droplets and glycogen-filled vacuoles in the cytoplasm.     

Male germ cell transplantation: promise and problems

Male germ cell transplantation is a novel technique in which donor male stem germ cells are surgically transferred to the seminiferous tubules of a recipient testis by direct injection or via the rete testis or efferent duct. All germ cells that are destined to become stem spermatogonia are defined as male stem germ cells, including primordial germ cells from the gonadal ridges, and gonocytes and stem spermatogonia from the testis, all of which are transplantable and capable of undergoing normal spermatogenesis. Xenotransplantation of male germ cells from one species into the testis of another species, including human testicular cells in the mouse, has so far proved to be unsuccessful. However, the immunodeficient mouse testis can support rat spermatogenesis and produce apparently normal rat spermatozoa. The underlying mechanisms remain elusive. The present mini-review will focus on the importance of stem spermatogonial transplantation for testicular stem cell biology and discuss the likelihood of immune rejection after transplantation, which may limit the success of all male germ cell transplantation.     

Testis functions and sexual potentia in langur monkey treated with a combination steroidal contraceptive formulation

Administration of a combination formulation of danazol (100 mg/day; oral) plus testosterone enanthate (TE) (50 mg/15 days; i.m.) for 30 to 60 days in adult male langur monkeys resulted in the reversible suppression of testicular function without affecting the sexual potentia. Testicular weight and volume decreased significantly along with the mass atrophy of germinal epithelium and impaired morphology of Leydig and Sertoli cells. A conspicuous shrinkage of seminiferous tubules, Leydig cell nuclei and Sertoli cell nuclei was noted. Elevation of testicular cholesterol, total lipids, glycogen and phosphatases activity with the depletion of total proteins, nucleic acid, sialic acid and fructose was noteworthy. All changes were maintained during maintenance dose studies (danazol: 50 mg/day; oral plus TE: 50 mg/15 days; i.m.) for 60 days. Resumption of all measures to normal was evident following 120 days of recovery. It can be concluded that the exogenous TE substitutes the serum testosterone levels to maintain extratesticular androgen actions even after interference by danazol of Leydig cell function along with spermatogenesis inhibition.     

Histopathological and biochemical effects of gossypol acetate on pituitary-gonadal axis of male albino rats.

Histopathological and biochemical effects of gossypol acetate (GA) on pituitary-gonadal axis were investigated. 10 and 25 mg GA/kg were administered orally to sexually mature adult male Wistar rats for 4 and 5 weeks, respectively. STH and LTH/PRL cells showed no significant changes as compared to those of controls while TSH cells showed hypertrophy, hyperplasia and degranulation in both experimental groups. Pituitary FSH, LH/ICSH cells showed progressive regression. Gonosomatic indices of sex accessory glands at 25 mg showed significant reduction in the experimental animals as compared to those of controls. The diameter of seminiferous tubules reduced and azoospermia developed. Sertoli and Leydig cells also regressed. At 10 and 25 mg GA treatment, spermatogenesis ceased at secondary spermatocytes and spermatogonia stages, respectively. Epididymis and prostate regressed. Seminal vesicle showed no significant histological variations as compared to that of control except reduction in secretion. Biochemical observations revealed increased levels of acid phosphatase, fructose and citric acid and significant reduction in glycerylphosphoryl choline in reproductive glands of both experimental groups as compared to those of controls. Possible mechanism of antifertility action of GA is discussed.     

Immunolocalization of neurotrophic factors and their receptors in the Leydig cells of rat during postnatal development

The aim of the present study was to localize the neurotrophic factors glial cell line-derived neurotrophic factor (GDNF), nerve growth factor receptor (NGFr) and low sensitive receptor for NGF (p75LNGFR) in rat testes during the postnatal development. Rat testes obtained at different stages of the postnatal development--day 5, 10, 15, 20, 24 and 27 after birth were used. Amplification immunocytochemical technique, which includes a combination of the peroxidase-antiperoxidase method (PAP) and the avidin-biotin peroxidase complex (ABC) methods, was applied. Immunoreactivity for the tested antigens was established in the Leydig cell with characteristic fluctuation in the intensity of the immune reaction at the different stages of the postnatal development. Positive immunostaining was seen in the Sertoli and some of the germ cells (spermatocytes and spermatids). The results obtained show that neurotrophic factors play a role in the processes of postnatal differentiation of the Leydig cells and in the regulation of their functional activity.