Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization

A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent).     

Identification and immunochemical characterization of spermatogenic cell surface antigens that appear during early meiotic prophase

Three spermatogenic cell populations isolated from prepuberal mice--type B spermatogonia, preleptotene spermatocytes, and leptotene/zygotene spermatocytes--were used to elicit distinct polyclonal antisera. Surface binding specificities were determined for purified IgGs by indirect immunofluorescence and rosette assays on live cells. Binding activities were assayed both before and after absorptions with a variety of somatic and spermatogenic cells. Each of these antisera binds to surface antigens that are present on germ cells throughout spermatogenesis and are not shared by splenocytes, thymocytes, and erythrocytes. Only the antiserum raised against leptotene and zygotene spermatocytes (ALZ) recognizes a stage-specific subset of surface determinants. After appropriate absorptions, ALZ binds to the surface of early pachytene spermatocytes and germ cells at subsequent stages of differentiation, including vas deferens spermatozoa. Antigens which react with this absorbed IgG are not detected on the surface of spermatogonia or meiotic cells prior to pachynema, including leptotene and zygotene spermatocytes. The observed binding specificities may result from the synthesis of one or more surface molecules during the early meiotic stages, followed by delayed insertion into the plasma membrane during the pachytene stage of meiotic prophase. Stage-specific antigens recognized by ALZ, including both protein and probably lipid, have been localized immunochemically on nitrocellulose blots from one-dimensional SDS gels. A dithiothreitol-sensitive constituent (Mr approximately 39,000) recognized by ALZ has been identified as the major protein determinant present in early meiotic cells but absent in 8-day-old seminiferous cell suspensions containing spermatogonia and Sertoli cells. This determinant is present in populations of preleptotene, leptotene/zygotene, and early pachytene spermatocytes isolated from 17-day-old animals, an observation consistent with the hypothesis of delayed insertion into the plasma membrane.     

Enrichment of the stages of the seminiferous epithelium in vitamin A-replaced-vitamin A-deficient rats

Morphometric study revealed that, at 40 days after the start of vitamin A replacement, A1 spermatogonia and preleptotene spermatocytes appeared in more than 70% of the whole mounts of seminiferous tubules of vitamin A-deficient rats. By 42 days, the appearance of these cell types was reduced by 50%, and A2 and A3 spermatogonia were predominant. By 46 days, A1-A3 spermatogonia appeared in less than 30% of the tubular length while A4, intermediate and B spermatogonia became the major cell types in the basement compartment of seminiferous tubules. The predominance of spermatogonia noted at given times was corroborated by higher frequencies of tubular cross-sections of stages in which that particular type of spermatogonium resides. These results indicate that seminiferous tubules of vitamin A-replaced-vitamin A-deficient rats are 'enriched' for particular stages. Tracing the development of [3H]thymidine-labelled preleptotene spermatocytes revealed normal kinetics of germ cell differentiation in these animals. Furthermore, the spermatogonial proliferations in the vitamin A-replaced-vitamin A-deficient rats were quantitatively normal. We suggest that vitamin A replacement may result in temporal suppression of the differentiation of A2-B spermatogonia, leading to a stimulation or synchronization of certain groups of undifferentiating spermatogonia which undergo active proliferation simultaneously. These synchronized populations of spermatogonia continue to proliferate and differentiate, thus resulting in the stage-enrichments noted at later times.     

Effect of testosterone on nucleic acid synthesis in spermatogenic epithelial cells of irradiated animals

A study was made of changes in nucleic acid synthesis in proliferating germ cell populations of animals irradiated with doses of 2 and 4 Gy. The administration of exogenous testosterone was shown to stimulate DNA synthesis, inhibited by the effect of radiation, in spermatogonium populations of A1-4, intermediate, and B types, and in preleptotene primary spermatocytes; RNA synthesis was increased in the same populations of spermatogonia and pachytene primary spermatocytes. The obtained results indicate that exogenous testosterone exerts a beneficial effect on spermatogenesis damaged by ionizing radiation.     

Biochemical evidence of haploid gene activity in spermatogenesis of the mouse

Mice were exposed to two X-ray doses of 300 and 100 R with 4 days interval in order to deplete the testes of spermatogonia and early meiotic cells. After X-ray treatment, the seminiferous tubules were labelled in culture with radioactive RNA precursors, dispersed into single cells by trypsin treatment and these were fractionated into several cell classes by velocity sedimentation at unit gravity in a Ficoll gradient. With this method quasi-homogeneous populations of middle-late pachytene spermatocytes and round spermatids (steps 1–8 of spermiogenesis) were obtained. RNA was extracted from these two cell types and analysed by linear sucrose gradient fractionation and by affinity chromatography on a poly(U)-Sepharose column. The results showed that round spermatids, as well as pachytene spermatocytes, synthesize both ribosomal RNA (rRNA) and poly(A)⁺ RNA (presumptive messenger RNA) (mRNA). The post-meiotic synthesis of RNA ceases completely in mid-spermiogenesis after nuclear elongation in spermatids has set in.     

Gene expression during mammalian spermatogenesis. II. Evidence for stage-specific differences in mRNA populations

Gene expression during murine spermatogenesis has been studied using highly enriched populations of cells obtained by velocity sedimentation at unit gravity and further purified by density gradient centrifugation through Percoll. Polypeptides whose synthesis was directed by total cytoplasmic RNA from round spermatids, pachytene spermatocytes, primitive type A spermatogonia, and Sertoli cells in cell-free translation systems have been compared by two-dimensional polyacrylamide gel electrophoresis, followed by fluorography. At the level of detection provided by the electrophoretic methods used, each population of cells contained mRNAs encoding over 200 polypeptides, many of which were present in high abundance in all four cell types. However, for each cell type examined, a minimum of 5-10% of these polypeptides appear to be either specific to or greatly enriched within a particular cell type. Analysis of the polysomal and nonpolysomal cell fractions from pachytene spermatocytes and round spermatids revealed that the two compartments share many identical mRNAs but specific mRNAs are selectively compartmentalized between the cell fractions and between the two cell types. Movement between compartments was seen; e.g., some polypeptides encoded by mRNA found primarily in the nonpolysomal fraction of pachytene cells were later seen in the polysomal fraction from round spermatids. Virtually every other combination was also observed. These results suggest that the control of gene expression at the level of selective production of mRNA and selective utilization of mRNA are among the mechanisms involved in regulation of spermatogenic cell differentiation.     

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.     

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.     

Spermatogenic cell differentiation in vitro

Culture conditions that support the in vitro development of many spermatogenic stages from the frog Xenopus laevis are described. Spermatogenic cells were dissociated with collagenase and preelongation stages aseptically isolated by density gradient centrifugation in Metrizamide. The cells were then cultured in modified forms of defined nutrient oocyte medium (DNOM). The development of spermatogenic cells was affected significantly by changes in fetal calf serum concentration, cell density, energy sources, and NaCl concentration. Optimum in vitro spermatid development was obtained when spermatogenic cells were cultured at relatively high densities (3–7 × l0⁷ cells/25 cm²) in DNOM modified to contain 10% heat-inactivated, dialyzed fetal calf serum, 2 mM 1-glutamine, 0.1 % glucose, 15 mM HEPES buffer (pH 7.4), and 38.3–48.3 mM NaCl. These culture conditions also supported the differentiation of preelongation spermatids and spermatocytes isolated by density-gradient centrifugation in Metrizamide and subsequent unit gravity sedimentation in gradients of bovine serum albumin. Approximately 95 % of such isolated spermatids and spermatocytes continued differentiating in vitro for 14 days at in vivo rates. Phase-contrast and electron microscopy of the cultured cells demonstrated that in vitro differentiation was morphologically normal between the leptotene and elongate spermatid stages. Autoradiographic studies of preleptotene development demonstrated that spermatogonia proliferated and preleptotene spermatocytes developed to zygotene in 12-day cultures. The results suggest that many spermatogenic stages in Xenopus can develop independent of Sertoli cells, and demonstrate that spermatogenic cell cultures can now be used for in vitro studies of spermatogenesis.     

A quantitative analysis of germ cells and the histone variants in the testes of vitamin A-deficient rats and during subsequent repletion with vitamin A

A quantitative analysis of the different types of germ cells present in the seminiferous tubules of vitamin A-deficient-retinoate maintained rats revealed that the number of pachytene spermatocytes and spermatogonia was greatly reduced in the deficient rats. Spermatids were virtually absent in the deficient tubules which contained mostly spermatogonia and preleptotene spermatocytes along with the Sertoli cells. There was no change in the number of Sertoli cells present in the tubules of deficient rats as compared to that of normal rats. Following supplementation of retinyl acetate to vitamin A-deficient-retinoate maintained rats, there was an immediate thinning of the germinal epithelium resulting from the sloughing off of the damaged spermatocytes which were beyond repair. However, after 12 days of vitamin A supplementation fresh batch of pachytene spermatocytes started appearing while by day 16 round spermatids could be seen. Analysis of the acid soluble proteins from nuclei on different types of Polyacrylamide gel electrophoretic systems has revealed that the levels of the testis specific histone variants Hlt, TH2A and TH2B, synthesized predominantly in the pachytene spermatocytes were greatly reduced in the testes of retinoate maintained rats. Following supplementation of retinyl acetate for either 4 days or 8 days the levels of these histone variants further decreased which correlated with the decrease in the number of pachytene spermatocytes. However, by day 12 of supplementation onwards, their levels started increasing and reached near normal levels by day 24 of vitamin A-supplementation     

Expression of heat shock proteins by isolated mouse spermatogenic cells.

Proteins of the hsp70 family are abundant in mouse spermatogenic cells. These cells also synthesize relatively large amounts of a 70,000-molecular-weight protein (P70) that appears to be a cell-specific isoform of hsp70, the major heat-inducible protein (R.L. Allen, D.A. O'Brien, and E.M. Eddy, Mol. Cell. Biol. 8:828-832, 1988). In this study, proteins of unstressed and heat-stressed spermatogenic cells consisting of purified preparations of preleptotene, leptotene-zygotene, pachytene spermatocytes, and round spermatids were analyzed by two-dimensional polyacrylamide gel electrophoresis. Unstressed preleptotene and leptotene-zygotene spermatocytes contained little P70, whereas relatively large amounts of P70 were present in pachytene spermatocytes and round spermatids. Labeling studies showed that P70 was synthesized primarily in pachytene spermatocytes and that little synthesis occurred in round spermatids or in preleptotene and leptotene-zygotene stages of spermatogenesis. Synthesis of hsp70 was not detectable in unstressed cells but was induced in all stages of isolated germ cells following heat stress. These results indicate that P70 is expressed in a stage-specific manner during cell differentiation, whereas hsp70 is synthesized in response to stress in all populations of isolated spermatogenic cells examined.     

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.     

Histone variants in rat spermatogonia and primary spermatocytes

The levels and synthesis of histone variants have been directly measured in spermatogonia and in various stages of primary spermatocytes purified from the rat testis. These measurements were made possible by the development of a procedure, employing centrifugal elutriation and density gradient centrifugation, to separate highly enriched populations of such cells from immature rat testes at the early stages of spermatogenesis. The results show a difference in regulation of the synthesis and accumulation of testis-specific histones H1t, TH2A, TH2B, and TH3. TH3 is present and actively synthesized in A and B spermatogonia. The testis-enriched variants, H2A.X and H1a, are also present at their maximal levels in A spermatogonia. No detectable amounts of H1t, and at most, low levels of TH2A and TH2B could be found in spermatogonia. While TH2A and TH2B are already present and actively synthesized in early primary spermatocytes (around the preleptotene stage), H1t does not accumulate until the pachytene stage.     

Follicle-stimulating hormone regulates both Sertoli cell and spermatogonial populations in the adult photoinhibited Djungarian hamster testis

The hormones that regulate spermatogonial development are ill defined, in part due to lack of appropriate experimental models. The photoinhibited hamster model provides a rich source of spermatogonia, thus making it an ideal model to study their control. This study aimed to assess the effects of FSH, in the absence of testosterone, on the reinitiation of Sertoli cell and spermatogonial development in the photosensitive adult Djungarian hamster. Hamsters raised under long photoperiods (LD, 16L:8D) were exposed to short photoperiods (SD, 8L:16D) for 11 wk, leading to suppression of gonadotropins and regression of testicular function. Groups of 10 animals then received FSH alone or in combination with the antiandrogen, flutamide, for 7 days. Two control groups maintained either under long or short photoperiods were treated with vehicle. Sertoli and germ cell number were then determined using the optical disector (sic) stereological technique. The number of Sertoli cells, type A spermatogonia, type B spermatogonia/preleptotene spermatocytes, and leptotene/zygotene spermatocytes were suppressed in SD controls to 66%, 34%, 19%, and 10% (all P < 0.01) of long-day control values, respectively. Later germ cell types were not detected. FSH treatment, with or without flutamide, increased Sertoli cell number (P < 0.01) to normal long-day values. Similarly, FSH treatment in the absence/presence of flutamide increased type A spermatogonia, type B spermatogonia/preleptotene spermatocytes, and leptotene/zygotene spermatocytes to approximately 85%, 69%, and 80% (all P < 0.01) of long-day controls, respectively. Our data demonstrate that the reinitiation of spermatogonial maturation in this model is dependent on FSH in the presence of an antiandrogen. Surprisingly, the adult Sertoli cell population in this model is also hormone dependent. This naturally occurring model provides a unique opportunity to understand the mechanisms (apoptotic and/or proliferative) by which FSH regulates Sertoli and germ cell development in the adult animal.     

Clastogenic effects of cis-diamminedichloroplatinum. II. Induction of chromosomal aberrations in primary spermatocytes and spermatogonial stem cells of mice

The clastogenic effect of the anticancer drug cis-diamminedichloroplatinum (II) (cisplatin) on meiotic prophase in primary spermatocytes and on spermatogonial stem cells of male (101/E1 x C3H/E1)F1 mice was studied. The intraperitoneal doses of cisplatin tested were 5.0, 7.5 and 10.0 mg/kg. Chromosomal aberrations were examined at diakinesis-metaphase 1 of meiosis 1-13 days after treatment, representing cells treated at diplotene, pachytene, zygotene, leptotene an preleptotene. Reciprocal translocations were evaluated 63-70 days after treatment, representing treated stem-cell spermatogonia. Cisplatin had a toxic effect in zygotene to preleptotene of meiosis, as indicated by the significant reduction in testicular weight. At diplotene, pachytene and zygotene no enhancement of aberrations was found. An increase in aberrant cells was observed during leptotene with preleptotene being the most sensitive stage. The dose-response relationship for aberrant cells was linear on day 13 after treatment. It is concluded that, like mitomycin C (Adler, 1976), cisplatin primarily caused aberrations during the premeiotic phase of DNA synthesis. No significant increase of translocation multivalents was found after treatment of stem-cell spermatogonia.     

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.     

Effect of antenatal action of Thio-Tepa on spermatogenesis of mature 101/N and CBA mice]

On pregnancy day 12 101/H and CBA mice were injected intraperitoneally 2.5 mg/kg bw thiophosphamide. 3.5-month-old male offspring were sacrificed. The drug effect on the testes was evaluated by karyologic analysis of the germ cell generations on stage 7 of the seminiferous epithelium cycle. A reliable reduction in the number of spermatogonia A, preleptotene and pachytene spermatocytes and spermatids at development stage 7 was found in 101/H mice. There were interspecific differences in spermatogenesis intensity in intact animals and recovery of germ cell pool after thiophosphamide action inducing toxicity.     

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.