Fibroblast growth factor 16 stimulates proliferation but blocks differentiation of rat stem Leydig cells during regeneration.

OBJECTIVES: We aim to investigate the effects of fibroblast growth factor 16 (FGF16) on Leydig cell regeneration in ethane dimethane sulphonate (EDS)-treated rat testis. METHODS: We intraperitoneally inject 75 mg/kg EDS to adult male Sprague Dawley rats and then intratesticularly inject FGF16 (0, 10 and 100 ng/testis/day) from post-EDS day 14 for 14 days. We investigate serum hormone levels, Leydig cell number, gene and protein expression in vivo. We also explore the effects of FGF16 treatment on stem Leydig cell proliferation in vitro. RESULTS: FGF16 lowers serum testosterone levels (21.6% of the control at a dose of 100 ng/testis) without affecting the levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) on post-EDS day 28 in vivo. FGF16 increases Leydig cell number at doses of 10 and 100 ng/mg without affecting Sertoli cell number, increases the percentage of PCNA-positive Leydig cells, and down-regulates the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1 and Hsd17b3) and Sertoli cell genes (Fshr, Dhh and Sox9) and their proteins in vivo. FGF16 increases phosphorylation of AKT1 and AKT2 as well as EKR1/2 in vivo, indicating that it possibly acts via AKT1/ATK2 and ERK1/2 pathways. FGF16 also lowers medium testosterone levels and down-regulates the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1 and Hsd17b3) but increases EdU incorporation into stem Leydig cells in vitro. CONCLUSIONS: These data suggest that FGF16 stimulates stem and progenitor Leydig cell proliferation but blocks their differentiation, thus lowering testosterone biosynthesis.

In vitro study evaluating the instantaneous treatment of ozonised olive oil on human sperm.

OBJECTIVE: The aim of our study was to evaluate the effects of ozonised olive oil (OOO) on human sperm in vitro. METHODS: Human sperm was incubated with OOO for 20 s in vitro. The lowest concentration that completely immobilised all the sperm in 20 s without subsequent recovery of motility was recorded as the minimum effective concentration (MEC). The effects of OOO at MEC on human sperm viability, mitochondrial and acrosomal status, DNA integrity and transmission electron microscopy were observed. RESULTS: Our findings demonstrate that OOO dose-dependently inhibits sperm motility. The MEC of OOO for 100% sperm immobilisation in 20 s was 6 µg/ml. Further experiments showed that sperm ultrastructure, function and DNA integrity were significantly affected after treatment with 6 µg/ml OOO in vitro. CONCLUSIONS: OOO has spermicidal potential and may be explored as a promising vaginal contraceptive agent.

Lonidamine-ethyl ester-mediated remodelling of the Sertoli cell cytoskeleton induces phosphorylation of plakoglobin and promotes its interaction with α-catenin at the blood-testis barrier.

Several compounds affect male fertility by disrupting the adhesion of germ cells to Sertoli cells, which results in the release of undeveloped germ cells into the seminiferous tubule lumen that are incapable of fertilising the ovum. Indazole carboxylic acids are one class of compounds exhibiting such effects and they have been investigated as non-hormonal contraceptives for potential human use. The aims of this study were to investigate the effects of lonidamine-ethyl ester, an indazole carboxylic acid, on spermatogenesis and cell junctions, in particular, desmosomes. We found two doses of lonidamine-ethyl ester at 50mg kg-1 to disrupt Sertoli-germ cell adhesion. By light and fluorescent microscopy, pronounced changes were observed in the distribution of actin microfilaments and intermediate filaments, as well as in the localisation of plakoglobin, a protein with structural and signalling roles at the desmosome and adherens junction at the blood-testis barrier. Furthermore, immunoblotting and immunoprecipitation experiments using testis lysates revealed a significant upregulation (P<0.01) of plakoglobin and Tyr-phosphorylated plakoglobin. Co-immunoprecipitation experiments showed an increase in the interaction between plakoglobin and fyn proto-oncogene, an Src family non-receptor tyrosine kinase, after treatment, as well as an increase in the interaction between plakoglobin and α-catenin. Taken collectively, these data indicate that a disruption of Sertoli cell and spermatocyte-spermatid adhesion in the seminiferous epithelium by lonidamine-ethyl ester results in the phosphorylation of plakoglobin, thereby promoting its interaction with α-catenin at the blood-testis barrier.

The Adverse Effects of Triptolide on the Reproductive System of Caenorhabditis elegans: Oogenesis Impairment and Decreased Oocyte Quality.

Previous studies have revealed that Triptolide damages female reproductive capacity, but the mechanism is unclear. In this study, we used Caenorhabditis elegans to investigate the effects of Triptolide on the germline and explore its possible mechanisms. Our data show that exposure for 4 h to 50 and 100 mg/L Triptolide reduced C. elegans fertility, led to depletion and inactivation of spermatids with the changes in the expression levels of related genes, and increased the number of unfertilized oocytes through damaging chromosomes and DNA damage repair mechanisms. After 24 and 48 h of the 4 h exposure to 50 and 100 mg/L Triptolide, we observed shrink in distal tip cells, an increase in the number of apoptotic cells, a decrease in the number of mitotic germ cells and oocytes in diakinesis stage, and chromatin aggregates in -1 oocytes. Moreover, expression patterns of the genes associated with mitotic germ cell proliferation, apoptosis, and oocyte quality were altered after Triptolide exposure. Therefore, Triptolide may damage fertility of nematodes by hampering the development of oocytes at different developmental stages. Alterations in the expression patterns of genes involved in oocyte development may explain the corresponding changes in oocyte development in nematodes exposed to Triptolide.

[IMMUNOCYTOCHEMICAL ANALYSIS OF THE DISTURBANCES IN THE STRUCTURE OF SYNAPTONEMAL COMPLEXES IN SPERMATOCYTE NUCLEI IN MICE UNDER EXPOSURE TO ROCKET FUEL COMPONENT].

There was performed an assessment of genotoxic effects of rocket fuel component--unsymmetrical dimethylhydrazine (UDMH, heptyl)--on forming germ cells of male mice. Immunocytochemically there was studied the structure of meiotic nuclei at different times after the intraperitoneal administration of UDMH to male mice. There were revealed following types of disturbances of the structure of synaptonemal complexes (SCs) of meiotic chromosomes: single and multiple fragments of SCs associations of autosomes with a sex bivalent, atypical structure of the SCs with a frequency higher than the reference level. In addition, there were found the premature desinapsis of sex bivalents, the disorder offormation of the genital corpuscle and ring SCs. Established disorders in SCs of spermatocytes, analyzed at 38th day after the 10-days intoxication of animal by the component of rocket fuel, attest to the risk of permanent persistence of chromosomal abnormalities occurring in the pool of stem cells.

Evaluation of atorvastatin efficacy and toxicity on spermatozoa, accessory glands and gonadal hormones of healthy men: a pilot prospective clinical trial.

BACKGROUND: Recommendations for cardiovascular disease prevention advocate lowering both cholesterol and low-density lipoprotein cholesterol systemic levels, notably by statin intake. However, statins are the subject of questions concerning their impact on male fertility. This study aimed to evaluate, by a prospective pilot assay, the efficacy and the toxicity of a decrease of cholesterol blood levels, induced by atorvastatin on semen quality and sexual hormone levels of healthy, normocholesterolaemic and normozoospermic men. METHODS: Atorvastatin (10 mg daily) was administrated orally during 5 months to 17 men with normal plasma lipid and standard semen parameters. Spermatozoa parameters, accessory gland markers, semen lipid levels and blood levels of gonadal hormones were assayed before statin intake, during the treatment, and 3 months after its withdrawal. RESULTS: Atorvastatin treatment significantly decreased circulating low-density lipoprotein cholesterol (LDL-C) and total cholesterol concentrations by 42% and 24% (p<0.0001) respectively, and reached the efficacy objective of the protocol. During atorvastatin therapy and/or 3 months after its withdrawal numerous semen parameters were significantly modified, such as total number of spermatozoa (-31%, p<0.05), vitality (-9.5%, p<0.05), total motility (+7.5%, p<0.05), morphology (head, neck and midpiece abnormalities, p<0.05), and the kinetics of acrosome reaction (p<0.05). Seminal concentrations of acid phosphatases (p<0.01), α-glucosidase (p<0.05) and L-carnitine (p<0.05) were also decreased during the therapy, indicating an alteration of prostatic and epididymal functions. Moreover, we measured at least one altered semen parameter in 35% of the subjects during atorvastatin treatment, and in 65% of the subjects after withdrawal, which led us to consider that atorvastatin is unsafe in the context of our study. CONCLUSIONS: Our results show for the first time that atorvastatin significantly affects the sperm parameters and the seminal fluid composition of healthy men.

Histomorphological and biochemical changes induced by triptolide treatment in male lesser bandicoot rat, Bandicota bengalensis.

Mature and healthy male lesser bandicoot rats, Bandicota bengalensis (n = 40) were fed on bait (mixture of cracked wheat and powdered sugar in 98:2) containing different concentrations of triptolide (0, 0.15, 0.20 and 0.25% w/w) for 15 days in two-choice trials. Results revealed no significant effect of triptolide treatment on weights of vital organs after 30 and 60 days of treatment withdrawal. A significant (P ≤ 0.05) increase in plasma levels of TP, ALP, ACP, ALT and AST in response to stress induced in groups of rats treated with 0.20 and 0.25% triptolide was observed after 30 days of treatment withdrawal. No significant effect of treatment was observed on histomorphology of liver. A significant (P ≤ 0.05) effect of triptolide treatment was, however, observed on testicular function in the form of reduced diameter of seminiferous tubules and number of various spermatogenic cells indicating effect on spermatogenesis and spermiogenesis. The cell stages affected did not recover fully within 60 days period following treatment withdrawal. The present study suggests the potential of triptolide in the reproductive management of B. bengalensis by way of affecting testicular function.

Reproductive toxicity of triptolide in male house rat, Rattus rattus.

The aim of study was to investigate the toxic effect of triptolide fed in bait on reproduction of male house rat, Rattus rattus. Feeding of cereal based bait containing 0.2% triptolide to male R. rattus for 5 days in no-choice feeding test, leading to mean daily ingestion of 20.45 mg/kg bw of triptolide, was found effective in significantly (P ≤ 0.05) reducing sperm motility and viability in cauda epididymal fluid by 80.65 and 75.14%, respectively, from that of untreated rats. Pregnancy rates were decreased by 100% in untreated cyclic female rats paired with male rats treated with 0.2% triptolide. Present studies suggest the potential of 0.2% triptolide bait in regulating reproductive output of R. rattus.

Effect of aqueous leaf extract of Dalbergia sissoo Roxb. on spermatogenesis and fertility in male mice.

OBJECTIVES: Antifertility effects of Dalbergia sissoo in male mice were investigated. METHODS: Adult Parkes strain male mice were orally administered aqueous leaf extract of Dalbergia sissoo (50 and 100 mg/kg body weight/day) or distilled water or no treatment (controls) for 35 days (n = 5/group). Motility, viability and number of spermatozoa in the cauda epididymidis; testis histology; serum level of testosterone; and toxicological parameters were evaluated. To assess reversibility, more mice were treated with 100 mg/kg body weight of Dalbergia sissoo or distilled water (n = 5/group) for 35 days and sacrificed 56 days later. Fertility was also assessed separately. RESULTS: Histologically, testes of Dalbergia-treated mice showed dissimilar degenerative changes in the seminiferous tubules. Significant reductions were noted (i) in epididymal sperm motility, viability and number, and (ii) in serum level of testosterone in Dalbergia-treated mice compared to controls. However, serum levels of alanine aminotransferase, aspartate aminotransferase and creatinine, and haematological parameters were not affected. Also libido of Dalbergia-treated males showed no change, but their fertility was markedly suppressed. By 56 days of treatment withdrawal, alterations induced in the above parameters returned to control levels. CONCLUSIONS: Dalbergia sissoo treatment caused reversible suppression of spermatogenesis and fertility in P mice, without eliciting detectable toxic effects.

Experimental endocrine manipulation by contraceptive regimen in the male marmoset (Callithrix jacchus).

Marmosets are used as preclinical model in reproductive research. In contrast to other primates, they display short gestation times rendering this species valid for exploration of effects on fertility. However, their peculiar endocrine regulation differs from a those of macaques and humans. We subjected male marmosets to previously clinically tested hormonal regimens that are known to effectively suppress spermatogenesis. Beside a control group, seven groups (each n=6) were investigated for different periods of up to 42 months: regimen I, (four groups) received testosterone undecanoate (TU) and norethisterone enanthate (NETE); regimen II, (two groups) received TU and NETE followed by NETE only; and regimen III, (one group) received NETE only. Testicular volume, cell ploidy and histology, endocrine changes and fertility were monitored weekly. TU and NETE and initial TU and NETE treatment followed by NETE failed to suppress spermatogenesis and fertility. Testicular volumes dropped, although spermatogenesis was only mildly affected; however, testicular cellular composition remained stable. Serum testosterone dropped when NETE was given alone but the animals remained fertile. Compared with controls, no significant changes were observed in sperm motility and fertility. Administration of TU and NETE affected testicular function only mildly, indicating that the regulatory role of chorionic gonadotrophin and testosterone on spermatogenesis is obviously limited and testicular function is maintained, although the endocrine axis is affected by the treatment. In conclusion, marmosets showed a different response to regimens of male contraception from macaques or men and have to be considered as a problematic model for preclinical trials of male hormonal contraception.

Emerging approaches to male contraception.

Despite significant interests in contraception by men, effective methods of male contraception are limited to vasectomy and condoms. Recently, there have been several promising advances in male contraceptive research. This review will update readers on recent research in both hormonal and nonhormonal approaches to male contraception. Hormonal approaches to male contraception have been stymied by adverse effects, formulations requiring injections or implants, a 5% to10% nonresponse rate, as well as poor understanding of user acceptability. In the last several years, research has focused on novel, orally bioavailable androgens such as dimethandrolone undecanoate and 11ß-methyl-19-nor-testosterone. Additionally, combinations of a topical testosterone gel combined with a gel containing segesterone acetate, a potent progestin, have shown promise in clinical trials recently. Simultaneously, significant preclinical progress has been made in several approaches to nonhormonal male contraceptives, including compounds that inhibit sperm motility such as eppin, compounds that inhibit retinoic acid binding or biosynthesis, and reversible approaches to obstruction of the vas deferens. It is imperative for these areas of research to continue making strides so that there is a gamut of contraceptive options for couples to choose from. Some of these approaches will hopefully reach clinical utility soon, greatly improving contraceptive choice for couples.

[Anti-fertility effect of Tongbi composition and its reversibility in male rats].

OBJECTIVE: To study the anti-fertility effect of maximum-dose Tongbi Composition and its reversibility in male rats. METHODS: Thirty-six male SD rats were equally randomized into a control group and a medication group, the former given normal saline at 10 ml/(kg x d), while the latter treated with Tongbi Composition at 10 g/(kg x d), both for 60 days. Half the rats of each group were sacrificed randomly at the cessation of treatment, and the rest killed at 72 days after it. The relative testis weight, testis volume, sperm concentration and sperm motility were measured, and the pathological changes in the testicular tissue observed under the optical microscope. RESULTS: After 60 days of treatment, no statistically significant differences were found between the two groups in the relative testis weight, testis volume and sperm concentration (P > 0.05) , and the sperm motility of the medication group dropped to zero, but it was restored to normal at 72 days after drug withdrawal. Almost no lesions were observed in the testis tissue of the medication group. CONCLUSION: The short-term use of Tongbi Composition at the maximum clinical dose has an obvious anti-fertility effect, but it is reversible.

Recrudescence of spermatogenesis in the dog following downregulation using a slow release GnRH agonist implant.

The present study examined the degree to which downregulation with a GnRH agonist impaired spermatogenesis and the time course of morphological and hormonal changes that occurred during recrudescence of spermatogenesis. Using a control group (group 1, n = 5) of dogs, the effect of a removable slow release GnRH-agonist implant was investigated in beagle dogs (group 2, n = 30). The implant was removed after 5 months (week 0) and three to four dogs were castrated at weeks 0, 3, 6, 9, 12, 15, 18, 21 and 24. The degree of downregulation and recrudescence of spermatogenesis was assessed by evaluation of 200 tubular cross-sections, resulting in an assigning of dogs of group 2 to testis developmental groups (DG) according to the most developed germ cell observed: DG A, spermatocytes; DG B, round spermatids; DG C, elongating spermatids and DG D, elongated spermatids. Downregulation led to an arrest of spermatogenesis at the level of spermatogonia/primary spermatocytes. The time course of recrudescence showed high individual variations and the number of dogs falling into DG A, B, C and D was 4, 3, 6 and 17 respectively. Spermatogenesis in group 2, DG D was not different from group 1 (control). In DG A, mean area of Leydig-cell nuclei was lower (p < 0.001) than in the other DG and group 1 and resembled that of juvenile dogs (group 3, n = 3); nuclei of Sertoli cells had changed from more flat/polygonal (group 1, group 2, DG C and D) to round/ovoid and had moved to a more luminal position. As indicated by basal testosterone (T), luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentrations at implant removal, full downregulation had been obtained. Testosterone, LH and FSH concentrations [X(g) (DF), ng/ml] increased (p < 0.05) from implant removal to DG B [T: 0.1 (1.24) vs 2.12 (2.31); LH: 0.2 (2.15) vs 1.11 (1.7); FSH: 0.37 (3.50) vs 6.37 (1.68)] and were more or less constant thereafter indicating that onset of spermatogenesis was related to an increase of plasma T occurring in a very narrow time window. Following GnRH implantation, the size of the testes and the prostate decreased by approximately 55% (p < 0.001), they increased to sizes similar to pre-treatment values following implant removal.

Ginsenoside Rg3 protects mouse leydig cells against triptolide by downregulation of miR-26a.

BACKGROUND: Ginsenoside Rg3 has been reported to exert protection function on germ cells. However, the mechanisms by which Rg3 regulates apoptosis in mouse Leydig cells remain unclear. In addition, triptolide (TP) has been reported to induce infertility in male rats. Thus, this study aimed to investigate the protective effect of Rg3 against TP-induced toxicity in MLTC-1 cells. METHODS: CCK-8, immunofluorescence assay, Western blotting and flow cytometry were used to detect cell proliferation and cell apoptosis, respectively. In addition, the dual luciferase reporter system assay was used to detect the interaction between miR-26a and GSK3ß in MLTC-1 cells. RESULTS: TP significantly inhibited the proliferation of MLTC-1 cells, while the inhibitory effect of TP was reversed by Rg3. In addition, TP markedly induced apoptosis in MLTC-1 cells via increasing the expressions of Bax, active caspase 3, Cyto c and active caspase 9, and decreasing the level of Bcl-2. However, Rg3 alleviated TP-induced apoptosis of MLTC-1 cells. Moreover, the level of miR-26a was obviously downregulated by Rg3 treatment. The protective effect of Rg3 against TP-induced toxicity in MLTC-1 cells was abolished by miR-26a upregulation. Meanwhile, dual-luciferase assay showed GSK3ß was the direct target of miR-26a in MLTC-1 cells. Overexpression of miR-26a markedly decreased the level of GSK3ß. As expected, upregulation of miR-26a could abrogate the protective effects of Rg3 against TP-induced cytotoxicity via inhibiting the expression of GSK3ß. CONCLUSION: These results indicated that Rg3 could protect MLTC-1 against TP by downregulation of miR-26a. Therefore, Rg3 might serve as a potential agent for the treatment of male hypogonadism.

Dysregulation of lncRNA and circRNA Expression in Mouse Testes after Exposure to Triptolide.

BACKGROUND: Triptolide has been shown to exert various pharmacological effects on systemic autoimmune diseases and cancers. However, its severe toxicity, especially reproductive toxicity, prevents its widespread clinical use for people with fertility needs. Noncoding RNAs including lncRNAs and circRNAs are novel regulatory molecules that mediate a wide variety of physiological activities; they are crucial for spermatogenesis and their dysregulation might cause male infertility. However, whether they are involved in triptolide-induced reproductive toxicity is completely unknown. METHODS: After exposure of mice to triptolide, the total RNAs were used to investigate lncRNA/circRNA/mRNA expression profiles by strand-specific RNA sequencing at the transcriptome level to help uncover RNA-related mechanisms in triptolide-induced toxicity. RESULTS: Triptolide significantly decreased testicular weight, damaged testis and sperm morphology, and reduced sperm motility and density. Remarkable deformities in sperm head and tail were also found in triptolide-exposed mice. At the transcriptome level, the triptolide-treated mice exhibited aberrant expression profiles of lncRNAs/circRNAs/mRNAs. Gene Ontology and pathway analyses revealed that the functions of the differentially expressed lncRNA targets, circRNA cognate genes, and mRNAs were closely linked to many processes involved in spermatogenesis. In addition, some lncRNAs/circRNAs were greatly upregulated or inducibly expressed, implying their potential value as candidate markers for triptolide-induced male reproductive toxicity. CONCLUSION: This study provides a preliminary database of triptolide-induced transcriptome, promotes understanding of the reproductive toxicity of triptolide, and highlights the need for research on increasing the medical efficacy of triptolide and decreasing its toxicity.

Effect of germ cell depletion on levels of specific mRNA transcripts in mouse Sertoli cells and Leydig cells.

It has been shown that testicular germ cell development is critically dependent upon somatic cell activity but, conversely, the extent to which germ cells normally regulate somatic cell function is less clear. This study was designed, therefore, to examine the effect of germ cell depletion on Sertoli cell and Leydig cell transcript levels. Mice were treated with busulphan to deplete the germ cell population and levels of mRNA transcripts encoding 26 Sertoli cell-specific proteins and 6 Leydig cell proteins were measured by real-time PCR up to 50 days after treatment. Spermatogonia were lost from the testis between 5 and 10 days after treatment, while spermatocytes were depleted after 10 days and spermatids after 20 days. By 30 days after treatment, most tubules were devoid of germ cells. Circulating FSH and intratesticular testosterone were not significantly affected by treatment. Of the 26 Sertoli cell markers tested, 13 showed no change in transcript levels after busulphan treatment, 2 showed decreased levels, 9 showed increased levels and 2 showed a biphasic response. In 60% of cases, changes in transcript levels occurred after the loss of the spermatids. Levels of mRNA transcripts encoding Leydig cell-specific products related to steroidogenesis were unaffected by treatment. Results indicate (1) that germ cells play a major and widespread role in the regulation of Sertoli cell activity, (2) most changes in transcript levels are associated with the loss of spermatids and (3) Leydig cell steroidogenesis is largely unaffected by germ cell ablation.

Leydig cell re-generation and expression of cell signaling molecules in the germ cell-free testis.

Leydig cells in the rat testis can be specifically ablated with ethane dimethane sulfonate (EDS) and will subsequently re-generate. In this study, we have characterized Leydig cell re-generation and expression of selected cell-signaling molecules in a germ cell-free model of EDS action. This model offers the advantage that re-generation occurs on a stable background without confounding changes from the regressing and repopulating germ cell population. Adult rats were treated with busulfan to remove the germ cell population and Leydig cells were then ablated with EDS. Testicular testosterone levels declined markedly within 24 h of EDS treatment and started to recover after 8 days. After EDS treatment there were marked declines in levels of Leydig cell-specific mRNA transcripts coding for steroidogenic enzymes cytochrome P450 11a1 (Cyp11a1), cytochrome P450 17a1 (Cyp17a1), 3beta-hydroxysteroid dehydrogenase type 1 (Hsd3b1), 17beta-hydroxysteroid dehydrogenase type 3 (Hsd17b3) and the LH receptor. Levels of all transcripts recovered within 20 days of EDS treatment apart from Hsd17b3, which remained undetectable up to 20 days. Immunohistochemical localization of CYP11A1 during the phase of early Leydig cell re-generation showed that the Leydig cell precursors are spindle-shaped peritubular cells. Studies on factors which may be involved in Leydig cell re-generation showed there were significant but transient increases in platelet-derived growth factor A (Pdgfa), leukemia inhibitory factor (Lif), and neurofilament heavy polypeptide (Nefh) after EDS, while desert hedgehog (Dhh) levels declined sharply but recovered by 3 days. This study shows that the Leydig cell precursors are peritubular cells and that expression of Pdgfa and Lif is increased at the start of the re-generation process when precursor proliferation is likely to be taking place.

Rat testicular germ cell type(s) targeted by anti-spermatogenic agents in vivo and their recovery on withdrawal of treatment--a flow cytometric study.

Spermatogenesis goes through very critically and precisely balanced ratios of germ cells with diverse DNA ploidies (1C, 2C and 4C). Antispermatogenic agents that reversibly interrupt spermatogenesis may have a contraceptive relevance. With a view to study the precise mechanism of action of antispermatogenic agents and identify the germ cell type(s) targeted by various agents in vivo, spermatogenic cells with diverse DNA ploidies were measured in rat testis during treatment and recovery with compounds CDRI-84/35, gossypol and estradiol, using Flow Cytometry. Rats were treated with either CDRI-84/35 (100mg/(kg day) for 15 days followed by 25mg/(kg day) for 55 days) or gossypol (20mg/(kg day) for 70 days) or estradiol benzoate (2.5microg/(rat day) for 70 days) and 3 rats from each group were sacrificed after 22, 41, 53 and 70 days of treatment to monitor the changes in population of 1C, 2C, S-phase and 4C germ cell types. Treatment with CDRI-84/35 resulted in a significant and rapid drop in 1C population with a concomitant and parallel rise in 2C population. In gossypol-treated animals 1C peak disappeared gradually and the arrest was seen predominantly at 2C stage and partially at 4C stage. At the end of the treatment most of the germ cells were arrested at 2C stage. Estradiol affected spermatogenesis differently with 1C population falling in complement to rise in both 2C and 4C peaks. Germ cells were mainly arrested at the 4C stage after the treatment. The data suggest that germ cells fail to enter meiosis in CDRI-84/35-treated rats. Few cells entering meiosis do not complete the cell division and remain arrested at 4C stage. However in case of estradiol and gossypol the meiotic 4C cells become incapable of further differentiation into haploid cells. After receiving 70 days of treatment a few rats were allowed to recover for 60, 90 and 120 days. The population of various germ cell types in the testis of recovery-group animals indicated that spermatogenesis resumes substantially in case of estradiol treatment and partially in case of treatment with the other two agents.

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.