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).     

Distribution of repetitious DNA in randomly growing and synchronized Chinese hamster cells.

Early spermatogenic cells from the testes of 10-, 13-, 15-, 18-, 20- and 25-day-old rats were purified by sedimentation at unit gravity. Cell dissociation was accomplished in 5 mM EDTA or 0.1% trypsin in Ca-Mg-free phosphate-buffered saline (pH 7.35). Dissociation with trypsin resulted in more viable cells than with EDTA, while EDTA was more efficient for the dissociation of spermatogonia. The differential effects of the two dissociation media were particularly evident in cell preparations from the 10-day-old animal. Maximum purity of different cell types was obtained in different aged animals (spermatogonia, 98%, 10 days; preleptotene spermatocytes, 98%, 10 days; leptotene spermatocytes, 75%, 13 days; zygotene spermatocytes, 68%, 18 days; pachytene spermatocytes, 75%, 25 days). Purity of particular types was correlated with the age of the animal. Earlier stages were purified to a greater extent in younger animals and later stages to a greater extent in older animals. Later stages exhibited increasing sedimentation at unit gravity in correlation with the increase in cell size as differentiation proceeded to pachytene spermatocyte. Two early germinal cell types, spermatogonia and preleptotene spermatocytes, were greatly purified with this technique.     

Flow-cytometric isolation of testicular germ cells from rainbow trout (Oncorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions

There is a need to isolate different populations of spermatogenic cells to investigate the molecular events that occur during spermatogenesis. Here we developed a new method to identify and purify testicular germ cells from rainbow trout (Oncorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions (pvasa-GFP) at various stages of spermatogenesis. Rainbow trout piwi-like (rtili), rainbow trout scp3 (rt-scp3), and rainbow trout shippo1 (rt-shippo1) were identified as molecular markers for spermatogonia, spermatocytes, and spermatids, respectively. The testicular cells were separated into five fractions (A-E) by flow cytometry (FCM) according to their GFP intensities. Based on the molecular markers, fractions A and B were found to contain spermatogonia, while fractions C and D contained spermatocytes, and fraction E contained spermatids. We also classified the spermatogonia into type A, which contained spermatogonial stem cells (SSCs), and type B, which did not. As none of the molecular markers tested could distinguish between the two types of spermatogonia, we subjected them to a transplantation assay. The results indicated that cells with strong GFP fluorescence (fraction A) colonized the recipient gonads, while cells with weaker GFP fluorescence (fraction B) did not. As only SSCs could colonize the recipient gonads, this indicated that fraction A and fraction B contained mainly type A and type B spermatogonia, respectively. These findings confirmed that our system could identify and isolate various populations of testicular cells from rainbow trout using a combination of GFP-dependent FCM and a transplantation assay.     

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.     

DNA flow-cytometric analysis of testicular germ cell populations of the bonnet monkey (Macaca radiata) as a function of sexual maturity

Testicular germ cell populations of biopsies from 32 male bonnet monkeys in 5 different age groups were quantitated in a flow cytometer after labelling of germ cell DNA with the specific fluorochrome, 4,6-diamidino phenyl indole. The 5 quantifiable populations were spermatogonia (2C), preleptotene spermatocytes (S phase), primary spermatocytes (4C), round spermatids (1C) and elongate spermatids (HC). The seminiferous tubules of immature 3-4-year-old monkey had only Sertoli cells and spermatogonia (2C). At 5-6 years, germ cells in S-phase (9.5%), 4C (11.1%), 1C (41.8%) and HC (17.1%) stages of maturation appeared for the first time but at 7-8 years of age and beyond all cell types except HC decreased while 1C remained relatively constant. Histometric analysis correlated well with the flow-cytometric data. The decrease in cells of 2C, S-phase and 4C stages was associated with an increase in mitotic index, signifying acceleration in the kinetics of germ cell transformation into subsequent cell types. The total turnover in cell transformation (1C:2C) was significantly (P less than 0.01) increased at and beyond 7-8 years. Maximum transition from 2C to 4C occurred at 5-6 years (4C:2C ratio 0.8 at 5-6 years and 0.6 at 7-8 years). The ratio HC:1C (kinetics of cell transformation during spermiogenesis) attained near total efficiency only by 10 years of age (1.08 at 10-14 years; 0.9 at 18-20 years). Also, the cell associations within the seminiferous tubules of monkeys greater than or equal to 10 years of age were better defined than those of younger animals. The changes in germ cell ratios correlated well with alterations in testicular volume, sperm numbers in the ejaculate and surges of testosterone and increments in FSH in the serum, characteristic of development of sexual maturity. It is apparent from this study that DNA flow cytometry of testicular germ cell populations reveals subtle changes in spermatogenic status of bonnet monkeys with a high degree of sensitivity.     

Increased apoptosis occurring during the first wave of spermatogenesis is stage-specific and primarily affects midpachytene spermatocytes in the rat testis

The physiological apoptosis that occurs in immature testis appears to be necessary for the maturation of this tissue. Thus, inhibition of the early apoptotic wave associated with the first round of spermatogenesis is followed by accumulation of spermatogonia and infertility later in life. To identify the cell types undergoing apoptosis in immature rat testis and to characterize the relationship between this apoptosis and progression of the first wave of spermatogenesis, sequential viable segments of seminiferous tubules from 8-, 18-, and 26-day-old rats were examined under a phase-contrast microscope. One novel observation was the existence of pronounced stage-specificity during the peak of apoptosis at the very early postnatal ages of 18 and 26 days. Increased apoptosis of pachytene spermatocytes in stages VII-VIII was the major feature that distinguished immature spermatogenesis from the corresponding adult process. The frequency of apoptosis among type A spermatogonia in immature stages IX-I was also elevated in comparison to the corresponding mature stages. The age-related peak of apoptosis was mediated by caspase 3; furthermore, stage-dependent expression of Bax in midpachytene spermatocytes was observed in the 18- and 26-day-old testis. These observations suggest that this Bax-regulated, caspase 3-mediated, increased apoptosis of midpachytene spermatocytes during the first wave of immature spermatogenesis represents a major difference in comparison to apoptosis occurring in the mature testis, and it may play an important regulatory role in establishing spermatogenesis in the rat testis.     

Role of spermatogonia in the stage-synchronization of the seminiferous epithelium in vitamin-A-deficient rats

After 20-day-old rats are placed on a vitamin-A-deficient diet (VAD) for a period of 10 weeks, the seminiferous tubules are found to contain only Sertoli cells and a small number of spermatogonia and spermatocytes. Retinol administration to VAD rats reinitiates spermatogenesis, but a stage-synchronization of the seminiferous epithelium throughout the testis of these rats is observed. In order to determine which cell type is responsible for this synchronization, the germ cell population has been analyzed in whole mounts of seminiferous tubules dissected from the testes of rats submitted to the following treatments. Twenty-day-old rats received a VAD diet for 10 weeks and then were divided into three groups of six rats. In group 1, all animals were sacrificed immediately; in group 2, the rats were injected once with retinol and sacrificed 3 hr later; in group 3, the rats were injected once with retinol, placed on a retinol-containing diet for 7 days and 3 hr, and then sacrificed. Three rats from each group had one testis injected with 3H-thymidine 3 hr (groups 1 and 2) or 7 days and 3 hr (group 3) before sacrifice. Three normal adult rats (approximately 100 days old) served as controls. Labeled and unlabeled germinal cells were mapped and scored in isolated seminiferous tubules. In group 1, type A1 and type A0 spermatogonia as well as some preleptotene spermatocytes were present; type A2, A3, A4, In, and B spermatogonia were completely eliminated from the testis. Neither type A1 mitotic figures nor 3H-thymidine-labeled-type A1 nuclei were seen.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA polymerases in mouse spermatogenic cells separated by sedimentation velocity.

Activity levels of DNA polymerase alpha and DNA polymerase beta have been measured in mouse spermatogenic cells separated by sedimentation velocity. Testes from prepuberal (17 day old) and sexually mature mice were dissociated and separated by unit gravity sedimentation into 6 populations of cells. Phase contrast microscopy and [3H]thymidine labeling kinetics revealed that at least 85% of the cells in fraction A were pachytene-stage primary spermatocytes, fraction B was enriched for primary spermatocytes and round spermatids, fraction C contained spermatogonia and/or pre-leptotene primary spermatocytes and later stages of spermatids (no spermatids were present in fraction C from the testes of 17 day old mice) and fractions D to F contained mixed populations of cells, many in later stages of spermiogenesis. When expressed as activity in 10(6) cells or as a specific activity, fractions A, B, and C from mature animals population initially loaded onto the gradient while fractions D, E and F had activity levels similar to or below the population of dissociated cells. The ratio of activity between the DNA polymerases was constant in fractions A, B, and C, but in fractions D, E, and F, the ratio decreased due to a more rapid decline of activity of polymerase alpha. A comparison of activity levels in fraction C from prepuberal and sexually mature mice revealed an increase in DNA polymerase alpha activity and a decrease in the activity of DNA polymerase beta in the cells from the 17 day old animals.     

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.     

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.     

Immunoreactive sites and accumulation of somatomedin-C in rat Sertoli-spermatogenic cell co-cultures

Sertoli-spermatogenic cell co-cultures prepared from sexually immature rats (20-22 days old) and maintained in serum-free, hormone/growth factor-supplemented medium were used to determine the cell-specific localization of the growth factor somatomedin-C (SM-C). SM-C localization studies were carried out by indirect immunofluorescence using a monoclonal antibody (sm-1.2) to SM-C. In cultured rat hepatocytes, Sertoli and testicular peritubular cells, SM-C immunoreactivity was observed as a diffuse distribution of discrete immunofluorescent granules. Radio-immunoassay experiments using a rabbit antibody against human SM-C showed that testicular peritubular cells and Sertoli cells in primary culture accumulated SM-C in the medium. In spermatogenic cells co-cultured with subjacent Sertoli cells, immunoreactive SM-C was associated with pachytene spermatocytes but not with spermatogonia or early meiotic prophase spermatocytes (leptotene or zygotene). Both Sertoli cells and pachytene spermatocytes displayed binding sites for exogenously added SM-C. SM-C6 binding to spermatocytes reaching an advanced stage of meiotic prophase suggests a possible role of this growth factor in the meiotic process.     

Evaluation of Sycp3, Plzf and Cyclin B3 expression and suitability as spermatogonia and spermatocyte markers in zebrafish

Recent studies in mammals have revealed the heterogeneity of spermatogonial populations which contain differentiated and undifferentiated cells that further divide into actual stem cells and potential stem cells. In fish however, there are no functional definitions, and very few molecular markers, for germ cells. In our present study, specific antibodies were raised against Sycp3, Plzf and Cyclin B3 in zebrafish and then used to determine the localization of these proteins in the testis. We wished to confirm whether these molecules were potential markers for spermatocytes and spermatogonia. Immunohistochemical observations revealed that Sycp3 is specifically localized in spermatocytes in typical nuclear patterns at each meiotic stage. Plzf was found to be localized in the nucleus of both type A and type B spermatogonia until the 8-cell clone, similar to the pattern in Plzf-positive A(single)-A(aligned) undifferentiated spermatogonia in rodents. In addition to Plzf, the localization of Cyclin B3 was predominantly detected in the nuclei of type A and early type B spermatogonia until the 16-cell clone. Additionally, Cyclin B3 protein signals were detected in germ cells in large cysts, possibly corresponding to spermatocytes at the preleptotene stage. Our present data thus show that these molecules have properties that will enable their use as markers of spermatocytes and early spermatogonia in zebrafish.     

Separation of mouse testis cells by equilibrium density centrifugation in Renografin gradients

Mouse testis cells have been separated by equilibrium density centrifugation in gradients of Renografin. Intact testis cells were not damaged by the separation procedure provided that, following separation, the osmolarity was reduced gradually. The various cell types were identified microscopically and by ³H-thymidine labelling with similar results. The present technique has demonstrated that significant variations in cell density occur during spermatogenesis. Approximately ten-fold enrichments of nearly all testis cell types were achieved by equilibrium density separation of testis cell suspensions. More homogeneous cell populations were prepared by density gradient centrifugation of cell fractions obtained from velocity sedimentation separations. Overall enrichments of spermatogonia, by 29-fold; pachytene spermatocytes, 45-fold; dividing meiotic cells, 170-fold; round spermatids, 30-fold; step 11–13 elongating spermatids, 12-fold; Leydig cells, 70-fold; and cytoplasmic fragments, 55-fold, were obtained. In this study, a method for preparation of cell suspensions was also developed to produce higher yields of spermatogonia and young primary spermatocytes; however, the density distribution of these cells was altered.     

Expression of p57 in mouse and human testes

The expression of cyclin-dependent kinases inhibitors, p57kip2, was investigated during the postnatal development of mouse testis, and in adult human testis. Expression of p57kip2 mRNA was higher in immature than pubertal or adult mouse testes. In postnatal day 7 (PND7) testes, moderate p57kip2 immunoreactivity was found in spermatogonia, but signal was heterogeneous among the spermatogonia. In PND14 testes onward, strong immunoreactivity of p57kip2 was found in the nuclei of early spermatocytes but not in the late pachytene stage onward. In PND28 and PND50 testes, p57kip2 immunoreactivity was varying among the seminiferous tubules. There was no visible signal in late pachytene stage onward. In Leydig cells, heterogeneous immunoreactivity of p57kip2 was found in immature testis and the signal intensity was higher in adult testis than immature ones. In Sertoli cells, weak or negligible immunoreactivity of p57kip2 was found. In human seminiferous tubule, strong immunoreactivity of p57kip2 was found in the nucleus of early spermatocytes, but not in the late pachytene spermatocytes onward and Sertoli cells. These results suggest the possible role of p57kip2 in the regulation of early spermatogonial proliferation, meiotic progression of early spermatocytes and differentiation of Leydig cells in testis.     

Telomere length in male germ cells is inversely correlated with telomerase activity

Telomeres, the noncoding sequences at the ends of chromosomes, progressively shorten with each cellular division. Spermatozoa have very long telomeres but they lack telomerase enzymatic activity that is necessary for de novo synthesis and addition of telomeres. We performed a telomere restriction fragment analysis to compare the telomere lengths in immature rat testis (containing type A spermatogonia) with adult rat testis (containing more differentiated germ cells). Mean telomere length in the immature testis was significantly shorter in comparison to adult testis, suggesting that type A spermatogonia probably have shorter telomeres than more differentiated germ cells. Then, we isolated type A spermatogonia from immature testis, and pachytene spermatocytes and round spermatids from adult testis. Pachytene spermatocytes exhibited longer telomeres compared to type A spermatogonia. Surprisingly, although statistically not significant, round spermatids showed a decrease in telomere length. Epididymal spermatozoa exhibited the longest mean telomere length. In marked contrast, telomerase activity, measured by the telomeric repeat amplification protocol was very high in type A spermatogonia, decreased in pachytene spermatocytes and round spermatids, and was totally absent in epididymal spermatozoa. In summary, these results indicate that telomere length increases during the development of male germ cells from spermatogonia to spermatozoa and is inversely correlated with the expression of telomerase activity.     

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.     

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.     

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.     

Selective partitioning of plasma membrane antigens during mouse spermatogenesis

The selective partitioning of cell membrane components during mouse spermatogenesis has been examined using a heterologous antibody raised against isolated type B spermatogonia. The anti-type B spermatogonia rabbit IgG (ATBS) binds to isolated populations of mouse primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene spermatocytes, leptotene/zygotene spermatocytes, pachytene spermatocytes, round spermatids, residual bodies, and mature spermatozoa. Although immunofluorescent labeling is uniformly distributed on the cell surface of early spermatogenic cells, a discrete topographical localization of IgG is observed on testicular, epididymal, and vas deferens spermatozoa. The convex surface of the acrosome, postacrosomal region, and tail are labeled. Antibody does not bind to a broad area corresponding to the concave region of the acrosome. The antibody also binds to mouse somatic cells including Sertoli cells, Leydig cells, thymocytes, and splenocytes, but not to mature spermatozoa of the vole, rat, hamster, guinea pig, rabbit, or human. ATBS, after absorption with mouse splenocytes or thymocytes, does not react with any somatic cells examined by fluorescence except with Sertoli cells. In addition, all reactivity with testicular, epididymal, and was deferens spermatozoa is abolished. However, spermatogenic cells at earlier stages of differentiation, including residual bodies, still react strongly with the absorbed antibody. The number of surface receptor sites per cell for absorbed ATBS ranges from approximately 3 million on primitive type A spermatogonia to 1 million on round spermatids and on residual bodies. Spermatozoa, however, have only 0.003 million binding sites for absorbed ATBS, in contrast to 10 million sites for the unabsorbed antibody. It appears that receptor sites for absorbed ATBS are not masked by components of epididymal secretions. These data imply, therefore, that specific mechanisms operate at the level of the cell membrane during spermiogenesis to insure that some surface components, not required in the mature spermatozoon, are removed selectively by partitioning to that portion of the spermatid membrane destined for the residual body.