Proliferation of spermatogonia and Sertoli cells in maturing mice

Summary Spermatogonial proliferation was studied in mice from day 13 p.p. when the seminiferous epithelium is incomplete, until week 12 p. p. when a steady state at adult levels has been attained. Counts of undifferentiated, A ₁ and intermediate spermatogonia and primary spermatocytes in stages IV and IX of the cycle of the seminiferous epithelium were made in whole mounted seminiferous tubules. Sertoli cell proliferation was studied in a separate series from 6 to 14 days p.p. employing the ³H-thymidine labeling index.It appeared that 1. Sertoli cell proliferation stops at day 12 whereafter the cells obtain their adult appearance; 2. The numbers of stem cell spermatogonia and the production of differentiating A ₁ spermatogonia increase almost twofold between day 13 and week 12; 3. The efficiency of the divisions of the differentiating A ₁-B spermatogonia is similar to that in the adult throughout this period; 4. At all ages studied, the cell counts revealed an almost constant numerical relationship between Sertoli cells and germ cells, which suggests a function of Sertoli cells in the regulation of spermatogonial proliferation.     

Spermatogonial stem cells in the albino rat

The existence of two classes of spermatogonial stem cells in the rat testis, i.e., reserve type A₀ spermatogonia and renewing, types A₁-A₄ spermatogonia, postulated by Clermont and Bustos-Obregon (′68), was reexamined in a quantitative analysis of type A spermatogonia in both whole mounts of tubules and in radioautographed sections of testes from animals killed at various times, up to 26 days, after one or multiple injections of ³H-thymidine. The cell counts obtained from whole mounts of tubules revealed that the number of isolated type A₀ cells per unit area of limiting membrane remained constant throughout the cycle of the seminiferous epithelium. Paired type A₀ spermatogonia also remained unchanged in number per unit area of basement membrane from stage I to stage VIII of the cycle. The low mitotic index of type A₀ spermatogonia (0.1%) indicated that these cells were not actively involved in the production of spermatogonia or spermatocytes during each cycle of the seminiferous epithelium and thus were considered as reserve stem cells. The type A₁ spermatogonia, which are formed during stage I of the cycle, remained resting until stage IX, when they undertook a series of four successive divisions resulting in the production of new type A₁ and Intermediate-type spermatogonia. An analysis of the labeling indices of type A spermatogonia obtained from cell counts in radioautographed testicular sections after a single or multiple ³H-thymidine injections indicated that the percentages of labeled type A cells corresponded to the percentages of type A₁-A₄ at each stage, whereas the percentages of unlabeled type A cells corresponded to the percentages of type A₀ spermatogonia obtained from counts of cells in whole mounts. This confirmed that type A₀ cells were generally non-proliferative throughout the cycle of the seminiferous epithelium while the type A₁-A₄ spermatogonia underwent complete renewal during each cycle. The present results thus support the concept of the existence of two classes of spermatogonial stem cells in rats.     

Renewal of spermatogonia in man

The spermatogonia of normal adult human testis were investigated in view of clarifying their mode of proliferation and renewal. Three main types of spermatogonia were identified: the dark type A spermatogonia (Ad) tentatively considered as the stem cells, the pale type A spermatogonia (Ap) and the type B spermatogonia (B), these being the more and more differentiated elements giving rise to preleptotene spermatocytes. The dark and pale type A spermatogonia were present in all stages of the cycle of the seminiferous epithelium, the type B spermatogonia were found in stages VI, I and II of the cycle and the preleptotene spermatocytes in stages III and IV of the cycle. The type A spermatogonia divided preferentially in stage V of the cycle and the type B spermatogonia in stage II of the cycle. Quantitative data on spermatogonia and preleptotene spermatocytes revealed that the cell ratio Ad: Ap: B: Pl was equal to 1:1:2:4. This indicated that the spermatogonial stem cells divided to produce equal numbers of new stem cells (Ad) and of the more differentiated pale type A spermatogonia (Ap). Each one of the latter gave rise to two type B spermatogonia which in turn produced four spermatocytes. The arrangement in pairs of the dark and pale type A spermatogonia throughout the duration of the cycle indicated that the mitoses of spermatogonial stem cells are “equivalent” in nature; therefore, the possibility of having “differential” mitoses to explain the renewal of spermatogonial stem cells should be abandoned. Lastly, the frequent arrangement of the two classes of type A spermatogonia in homogeneous clusters indicated that the impetus which facilitates the differentiation of stem cells into the more differentiated elements (Ap) may affect homogeneous and compact groups of stem cells.     

Role of spermatogonia in the repair of the seminiferous epithelium following X-irradiation of the rat testis

In the normal adult rat testis, type A₀ spermatogonia do not appear to participate to a significant extent in the production of spermatocytes, while type A₁ spermatogonia periodically initiate a series of divisions resulting in the production of spermatocytes and new type A₁ spermatogonia. The behavior of type A₀ and A₁ spermatogonia was investigated following administration of a single dose of x-rays (300 r) to the testis. Using whole mounts of seminiferous tubules, the type A₀ and A₁ cells were counted at various intervals after irradiation. At 8 and 13 days after irradiation, type A₁ spermatogonia reached lowest values, i.e., 6% and 3% of non-irradiated control, while type A₀ reached the lowest value, i.e., 62% of control at eight days. Thereafter the numbers of type A₀ and A₁ progressively increased to return to normal at 39 days. It was thus concluded that the type A₀ were comparatively more resistant to x-irradiation than type A₁ spermatogonia. To verify if the surviving type A₀ proliferated in the irradiated testis, animals were injected with ³H-thymidine three hours before they were sacrificed at various times after x-irradiation. In irradiated testes the labeling indices of the surviving type A (A₀, A₁–A₄) were the same as in the non-irradiated testes except in stages V-VI of the cycle of the seminiferous epithelium. While in the controls only 2% of type A cells were labeled at these two stages of the cycle, after irradiation the labeling index of type A reached a maximum of 31% at 13 days to return to control values by 39 days. Since at 13 days after irradiation type A₀ spermatogonia were the predominant component of the spermatogonial population, it was concluded that these cells must have incorporated ³H-thymidine and thereby contributed to the reconstruction of the spermatogonial population partially destroyed by irradiation.     

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 of 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. Three hr after retinol injection (group 2), type A₁ mitoses, but no labeled type A₁ nuclei were observed. At 7 days and 3 hr after retinol administration (group 3), type A₄ and In Spermatogonia as well as type A₁ spermatogonia were present. A few residual pachytene spermatocytes were found, and some type A₀ cells were labeled. These results indicate that VAD caused, in addition to an impairment of spermatogenesis at the preleptotene spermatocyte step, a selective momentary arrest of surviving type A₁ spermatogonia at the G₂ phase of their cell cycle. Following administration of vitamin A to VAD rats, these type A₁ cells reinitiated spermatogenesis synchronously and, after several cycless of proliferation and renewai, reconstituted the seminiferous epithelium in a stage-synchronized manner.     

Relative volume of sertoli cells in monkey seminiferous epithelium: A stereological analysis

Techniques of quantitative stereology have been utilized to determine the relative volume occupied by the Sertoli cells and germ cells in two particular stages (I and VII) of the cycle of the seminiferous epithelium. Sertoli cell volume ranged from 24% in stage I of the cycle to 32% in stage VII. Early germ cells occupied 3.4% in stage I (spermatogonia) and 8.7% in stage VII (spermatogonia and preleptotene spermatocytes). Pachytene spermatocytes occupied 15% (stage I) and 24% (stage VII) of the total volume of the seminiferous epithelium. In stage I the two generations of spermatids comprised 58% of the total epithelium by volume, whereas in stage VII, after spermiation, the acrosome phase spermatids occupied 35% of the total seminiferous epithelial volume.     

Re-examination of spermatogonial renewal in the rat by means of seminiferous tubules mounted “in toto”

Observations on dissected tubules, fixed in Carnoy, stained with hematoxylin and mounted “in toto” revealed that there were five distinct classes of type A spermatogonia. The type A₁ found in stages II–VIII of the cycle of the seminiferous epithelium had round, pale-stained nuclei, typically arranged in linear clusters of four or eight along the tubular wall. They all divided at stage IX to produce type A₂ cells. These in turn divided at stage XII to produce type A₃ spermatogonia. The type A₂ and A₃ cells had large ovoid nuclei containing globular masses of deeply stained chromatin and were randomly distributed in the space between Sertoli nuclei. The type A₃ spermatogonia divided at stage XIV to produce type A₄ cells. These had smaller nuclei, sometimes lobulated, containing more deeply stained chromatin granulation, free in the nucleus or adhering to the nuclear membrane. They divided in stage I of the cycle to yield two classes of spermatogonia: intermediate type and new type A₁. Hence, type A₁–type A₄ spermatogonia were considered as “renewing stem cells.” The fifth class of type A spermatogonia (A₀) was found at all stages of the cycle. Rare, isolated or in pairs, they had oval nuclei with deeply stained chromatin granulations. Seldom seen to divide, they did not appear to be actively involved in cell renewal and were tentatively considered as “reserve stem cells”.     

Duration of the cycle of the seminiferous epithelium and the spermatogonial renewal in the monkey Macaca arctoides

Monkeys were sacrificed at three hours and at 12 days-3 hours after an intraperitoneal injection with ³H-thymidine. Radioautographs were prepared of periodic acid-Schiff-hematoxylin-stained sections of the testes. At three hours, the most evolved labeled germ cells were leptotene spermatocytes in stage VIII of the cycle; at 12 days-three hours the most evolved labeled cells were pachytene spermatocytes in stage IX of the succeeding cycle. Thus, over a 12-day interval, the most evolved labeled spermatocytes had advanced through slightly more than one complete cycle. A quantitative analysis of tubular cross sections containing labeled spermatocytes permitted the calculation of the duration of one cycle of the seminiferous epithelium which turned out to be 11.6 days. The whole process of spermatogenesis which begins with the first spermatogonial mitoses taking place in stage VIII and terminates with the release of spermatozoa taking place in stage VI of the cycle, extends over the duration of 3.8 consecutive cycles and therefore requires approximately 44 days. On the same histological material counts of resting and dividing spermatogonia were performed at various stages of the cycle to determine their mode of proliferation and renewal. Six mitotic peaks were disclosed and were located in stages VIII, X, XII, II, IV and VI of the cycle. Cell counts and labeling indices indicated that the pale type A spermatogonia were the cells dividing during the first two of these six peaks of mitoses while the type B spermatogonia divided during the remaining four peaks of mitoses. The dark type A spermatogonia were not seen to divide during the cycle and may be considered as “reserve stem cells.” Cell counts and cell ratios indicated further that the pale type A spermatogonia divided in stage VIII of the cycle (and some in stage IX) to yield about twice their number of pale type A spermatogonia: of several possible schemes the authors believe that half of these entered a long interphase and became stem cells for the spermatogonia to be formed during the next cycle; these may be referred to as “renewing stem cells”; we consider that the other half of the pale type A spermatogonia arising from stage VIII peak of mitoses divided in stage X to produce type B spermatogonia. The latter cells then could enter the series of four consecutive mitoses during which they doubled their number each time to yield a generation of primary spermatocytes.     

Stages and duration of the seminiferous epithelium cycle in the bat Sturnira lilium

Knowledge of the stages that compose the seminiferous epithelium cycle (SEC) and determination of the duration of spermatogenic processes are fundamental for the accurate quantification of the dynamics of spermatogenesis. The aim of this study was to characterize the stages that compose the SEC of the bat Sturnira lilium, including evaluation of the average frequency of each of these stages throughout the year and calculation of the duration of the spermatogenic process. An ultrastructural characterization of the formation of the acrosomal cap was also performed. Testicular fragments were processed for morphological and immunohistochemical analysis as well as ultrastructural analysis using transmission electron microscopy. According to the tubular morphology method, the SEC in S. lilium is divided into eight stages, following the pattern found in other mammals. Primary spermatocytes were found at zygotene in stage 1 of the cycle. There was no variation in frequency of each of the stages over the seasons, with stage 1 being the most frequent, and stage 7 the least frequent. The duration of one seminiferous epithelium cycle was 3.45 days, and approximately 15.52 days were required for the development of sperm from spermatogonia. Ultrastructural characterization allowed the formation of the acrosomal cap in round spermatids to be monitored. In conclusion, the stages that compose the SEC in S. lilium are generally similar to those described for other mammals, but the duration of the spermatogenic process is shorter than previously recorded for mammals. The presence of primary spermatocytes at zygotene in stage 1 of the cycle is probably due to the longer duration of this stage.     

Histological organization of roe deer testis throughout the seasonal cycle: variable and constant components of tubular and interstitial compartment

Seasonally regulated breeding in roe deer, Capreolus capreolus, is associated with significant changes in testis mass, structure and function. This study has quantified seasonal changes of morphometric parameters and cellular composition in roe deer testis parenchyma. Tissue samples were collected bimonthly during a complete annual cycle. Morphometric parameters of seminiferous tubules were measured and the number of different cell types was counted using a computer-aided image-analyzing system. A scheme of eight tubular epithelium stages for active spermatogenesis was devised according to the spermatid development. Stage I is characterized by the occurrence of new round spermatids, stage IV by spermiation and stage VIII by the meiotic division of spermatocytes. The average diameter of seminiferous tubules varied between 88.4±3.6 µm (February) and 216.8±9.2 µm (June). Also numbers of spermatogonia, spermatocytes and spermatids per tubule cross-section showed considerable seasonal changes. In December and February the germinative epithelium mainly consists of Sertoli cells and spermatogonia. In February, the first differentiated spermatogonia enter meiosis, and in April even spermatids occasionally occur, which reach their highest numbers during the rut in August. Both the expansion and the proportion of tubular and interstitial compartment change seasonally and result in differing cell densities. Assuming numerically constant populations of Sertoli cells and interstitial cells during the entire year, the hypothetical cell numbers per mm² of the tubular and interstitial areas were calculated for the seasonally variable total areas of tissue cross-sections. The concordance of these theoretical values with measured cell densities provided evidence that the total numbers of Sertoli cells, as well as interstitial cells, remain really constant throughout the seasonal cycle. The exact quantification of variable and constant components provides basic data for characterization of cell type and stage-specific processes of spermatogenesis.     

The changes of stage distribution of seminiferous epithelium cycle and its correlations with Leydig cell stereological parameters in aging men

PurposeTo evaluate the changes of stage distribution of seminiferous epithelium cycle and its correlations with Leydig cell stereological parameters in aging men.MethodsPoint counting method was used to analyze the stereological parameters of Leydig cells. The stage number of seminiferous epithelium cycle was calculated in the same testicular tissue samples which were used for Leydig cell stereological analysis.ResultsThe aging group had shown more severe pathological changes as well as higher pathologic scores than the young group. Compared with the control group, the volume density (V_V) and surface density (N_A) of Leydig cells in the aging group were increased significantly. The stage number of seminiferous epithelium cycle in the aging group was decreased coincidently compared to the young group. Leydig cell V_v in the young group has a positive relationship with stages I, II, III, V and VI of seminiferous epithelium cycle, and Leydig cell N_A and numerical density (N_V) were positively related to stage IV. However, only the correlation between N_V and stage II was found in the aging group.ConclusionsThe stage number of seminiferous epithelium cycle was decreased in aging testes. Changes in the stage distribution in aging testes were related to the Leydig cell stereological parameters which presented as a sign of morphological changes.     

Degeneration of germ cells in normal,hypophysectomized and hormone treated hypophysectomized rats

In normal adult rats some germ cells degenerate at several vulnerable steps of spermatogenesis. These are the type A spermatogonia, midpachytene spermatocytes, primary and secondary spermatocytes which degenerate during their respective maturation divisions and step 7 and 19 spermatids. In the present study, these degenerating cells were examined under the electron microscope, and their frequency was determined in toluidine blue stained semithin sections of testes from normal, hypophysectomized (at 5.5 days after operation) and hypophysectomized rats injected with FSH and LH separately or in combination. With the exception of the step 19 spermatids, the degenerating germ cells underwent necrosis in vacuolated spaces delimited by Sertoli cells. In the case of the affected step 19 spermatids, an apical cytoplasmic process of the Sertoli cell initially ensheathed a long segment of their flagellum, and then each degenerating cell was drawn deep in the seminiferous epithelium where it was phagocytozed by the Sertoli cell. Soon after hypophysectomy the incidence of degenerating mid-pachytene spermatocytes, step 7 and 19 spermatids which are present in stages VII or VIII of the cycle of the seminiferous epithelium, increased significantly. In contrast the number of degenerating primary or secondary spermatocytes during the meiotic divisions seen in stage XIV of the cycle or of any other germinal cell was not significantly modified. While the injection of FSH alone had no influence on the number of degenerating cells in hypophysectomized rats, injections of LH at the two doses administered (0.7 μg or 20 μg) reduced significantly the number of degenerating cells seen in stages VII-VIII of the cycle; combined injections of FSH and LH (20 μg) reduced the number of these degenerating cells to the normal low values. Thus it appeared that the mid-pachytene spermatocytes and the step 7 and 19 spermatids, all present in the adluminal compartment of the seminiferous epithelium in stages VII or VIII of the cycle, were more sensitive to the presence of absence of gonadotropic hormones than the other germ cells present in the seminiferous epithelium.     

Seminiferous tubule stages in the prairie dog (Cynomys ludovicianus) during the annual breeding cycle

The male prairie dog (Cynomys ludovicianus) is an annual breeder with complete testicular regression between breeding periods. Knowledge of the seminiferous tubule cycle stages at all phases of the annual cycle is essential for evaluation of testicular effects of endogenous and exogenous hormones. Testis tubule diameter is directly correlated with testicular weight during the annual cycle. Seminiferous tubule stages found during testicular activity start with sperm release and round spermatids in the Golgi stage (I). Then they progress through the cap and acrosome stages (stages II to VI) until elongate spermatids are formed. During these stages preleptotene, leptotene and zygotene cells develop into pachytene cells which mature with the long spermatids (stage VII). Two distinct tubule associations (stages VIII, IX) follow during which the first and second meiotic metaphases occur. These stages are correlated with the middle and late phases of residual lobe retraction and condensation. The last stage (X) has final sperm development and is present with round spermatids that have no Golgi development. During regression changes are initially associated with the seminiferous tubule stages of active testes and end with relocation of Sertoli cell nuclei to a position above the basal layer of spermatogonia. Out of season testes are characterized by few spermatogonial mitoses and absence of viable spermatocytes. In recrudescent testes, Sertoli cell nuclei again become basal, spermatogonia resume mitoses and spermatocytes and spermatids progressively develop. After each cycle of proliferation of germ cells there is sloughing of the most differentiated spermatocytes and spermatids until the final tubule associations of the active testis are present. Anat. Rec. 247:355–367, 1997. © 1997 Wiley-Liss, Inc.     

Seminiferous epithelium damage after short period of busulphan treatment in adult rats and vitamin B12 efficacy in the recovery of spermatogonial germ cells

Several different strategies have been adopted in attempt to recover from chemotherapy‐damaged spermatogenesis that is often seen in oncologic patients. In this study, we have evaluated the impact of short period of exposure to busulphan on the haemogram and seminiferous epithelium of adult rats, focusing on spermatogonial depletion and Sertoli cell (SC) integrity. We then examined whether vitamin B₁₂ supplementation improves the haematological parameters and spermatogonia number. The animals received 10 mg/kg of busulphan (BuG) or busulfan+vitamin B₁₂ (Bu/B₁₂G) on the first and fourth days of treatment. In H.E.‐stained testicular sections, the areas of the seminiferous tubule (ST) and seminiferous epithelium were measured. The number of spermatogonia in H.E‐stained and PCNA‐immunolabelled testicular sections was quantified. The frequency of tubules with abnormal SC nuclei or TUNEL‐positive SC was evaluated. Vimentin immunofluorescence in ST was also evaluated. In BuG and Bu/B₁₂G, the animals showed leukopenia and thrombocytopenia, but the body weight reduced only in BuG. The areas of ST and seminiferous epithelium decreased in Bu/B₁₂G and BuG. In BuG, the number of H.E.‐stained and PCNA‐immunolabelled spermatogonia reduced significantly. The frequency of tubules containing abnormal SC nuclei and TUNEL‐positive SC increased and the vimentin immunoexpression pattern changed. In Bu/B₁₂G, the number of H.E.‐stained or PCNA‐immunolabelled spermatogonia increased fourfold in comparison with BuG. The structural changes in ST after 6 days of busulphan exposure may be associated with the potential effect of this anti‐neoplastic agent on SC. The increased number of spermatogonia in the busulphan‐treated animals receiving vitamin B₁₂ indicates that this vitamin can be an adjuvant therapy to improve the fertility in male cancer patients.     

Apoptotic response of spermatogenic cells to the germ cell mutagens etoposide,adriamycin, and diepoxybutane

In testis, apoptosis is a way to eliminate damaged germ cells during their development. In this study, we evaluated the ability of three germ cell mutagens to induce apoptosis (or programmed cell death) at specific stages of rat seminiferous epithelial cycle. These chemicals include the cancer chemotherapy drugs etoposide and adriamycin and the butadiene metabolite diepoxybutane. According to our results, etoposide is a very potent inducer of apoptosis in male rat germ cells and the cell types most sensitive to it include all types of spermatogonia, zygotene, and early pachytene spermatocytes and meiotically dividing spermatocytes. Also, adriamycin causes an increase in apoptosis at specific stages of seminiferous epithelial cycle and the most sensitive cell types are type A_3–4 spermatogonia, preleptotene, zygotene, and early pachytene spermatocytes. Die poxybutane does not cause any significant increase in the frequency of apoptosis in rat testis. In addition, we studied whether p53 is taking part in the apoptotic response of spermatogenic cells by studying the levels of p53 protein in testis before and after chemical treatment. No accumulation of p53 in testis was seen after treatment with these three chemicals. The expression of two p53-regulated genes, p21^WAF1 and mdm2, was also studied but no increase in the levels of mRNA of these genes was observed after treatment. The results indicate that apoptosis should be taken into consideration when the genotoxic effects of chemicals are evaluated in germ cells. Environ. Mol. Mutagen. 31:133–148, 1998 © 1998 Wiley-Liss, Inc.     

Morphometric studies on rat seminiferous tubules

The goal of this morphometric study was to obtain quantitative information on the seminiferous tubules of Sprague-Dawley rats, including changes seen at various stages of the cycle of the seminiferous epithelium. Tissue from perfusion-fixed testes was embedded in Epon-Araldite; and sections were subjected to morphometric measurements at the light microscopic level, using point counting for volume densities and the Floderus equation for numerical densities. Changes occur in the diameter of the seminiferous tubule, as well as in the volume of the seminiferous epithelium and tubule lumen, from stage to stage during the cycle. A significant constriction of the seminiferous tubule accompanies spermiation. The volume of the seminiferous epithelium per unit length of the tubule begins to increase after stage XIV, and peaks at stage V of the next cycle. The tubule lumen increases dramatically from stages V to VII, at the expense of the epithelium. The number of Sertoli cells is constant per unit length of the seminiferous tubule at all stages of the cycle. This is also true for primary spermatocytes of various developmental phases and for round spermatids from step 1 through step 10 of spermiogenesis. The average number of younger (preleptotene, leptotene, zytgotene) primary spermatocytes per Sertoli cell is 2.34 ± 0.082 (SEM), the number of older (pachytene, diplotene) primary spermatocytes per Sertoli cell is 2.37 ± 0.064, and the ratio of step 1–10 spermatids to Sertoli cells is 7.89 ± 0.27. By studying tangential views of serially sectioned seminiferous tubules at stage V, it is shown that the number of step-17 spermatids associated with each Sertoli cell averages 8.35 ± 0.128, although the counts ranged from 6 to 11. The only appreciable occurrence of cell death after the last spermatogonial mitosis appears to be a 15% loss during the first meiotic division. From our morphometric results, corrected for volume changes during preparation for microscopy, there are 15.7 million (± 0.99 million) Sertoli cells per gram of fresh rat testis. The length of seminiferous tubule per gram of testis is estimated to be 12.4 ± 0.56 meters, and the tubule surface area per gram testis is 119.7 ± 2.57 cm². The daily production of mature spermatids is 9.61 million (± 0.615 million) per gram of testis.     

Early G1 in the male rat meiotic cell cycle is hypersensitive to N-methyl-N-nitrosourea-induced micronucleus formation

The effects of the known carcinogenic and teratogenic agentN-methyl-N-nitrosourea (MNU) were studied on male rat meiosis.To examine possible cell-cycle delay, an immunohistochemicaltechnique based on 5-bromo-2'-deoxyuridine (BrdU) labellingof S-phase cells was developed. BrdU tablets were implantedsubcutaneously in adult male rats. A single i.p. injection of10 mg/kg of MNU was given simultaneously. After 16–22days, preparations of stage 1 of the seminiferous epitheliumwere made and stained immunohistochemically using anti-BrdUantibodies. MNU did not cause any significant meiotic delay,but did cause a slight non-significant reduction of the percentagesof BrdU-labelled step 1 spermatids at 18 days (80%) comparedto controls (95%). In addition, the induction of meiotic micronucleiwas studied after short (1–3 days: late meiotic stages)and long (16–22 days: early spermatocytes and B spermatogonia)exposure times. The peak induction occurs between 21 and 20days, indicating that the M–G₁ transition or the verybeginning of G₁ of the cell cycle of primary spermatocytes arethe most sensitive stages to the action of MNU. The number ofstep 1 spermatids decreased dramatically in animals treatedfor 22 days, denoting a highly toxic effect on type-B spermatogonia.No unscheduled DNA synthesis was detected in any meiotic stageof spermatogenesis by using this BrdU labelling method. Theresults indicate that the spermatid micronucleus test basedon microdissection of seminiferous tubules can accurately pointout the most sensitive stage for chemically induced clastogenesis.More-over, the BrdU-immunohistochemical application enablesthe simultaneous study of cell cycle kinetics. The probablereasons for hypersensitivity of spermatocytes to MNU in thebeginning of G₁ are discussed. ¹To whom correspondence should be addressed     

Spermatogenesis in the vasectomized monkey: Quantitative analysis

The seminiferous epithelium in mature vasectomized Macaca fascicularis was examined quantitatively to assess spermatogenesis. Monkeys were bilaterally vasectomized and controls were bilaterally sham operated. At postoperative periods of 10 and 18 months, groups of monkeys were castrated and their testes prepared for morphologic analysis. Diameters were measured in 100 cross sections of seminiferous tubules from each animal. Numbers of spermatogonia (Ad and Ap), preleptotene spermatocytes, pachytene spermatocytes, and step 7 spermatids, relative to Sertoli cell nucleoli, were counted in stage VII tubules. Tubule diameter and germ cell numbers per Sertoli cell nucleoli were not altered by vasectomy. Our study demonstrates quantitatively that spermatogenesis in the monkey is not inhibited up to 18 months following vasectomy.