Effects of various doses of testosterone propionate on intratesticular and plasma testosterone levels and maintenance of spermatogenesis in adult hypophysectomized rats.

Adult hypophysectomized rats were maintained on different regimens of testosterone propionate (TP) treatment for 27 days (0.2, 0.4, 0.6 and 1 mg/day) and autopsied 16 hours after the last injection. Blood samples were taken, sex organs were weighed and one testis from each animal was fixed in Bouins fluid for histologic analysis. The other testis and blood were used for testosterone (T) determinations. Both testicular and plasma T were below detectable levels in hypophysectomized control rats. The plasma T level showed a dose response relationship with increasing dose of TP but such was not the case for intratesticular T concentrations. Qualitative and quantitative evaluation of testis sections showed that spermatogenesis was incomplete in rats receiving 0.2 mg TP/day characterized by the absence of step 15 to 19 spermatids, degeneration of some pachytene spermatocytes and a significantly lower yield of B type spermatogonia. Analysis of testis sections from animals treated with 0.4 to 1 mg TP/day showed complete maintenance and maturation of pachytene spermatocytes, meiosis and spermiogenesis. However, even with the highest dose of TP (1 mg/day) the total yield of B type spermatogonia was only about 58% of the intact controls. It is concluded that at least 0.4 mg/day of exogenous TP is essential for qualitative maintenance of spermatogenesis in hypophysectomized rats with an intratesticular T concentration of 17 to 18 ng/gm testis.     

Effects of a highly purified antiserum to FSH on testicular function in immature rats

Effects of highly purified antiserum (AS) to follicle stimulating hormone (FSH) on testicular function was studied in immature rats. Treatment with FSHAS for 10 days, from 25-34, decreased weights of the testis (p .001) and increased weights of the epididymis (p .05). Numbers of the cell types in the seminiferous epithelium, particularly Type A spermatogonia pachytene spermatocytes and spermatids, were markedly reduced, possibly due to: 1) decreased division of the initial stem cells, 2) impairment of division of Type B spermatogonia and their transformation to pachytene spermatocytes, and 3) desquamation and degeneration of pachytene spermatocytes and spermatids. FSHAS also affected the sertoli cell function which was reflected in the decreased binding of androgens to supernatant fraction of the testis and epididymides. Treatment with luteinizing hormone-AS for 5 days did not affect testicular function but the binding of androgens to the supernatants of the caput and cauda epididymides and ventral prostate was significantly reduced (p .001). These data indicate that FSH is necessary for the maintenance of the cellular integrity of the seminiferous epithelium during the completion of the 1st wave of spermatogenesis.     

Effects of danazol on spermatogenesis in adult rats

Adult male Wistar rats were treated with Danazol (4 mg/day s.c.) for 52 days. The drug produced a marked, rapid drop in serum testosterone concentrations to very low levels and caused a slower decrease in serum FSH, LH and testis weight. Flow cytometric analysis of testicular cell suspensions showed a decline in the absolute numbers of haploid cells (spermatids), tetraploid cells (mainly pachytene spermatocytes) and of cells in the S-phase of the division cycle, suggesting that Danazol inhibited proliferation of spermatogonia and/or primary spermatocytes. Histological counting of the different types of spermatogonia, however, revealed no significant change in their numbers during Danazol treatment. It is concluded that Danazol inhibited spermatogenesis primarily after the preleptotene stage of primary spermatocytes.     

Reaction of the chromatoid body with a monoclonal antibody to a rat histocompatibility antigen

The monoclonal antibody OX3 against a polymorphic class II antigen encoded by the major histocompatibility locus of the rat has been shown to cross-react with the chromatoid body during spermatogenesis. Using an indirect immunofluorescence assay on frozen, fixed testis sections, the antibody revealed a pattern of fluorescent speckling that correlated with specific stages of spermatogenesis. The positive material first appeared in late pachytene spermatocytes as multiple small spots. Larger dots appeared in all regions containing round spermatids, but, as the spermatids matured, only fine dots were seen. Mature spermatids were negative, as were all early cells (spermatogonia to early pachytene spermatocytes). When suspension of fixed testicular cells were tested, the activity was clearly associated with the chromatoid body adjacent to the nucleus in round spermatids and with multiple smaller structures encircling the nucleus in primary spermatocytes. These associations were confirmed in observations on immature testes at various ages. No reactivity was seen in testes of animals whose testes had previously been irradiated to render them aspermatogenic, nor in grc/grc rats in which spermatogenesis is arrested at the primary spermatocyte stage. Because the expression of this reactivity was seen even in rats that do not express the OX3 antigen on their somatic cells, this antibody should prove useful in determining the structure of this body, its origin and fate, and any possible role it may have in spermiogenesis.     

Indirect Sertoli cell-mediated ablation of germ cells in mice expressing the inhibin-alpha promoter/herpes simplex virus thymidine kinase transgene

In the present study, we describe a novel mouse model for inducible germ cell ablation. The mice express herpes simplex virus thymidine kinase (HSV-TK) under the inhibin-alpha subunit promoter (Inhalpha). When adult transgenic (TG) mice were treated with famciclovir (FCV) for 4 wk, their spermatogenesis was totally abolished, with only Sertoli cells and few spermatids remaining in the seminiferous tubules. However, testicular steroidogenesis was not affected. Shorter treatment periods allowed us to follow up the progression of germ cell death: After 3 days, spermatogonia and preleptotene spermatocytes were no longer present. After a 1-wk treatment, spermatogonia, preleptotene, and zygotene spermatocytes were missing and the amount of pachytene spermatocytes was decreased. After a 2-wk treatment, round and elongating spermatids were present. During the third week, round spermatids were lost and, finally, after a 4-wk treatment, only Sertoli cells and few spermatids were present. Interestingly, the transgene is detected in Leydig and Sertoli cells but not in spermatogonia. This suggests that FCV is phosphorylated in Sertoli cells, and thereafter, leaks to neighboring spermatogonia, apparently through cell-cell junctions present, enabling trafficking of phosphorylated FCV. Because of the many mitotic divisions they pass through, the spermatogonia are very sensitive to toxins interfering with DNA replication, while nondividing Sertoli cells are protected. Using transillumination-assisted microdissection of the seminiferous tubules, the gene-expression patterns analyzed corresponded closely to the histologically observed progression of cell death. Thus, the model offers a new tool for studies on germ cell-Sertoli cell interactions by accurate alteration of the germ cell composition in seminiferous tubules.     

Early effect of the anticancer drug biocarbazin (DTIC synonym) on mice spermatogenesis

Biocarbazin (DTIC synonym) is an anticancer drug acting as a purine analogue, as an alkylating agent, as a SH-group blocker. Biocarbazin administration in doses 50 and 200 mg/kg of body weight to adult male BALB/c mice suppresses the process of spermatogenesis. Seminiferous tubule of the testis displayed dose-dependent histological alterations manifested with decrease of mitotically dividing cells and increase in the number of multinucleated spermatocytes and spermatids. RNA synthetic activity studied by means of autoradiography showed a tendency for a reduction in the spermatogonia and pachytene spermatocytes.     

Spermatogenesis and related plasma androgen levels in Atlantic halibut (Hippoglossus hippoglossus L.)

Spermatogenesis in male Atlantic halibut (Hippoglossus hippoglossus L.) was investigated by sampling blood plasma and testicular tissue from 15-39-month-old fish. The experiment covered a period in which all fish reached puberty and completed sexual maturation at least once. The germinal compartment in Atlantic halibut testis appears to be organized in branching lobules of the unrestricted spermatogonial type, because spermatocysts with spermatogonia were found throughout the testis. Spermatogenesis was characterized histologically, and staged according to the most advanced type of germ cell present: spermatogonia (Stage I), spermatogonia and spermatocytes (Stage II), spermatogonia, spermatocytes and spermatids (Stage III), spermatogonia, spermatocytes, spermatids and spermatozoa (Stage IV), and regressing testis (Stage V). Three phases could be distinguished: first, an initial phase with low levels of circulating testosterone (T; quantified by RIA) and 11-ketotestosterone (11-KT; quantified by ELISA), spermatogonial proliferation, and subsequently the initiation of meiosis marked by the formation of spermatocytes (Stage I and II). Secondly, a phase with increasing T and 11-KT levels and with haploid germ cells including spermatozoa present in the testis (Stage III and IV). Thirdly, a phase with low T and 11-KT levels and a regressing testis with Sertoli cells displaying signs of phagocytotic activity (Stage V). Circulating levels of 11-KT were at least four-fold higher than those of T during all stages of spermatogenesis. Increasing plasma levels of T and 11-KT were associated with increasing testicular mass throughout the reproductive cycle. The absolute level of, or the relation between, testis growth and circulating androgens were not significantly different in first time spawners compared to fish that underwent their second spawning season. These results provide reference levels for Atlantic halibut spermatogenesis.     

DNMT1 and HDAC1 gene expression in impaired spermatogenesis and testicular cancer

DNA methylation catalyzed by DNA methyltransferases (DNMTs) and histone deacetylation catalyzed by histone deacetylases (HDACs) play an important role for the regulation of gene expression during carcinogenesis and spermatogenesis. We therefore studied the cell-specific expression of DNMT1 and HDAC1 for the first time in human testicular cancer and impaired human spermatogenesis. During normal spermatogenesis, DNMT1 and HDAC1 were colocalized in nuclei of spermatogonia. While HDAC1 was additionally present in nuclei of Sertoli cells, DNMT1 was restricted to germ cells exhibiting a different expression pattern of mRNA (in pachytene spermatocytes and round spermatids) and protein (in round spermatids). Interestingly, in infertile patients revealing round spermatid maturation arrest, round spermatids lack DNMT1 protein, while pachytene spermatocytes became immunopositive for DNMT1. In contrast, no changes in the expression pattern could be observed for HDAC1. This holds true also in testicular tumors, where HDAC1 has been demonstrated in embryonal carcinoma, seminoma and teratoma. Interestingly, DNMT1 was not expressed in seminoma, but upregulated in embryonal carcinoma. Olufunmilade A. Omisanjo is a scholarship holder of the German Academic Exchange Service (DAAD). Sonja Hartmann is a member of the German Research Foundation (DFG) Research Training Group 533 Cell–cell-Interaction in Reproduction.     

Characterization of high mobility group protein levels during spermatogenesis in the rat

The distribution, quantitation, and synthesis of high mobility group (HMG) proteins during spermatogenesis in the rat have been determined. HMG1, -2, -14, and -17 were isolated from rat testes by Bio-Rex 70 chromatography combined with preparative gel electrophoresis. Amino acid analysis revealed that each rat testis HMG protein was similar to its calf thymus analogue. Tryptic peptide maps of somatic and testis HMG2 showed no differences and, therefore, failed to detect an HMG2 variant. Testis levels of HMG proteins, relative to DNA content, were equivalent to other tissues for HMG1 (13 micrograms/mg of DNA), HMG14 (3 micrograms/mg of DNA), and HMG17 (5 micrograms/mg of DNA). The testis was distinguished in that it contained a substantially higher level of HMG2 than any other rat tissue (32 micrograms/mg of DNA). HMG protein levels were determined from purified or enriched populations of testis cells representing the major stages of spermatogenesis; spermatogonia and early primary spermatocytes, pachytene spermatocytes, early spermatids, and late spermatids; and testicular somatic cells. High levels of HMG2 in the testis were due to pachytene spermatocytes and early spermatids (56 +/- 4 and 47 +/- 6 micrograms/mg of DNA, respectively). Mixtures of spermatogonia and early primary spermatocytes showed lower levels of HMG2 (12 +/- 3 micrograms/mg of DNA) similar to proliferating somatic tissues, whereas late spermatids had no detectable HMG proteins. The somatic cells of the testis, including isolated populations of Sertoli and Leydig cells, showed very low levels of HMG2 (2 micrograms/mg of DNA), similar to those in nonproliferating somatic tissues. HMG proteins were synthesized in spermatogonia and primary spermatocytes, but not in spermatids. Rat testis HMG2 exhibited two bands on acid-urea gels. A "slow" form comigrated with somatic cell HMG2, while the other "fast" band migrated ahead of the somatic form and appeared to be testis-specific. The "fast" form of HMG2 accounted for the large increase of HMG2 levels in rat testes. These results show that the very high level of HMG2 in testis is not associated with proliferative activity as previously hypothesized.     

Effect of testosterone oenanthate on spermatogenesis and serum testosterone concentrations in adult mice

Administration of 80 or 160 micrograms testosterone oenanthate s.c. three times per week for 8 or 12 weeks reduced testis weight and increased seminal vesicle weight in mice. Radioimmunoassay indicated that treatment increased serum testosterone concentrations. Treatment with testosterone oenanthate decreased the number of step 16 and step 7 spermatids, pachytene spermatocytes and type A spermatogonia, and particularly reduced the proportion of step 7 spermatids which matured to form step 16 spermatids.     

DNA double-strand breaks and gamma-H2AX signaling in the testis

Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated at serine 139 and forms gamma-H2AX foci at the sites of damage. These foci then play a role in recruiting DNA repair and damage-response factors and changing chromatin structure to accurately repair the damaged DNA. These gamma-H2AX foci appear in response to irradiation and genotoxic stress and during V(D)J recombination and meiotic recombination. Independent of irradiation, gamma-H2AX occurs in all intermediate and B spermatogonia and in preleptotene to zygotene spermatocytes. Type A spermatogonia and round spermatids do not exhibit gamma-H2AX foci but show homogeneous nuclear gamma-H2AX staining, whereas in pachytene spermatocytes gamma-H2AX is only present in the sex vesicle. In response to ionizing radiation, gamma-H2AX foci are generated in spermatogonia, spermatocytes, and round spermatids. In irradiated spermatogonia, gamma-H2AX interacts with p53, which induces spermatogonial apoptosis. These events are independent of the DNA-dependent protein kinase (DNA-PK). Irradiation-independent nuclear gamma-H2AX staining in leptotene spermatocytes demonstrates a function for gamma-H2AX during meiosis. gamma-H2AX staining in intermediate and B spermatogonia, preleptotene spermatocytes, and sex vesicles and round spermatids, however, indicates that the function of H2AX phosphorylation during spermatogenesis is not restricted to the formation of gamma-H2AX foci at DNA double-strand breaks.     

Immunohistochemical distribution of sulfhydryl oxidase in the human testis

Summary Sulfhydryl oxidase (SOx) is an enzyme that catalyzes the oxidation of sulfhydryl compounds. It is present in mitochondria of certain testicular cells at specific stages of functional activation. In the mature human testis moderate SOx immunoreactivity is found in Leydig cells, and lacking in Sertoli and in peritubular cells. The A_dark spermatogonia usually contain immuno-reactive mitochondria, while in A_pale spermatogonia immunoreactivity is mostly low. In stage V of spermatogenesis, A_pale spermatogonia were found containing immunoreactive material. Leptotene (stages IV and V) and zygotene (stage VI) primary spermatocytes display a moderate immunoreaction. It is strongest in pachytene spermatocytes of stages I–IV, decreases in stage V, and is low during diakinesis and in secondary spermatocytes. Late spermatids usually show a stronger immunoreactivity than early spermatids. At stage V of spermatogenesis the late spermatids contain only few immunoreactive particles. Spermatozoa are free of SOx-immunoreactive mitochondria. In residual bodies small amounts of SOx-immunoreactive particles are seen. Compared to rat and hamster testis, SOx immunoreactivity of the human testis is less clearly stage-dependent and it is not confined to certain germ cell stages. As deduced from the findings in patients with spermatogenic disorders, the SOx immunoreactivity of spermatogonia in human testis seems to be of diagnostic relevance.     

Immunohistochemical study of calmodulin in developing mouse testis

The purpose of this study was to determine the localization of calmodulin in the developing mouse testis by the indirect immunoperoxidase method. In addition, the amount of calmodulin in pachytene spermatocytes, spermatids, and residual bodies isolated from the mouse testis and epididymal spermatozoa was quantitated by the adenylate cyclase activation assay and by enzyme immunoassay. The relative levels of calmodulin in the developing mouse testis and in the isolated testicular germ cells were confirmed by western transfer staining. The level of immunoreactive calmodulin was very low in the testes from immature animals. In testes from the mature mouse, calmodulin was found to be localized in spermatocytes and spermatids, but was not found in spermatogonia, Sertoli cells, and interstitial cells. By contrast, immunochemical staining of tubulin was extremely intense in Sertoli cells. Biochemical determinations also showed that pachytene spermatocytes, round spermatids, spermatozoa, and residual bodies contained 14.9 micrograms, 15.8 micrograms, 2.3 micrograms and 5.2 micrograms of calmodulin per mg of protein, respectively. Both the immunochemical and the biochemical studies revealed that levels of calmodulin were high in the spermatocytes and in the round spermatids, as compared to the level in spermatozoa. This fact strongly suggests that the large amount of calmodulin in mammalian testes may be associated primarily with meiotic divisions and/or spermatogenesis.     

Temporal expression of membrane antigens during mouse spermatogenesis.

The temporal expression of cell surface antigens during mammalian spermatogenesis has been investigated using isolated populations of mouse germ cells. Spermatogenic cells at advanced stages of differentiation, including pachytene primary spermatocytes, round spermatids, and residual bodies of Regaud and mature spermatozoa, contain common antigenic membrane components which are not detected before the pachytene stage of the first meiotic prophase. These surface constituents are not detected on isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, or leptotene and zygotene primary spermatocytes. These results have been demonstrated by immunofluorescence microscopy, by complement-mediated cytotoxicity, and by quantitative measurements of immunoglobulin (Ig) receptors on the plasma membrane of all cell populations examined. The cell surface antigens detected on germ cells are not found on mouse thymocytes, erythrocytes, or peripheral blood lymphocytes as determined by immunofluorescence and by cytotoxicity assays. Furthermore, absorption of antisera with kidney and liver tissue does not reduce the reactivity of the antibody preparations with spermatogenic cells, indicating that these antigenic determinants are specific to germ cells. This represents the first direct evidence for the ordered temporal appearance of plasma membrane antigens specific to particular classes of mouse spermatogenic cells. It appears that at late meiotic prophase, coincident with the production of pachytene primary spermatocytes, a variety of new components are inserted into the surface membranes of developing germ cells. The further identification and biochemical characterization of these constituents should facilitate an understanding of mammalian spermatogenesis at the molecular level.     

Stage-specific nuclear antigen is expressed in rat male germ cells during early meiotic prophase

A germ cell nuclear antigen with approximately 44-kDa molecular weight was identified by a novel monoclonal antibody designated as Mab 2F2 from the library we have accumulated against rat testicular cells. In immature 20-day-old and adult rat testis the recognized antigen was expressed in the nuclei of early meiotic cells from preleptotene to early pachytene spermatocytes exhibiting a stage-specific appearance in the cycle of the seminiferous epithelium. The immunoreactivity was clearly associated with the meiotic chromosomes. The antigen was not detected in the late pachytene spermatocytes and more advanced stages of spermatogenesis. No labeling was observed in spermatogonia and somatic Sertoli and Leydig cells. The pattern of expression of the recognized antigen during early meiotic stages of spermatogenesis but not in mitotically dividing spermatogonia could strengthen its possible role in meiotic division.