Stages and duration of the cycle of the seminiferous epithelium in oncilla (Leopardus tigrinus, Schreber, 1775)

Six adult Leopardus tigrinus (oncilla) were studied to characterize stages of the seminiferous epithelium cycle and its relative frequency and duration, as well as morphometric parameters of the testes. Testicular fragments were obtained (incisional biopsy), embedded (glycol methacrylate), and histologic sections examined with light microscopy. The cycle of the seminiferous epithelium was categorized into eight stages (based on the tubular morphology method). The duration of one seminiferous epithelium cycle was 9.19 d, and approximately 41.37 d were required for development of sperm from spermatogonia. On average, diameter of the seminiferous tubules was 228.29 μm, epithelium height was 78.86 μm, and there were 16.99 m of testicular tubules per gram of testis. Body weight averaged 2.589 kg, of which 0.06 and 0.04% were attributed to the testis and seminiferous tubules, respectively. In conclusion, there were eight distinct stages in the seminiferous epithelium, the length of the seminiferous epithelium cycle was close to that in domestic cats and cougars, and testicular and somatic indexes were similar to those of other carnivores of similar size.     

Spermatogenesis in white-lipped peccaries (Tayassu pecari)

We describe here morphological and functional analyses of the spermatogenic process in sexually mature white-lipped peccaries. Ten sexually mature male animals, weighing approximately 39 kg were studied. Characteristics investigated included the gonadosomatic index (GSI), relative frequency of stages of the cycle of seminiferous epithelium (CSE), cell populations present in the seminiferous epithelium in stage 1 of CSE, intrinsic rate of spermatogenesis, Sertoli cell index, height of seminiferous epithelium and diameter of seminiferous tubules, volumetric proportion of components of the testicular parenchyma and length of seminiferous tubules per testis and per gram of testis. The GSI was 0.19%, relative frequencies of pre-meiotic, meiotic and post-meiotic phases were, respectively 43.6%, 13.8% and 42.6%, general rate of spermatogenesis was 25.8, each Sertoli cell supported an average 18.4 germinative cells, height of seminiferous epithelium and diameter of seminiferous tubules were, respectively, 78.4 microm and 225.6 microm, testicular parenchyma was composed by 75.8% seminiferous tubules and 24.2% intertubular tissue, and length of seminiferous tubules per gram of testis was 15.8m. These results show that, except for overall rate of spermatogenesis, the spermatogenic process in white-lipped peccaries is very similar to that of collared peccaries, and that Sertoli cells have a greater capacity to support germinative cells than most domestic mammals.     

Cycle and duration of the seminiferous epithelium in puma (Puma concolor)

Puma or sussuarana (Puma concolor) is the second largest feline in the American continent and has an ample latitudinal distribution in very diverse habitats. In relation to its conservation status, the puma is considered an extinction-threatened species. The study of the testis morphology and the spermatogenic process in a species is fundamental for establishing the physiologic patterns that will make possible the selection of the protocols for assisted reproduction. A number of peculiarities associated with the reproductive biology of specific species such as the duration of spermatogenic process can be used to determine the frequency of sperm collection. Nine adult male pumas maintained in captivity were used to determine the relative frequency of stages in the seminiferous epithelium cycle. Three of them received intra-testicular injections of 0.1ml tritiated thymidine to determine the duration of the seminiferous epithelium cycle, and were subjected to biopsy 7 days later. The cycle of the seminiferous epithelium in puma was didactically described into eight stages by the tubular morphology method. The total duration of one seminiferous epithelium cycle in puma was calculated to be 9.89 days, and approximately 44.5 days are required for development of spermatozoon from spermatogonia. The duration of spermiogenesis, prophase and other events of meiosis were 14.08, 15.20 and 1.79 days, respectively. The relative frequency of the pre-meiotic, meiotic and post-meiotic phases were 3.98, 1.79 and 4.12 days, respectively.     

Evaluation of the seminiferous epithelial cycle, spermatogonial kinetics and niche in donkeys (Equus asinus)

Kinetics of spermatogonia as well as localization in niches have been described in rodents, but rarely in large animals or in species of economical interest. In this regard, and envisioning the possibility of spermatogonial transplantation from donkeys (Equus asinus) to mules (Equus mulus mulus), many variables that may contribute for an enhanced understanding of the spermatogonial biology in donkeys were investigated. Testes from five adult donkeys were routinely processed for high-resolution light microscopy. Donkey seminiferous epithelium can be divided in XII stages based on the development of the acrosomal system. In addition, spermatogonial morphology and morphometric analysis were performed allowing the characterization of two groups of spermatogonia: undifferentiated (A_und) and differentiating (A₁, A₂, A₃, B₁ and B₂). A_und spermatogonia were present along all XII stages of the seminiferous epithelium cycle of this species, whereas differentiating spermatogonia were only at specific stages. Number of differentiating spermatogonia gradually increased as the cycle progressed, despite the apparent rigid regulation of the balance between mitosis and apoptosis throughout the spermatogenic process. Understanding of spermatogonial biology and kinetics in donkeys, revealed that type A_und spermatogonia are located in specific microenvironments, the spermatogonial niches. The present results enhance understanding of spermatogonial biology in donkeys providing information about subtypes, morphology, number and mitosis/apoptosis along the seminiferous epithelium cycle.     

Duration of the cycle of the seminiferous epithelium and its stages in the rhesus monkey (Macaca mulatta)

Doses of 1 Gy or more of X-irradiation killed all B spermatogonia present in the testis, and during the first 3 weeks after irradiation, virtually no new B spermatogonia were formed. The number of Apale spermatogonia decreased during the first cycle of the seminiferous epithelium while the number of Adark spermatogonia only began to decrease during the second cycle after irradiation. In this study, the duration of the cycle of the seminiferous epithelium in the rhesus monkey was estimated to be 10.5 days (SE = 0.2 days). This was determined following the depletion of germinal cells in the seminiferous epithelium during the first 3 weeks after irradiation. The duration of each of the 12 stages of the cycle was also determined. Our observations of the progress of germinal cell depletion revealed that after a dose of X-irradiation sufficient to kill all B spermatogonia, all spermatocytes disappeared from the testis within about 17 days, and all spermatids within about 31 days.     

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.     

Synchronization of the seminiferous epithelium after vitamin A replacement in vitamin A-deficient mice

The effect of vitamin A deficiency and vitamin A replacement on spermatogenesis was studied in mice. Breeding pairs of Cpb-N mice were given a vitamin A-deficient diet for at least 4 wk. The born male mice received the same diet and developed signs of vitamin A deficiency at the age of 14-16 wk. At that time, only Sertoli cells and A spermatogonia were present in the seminiferous epithelium. These spermatogonia were topographically arranged as single and paired cells and as clones of 4, 8 and more cells. A few mitoses of single, paired, and clones of 4 A spermatogonia were found, which were randomly distributed over the seminiferous epithelium. When vitamin A-deficient mice were treated with retinol-acetate combined with a normal vitamin A-containing diet, spermatogenesis restarted again synchronously. Only a few successive stages of the cycle of the seminiferous epithelium were present up to at least 43 days after vitamin A replacement. After 20 days, 98.3% of the seminiferous tubules were synchronized, showing pachytene spermatocytes as the most advanced cell type, mostly being in epithelium stages IX-XII. After 35 and 43 days, spermatogenesis was complete in 99.6% of the tubular cross sections, and most tubular cross sections were in stages IV-VII of the cycle of the seminiferous epithelium. The degree of synchronization was comparable or even higher than found in rats. The rate of development of the spermatogenic cells between 8 and 43 days after vitamin A replacement seemed to be similar to that in normal mice. Assuming that the rate of development of the spermatogenic cells is also normal during the first 8 days after vitamin A replacement, it can be deduced that the preleptotene spermatocytes, present after 8 days, were A spermatogonia in the beginning of stage VIII at the moment of vitamin A replacement. These results indicate that the mouse can be used as a model to study epithelial stage-dependent processes in the testis.     

Reproduction in the male llama (Lama glama), a South American camelid. I. Spermatogenesis and organization of the intertubular space of the mature testis

The cycle of the seminiferous epithelium of the llama was divided into eight stages, using as criteria the shape and distribution of the germ cell nuclei, the location of the spermatids, the presence of meiotic figures and the release of spermatozoa from the tubular wall. Cell populations making up each stage are described. The relative frequencies of stages 1 through 8 were: 9.86, 12.46, 17.65, 14.12, 5.81, 8.09, 13.04 and 18.89%, respectively. In the seminiferous epithelium, spermatogonia of the A and B type are present and twelve spermiogenic steps can be recognized. Interstitial (Leydig) cells are packaged together forming large masses and elongated cords and share the intertubular space with one or two central great lymphatic vessels and few capillaries. Season male Leydig cells contain lipid droplets in their cytoplasm and show a marked immunoreactive testosterone reaction.     

Cyclic formation and decay of the blood-testis barrier in the mink (Mustela vison), a seasonal breeder

The correlations between the germ cell population and the blood-testis barrier were studied during puberty and throughout the reproductive cycle in a seasonal breeder, the mink. A classification of 12 stages, corresponding to the cellular associations appearing during the cycle of the seminiferous epithelium, was proposed and used to identify the stages of the cycle in pubertal mink. In adult mink, the reproductive cycle was divided into two spermatogenic phases--an active phase lasting 9 months, and an inactive phase lasting 3 months. The active spermatogenic phase was broken down into three distinct periods: the first spermatogenic wave, the peak of spermatogenic activity, and the last spermatogenic wave. Degenerating germ cells were found in comparable and relatively low proportions during puberty and during the first and last spermatogenic waves of the adult reproductive cycle. The permeability of the blood-testis barrier to intravascularly infused electron-opaque tracers (i.e., horseradish peroxidase and lanthanum) was tested at the time of the first spermatogenic wave at puberty and throughout the reproductive cycle of the adult. The relationship between epithelial permeability and germ cell populations prevailing during puberty and during the first and last spermatogenic waves of the adult active phase was the same. During puberty, the establishment of the blood-testis barrier did not coincide with the appearance of a particular step of meiosis but was correlated with the development of a tubular lumen. In adult mink, the barrier cyclically decayed during the last wave of the active spermatogenic phase and reformed during the first wave of the next active phase. The decay and the reformation of the barrier were not coincident with the appearance or disappearance of a particular generation of the germ cell population from the seminiferous epithelium but were correlated with cyclic cytological changes in Sertoli cells and the rhythmic development and occlusion of the lumen. During the peak months of the active spermatogenic phase, however, a blood-testis barrier secluded spermatogonia and young spermatocytes from older generations of germ cells. It is concluded that during puberty and also during the first and last spermatogenic wave of the adult mink reproductive cycle, the development of germ cells is possible in the absence of a competent, impermeable blood-testis barrier, and the transient presence of a permeable epithelial barrier does not initiate an autoimmune response of sufficient magnitude to cause destruction of the seminiferous epithelium.     

Repopulation of the seminiferous epithelium of the rhesus monkey after X irradiation

Repopulation of the seminiferous epithelium became evident from Day 75 postirradiation onward after doses of 0.5, 1.0, and 2.0 Gy of X rays. Cell counts in cross sections of seminiferous tubules revealed that during this repopulation the numbers of Apale (Ap) spermatogonia, Adark (Ad) spermatogonia, and B spermatogonia increased simultaneously. After 0.5 Gy the number of spermatogonia increased from approximately 10% of the control level at Day 44 to 90% at Day 200. After 1.0 and 2.0 Gy the numbers of spermatogonia increased from less than 5% at Day 44 to 70% at Days 200 and 370. The number of Ad and B spermatogonia, which are considered to be resting and differentiating spermatogonia, respectively, already had increased when the number of proliferating Ap spermatogonia was still very low. This early inactivation and differentiation of a large part of the population of Ap spermatogonia slows down repopulation of the seminiferous epithelium of the primates. By studying repopulating colonies in whole mounts of seminiferous tubules various types of colonies were found. In colonies consisting of only A spermatogonia, 40% of the A spermatogonia were found to be of the Ad type, which indicates that even before the colony had differentiated, 40% of the A spermatogonia were inactivated into Ad. Differentiating colonies were also found in which one or two generations of germ cells were missing. In some of those colonies it was found that the Ap spermatogonia did not form any B spermatogonia during one or two cycles of the seminiferous epithelium, while in other colonies all Ap spermatogonia present had differentiated into B spermatogonia. This indicates that the differentiation of Ap into B spermatogonia is a stochastic process. When after irradiation the density of the spermatogonia in the epithelium was very low, it could be seen that the populations of Ap and Ad spermatogonia are composed of clones of single, paired, and aligned spermatogonia, which are very similar to the clones of undifferentiated spermatogonia in non-primates.     

Cell proliferation in the seminiferous and epididymal epithelia of Sus domesticus

It is important to understand the proliferative activity of the different structures of the male reproductive apparatus in livestock species, such as Sus domesticus, to ensure reproductive efficiency. The main aims of this study were (a) to evaluate the proliferative activity of the spermatogonia in the different stages of the seminiferous cycle and (b) to study the cell proliferation in the epididymal epithelium in each region, identifying the different cells involved. For this, the testes and epididymis of three healthy, sexually mature Sus domesticus boars were used. The organs were processed for light microscopy, and immunohistochemical techniques were used to detect proliferating cell nuclear antigen. The cells immunostaining positively and negatively for proliferating cell nuclear antigen were counted and several parameters and indexes were calculated to evaluate the proliferation in both epithelia, taking into account the stage of the seminiferous epithelium cycle, and, in the case of the epididymal epithelium, the different regions and cells are the same. Finally, a contrast analysis of equality between pairs of means was carried out followed by a least significant differences test, in which differences were considered significant at P < 0.05. In the seminiferous epithelium, the greatest total number of spermatogonia and proliferating spermatogonia was observed in the postmeiotic stages (mainly VII and VIII). The proliferation index of the spermatogonia increased from the meiotic to postmeiotic stages. As regards the epididymal epithelium, the total proliferation index was higher in the caput. In each region, the clear and principal cells showed the highest proliferation index with respect to the total number of cells counted, whereas the proliferation index of each cell with respect to the same type was higher in the clear cells, followed by the narrow and principal cells. In conclusion, the proliferative activity of spermatogonia in the seminiferous epithelium of Sus domesticus is stage-dependent, and mainly occurs in the postmeiotic stages. In the epididymal epithelium, proliferative activity takes place in several cell types and is dependent on the anatomical region of the epididymis. We think that these results may be of importance for understanding the pathologic or reproductive processes in which cell proliferation is involved in the male reproductive system.     

Renewal of spermatogonia in the monkey (Macaca fascicularis)

Populations of different types of spermatogonia and their mitotic activity were analyzed in the monkey Macaca fascicularis: 3 adults aged 5-6 yr and 3 young aged 2-3 mo. Two young and two adult monkeys received injections of 3H-thymidine for radioautographic study of the relationships between Type A spermatogonia: dark Type A (Ad), pale Type A (Ap) and transition Type A (At). In the adult the number of Ad and At spermatogonia did not change significantly throughout the seminiferous epithelium cycle. The number of Ap spermatogonia doubled at Stage VII, and half divided at Stage IX to give rise to B1 spermatogonia. The durations of the seminiferous epithelium cycle and spermatogenesis were estimated as 10.5 days and 42 days respectively. In the young and adult monkeys, some Ap spermatogonia and a lesser number of At spermatogonia were labeled one h after injection of precursor. At longer intervals after injection, the number of labeled At spermatogonia increased significantly, and some Ad as well as Ap spermatogonia were also labeled. These results indicate that Ap spermatogonia are renewal stem cells, and Ad spermatogonia are reserve stem cells. The differences in labeling after isotope exposure suggest that Ap cells may give rise successively to At and Ad cells.     

Kinetics of spermatogenesis in megachiropteran bat, Rousettus leschenaulti (Desmarset): seminiferous epithelial cycle, frequency of stages, spermatogonial renewal and germ cell degeneration.

The paper describes in detail the cytomorphology of different types of germ cells, the 10 typical cellular associations or stages of the cycle of seminiferous epithelium (CSE), frequency of appearance of these stages, pattern of spermatogonial stem cell renewal and per cent degeneration of various germ cells in R. leschenaulti. Of the 14 steps of spermiogenesis (stained with PAS-haematoxylin) the first 10 were associated with the stages I-X, whereas, the remaining were found in association with one of the first six stages. The frequency of appearance of the various stages ranged from 3.84% (stage V) to 19.84% (stage I). These observations indicate that stage V is of shortest duration and stage I is of the longest duration in the bat. Five types of spermatogonia (A1, A2, A3, In and B) were identified based on their shape, size and nuclear morphology. Type A spermatogonia are oval with a large nucleus containing 1 or 2 nucleoli. The chromatin showed progressive condensation from A1 to A3 so that the latter appeared darkest among all the A type spermatogonia. The In type derived from A3 are smaller but appear darker than A3 due to heterochromatin crusts along the inner border of the nucleus. The B type spermatogonia derived from In are round and possess single nucleolus. The B type spermatogonia divided mitotically before entering meiosis or the actual production of the primary spermatocytes. The various spermatogonia divided mitotically at fixed stages of the cycle giving rise to their next generations.(ABSTRACT TRUNCATED AT 250 WORDS)     

THE SPERMATOGONIAL STEM CELL POPULATION IN ADULT RATS

Radioautographed whole mounted seminiferous tubules from adult rat testes were used to analyse undifferentiated type A spermatogonia at various intervals up to 81 hr following a single injection of ³H-TdR. the data obtained led to the identification of the spermatogonial stem cell and to the formulation of a new model for spermatogonial renewal and differentiation. Undifferentiated type A cells were morphologically alike, but were topographically classified as (1) isolated or (2) paired and aligned. Although labeled isolated A cells were scattered over most stages of the seminiferous epithelium, their proliferative activity varied with the stage; their labeling index was 20-30% in stages I and II, but less than 1% in stages VII and VIII. By tracing the labeled divisions of isolated A spermatogonia in time, it was seen that some daughter cells became separated from one another to form two new isolated cells, while others remained together as paired A spermatogonia. Analysis of two successive waves of labeled mitoses revealed that most paired A spermatogonia continued to proliferate forming four aligned A cells, many of which divided again to produce a chain of eight and so on. the greatest incidence of labeling among paired and aligned A spermatogonia occurred in stages XIII-III. In stage I, where the labeling index was 50%, the calculated proliferative fraction was 1 for these spermatogonia. Between stages II and V, they began to leave mitotic cycle, and during stage V this entire cohort morphologically transformed into A₁ spermatogonia. Labeled metaphase curves for undifferentiated A spermatogonia were distinct from any of the curves previously constructed for the six classes of differentiating spermatogonia, especially because of particularly long S and G₂ phases in the former. the cell cycle time of paired and aligned A cells was 55 hr, compared to an average of 42 hr for differentiating types A₂ to B.     

Feedback regulation of the proliferation of the undifferentiated spermatogonia in the Chinese hamster by the differentiating spermatogonia

In the seminiferous epithelium the differentiating spermatogonia proliferate following a very strict synchronous pattern, and undergo the S phase during parts of particular epithelial stages. The undifferentiated spermatogonia do not divide synchronously and display maximum proliferative activity in stages XI-III. Hence the S-phase-specific cytotoxic agent Ara-C kills different proportions of these two cell types dependent on the epithelial stage. We have studied the effect of several combinations of degrees of cell loss to both compartments on proliferation of the undifferentiated spermatogonia. It was found that when the differentiating spermatogonia are removed, the proliferation of the undifferentiated spermatogonia is not inhibited at epithelial stage III, as seen in controls. However, when the undifferentiated spermatogonia were already arrested in G1, removal of the differentiating spermatogonia did not evoke proliferation again. When the population of undifferentiated spermatogonia was reduced in an area where the differentiating spermatogonia were left intact, the inhibition of the proliferation of undifferentiated spermatogonia took place around stage III as usual. It is concluded that in the normal adult seminiferous epithelium, the length of the period of active proliferation of the undifferentiated spermatogonia is regulated by negative feedback from the differentiating spermatogonia.     

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.     

Effect of testicular extracts on proliferation of spermatogonia in the mouse

Two intraperitoneal injections with an interval of 4 h between them, of rat testicular extract into adult male mice causes a decrease in the production of A spermatogonia in the compartment of undifferentiated A (As, Apr and Aal) spermatogonia. A significant decrease in the total number of A spermatogonia in stages VII and VIII of the cycle of the seminiferous epithelium was found at 2, 4 and especially 5, 7 and 8 days after treatment. Extracts of rat liver and rat spleen were without effect. In addition, an extract of rat testis containing very few spermatogonia had no effect. It was concluded that the active substance in the extract is synthesized and/or specifically accumulated in the spermatogonial compartment of the testis. Thus the active substance is tissue-specific but not species-specific, since extracts of both rat and bull testes were effective after injection into mice. It is inferred from the data that the effect of injection of testicular extracts is unlikely to be due to cytotoxicity, hormonal changes in the tubular environment or to an immunologic reaction, but is probably due to a spermatogonial chalone. This chalone partially inhibits proliferation of early type A spermatogonia in the normal mouse testis.     

Late morfofunctional alterations of the Sertoli cell caused by doxorubicin administered to prepubertal rats

ABSTRACT: BACKGROUND: Doxorubicin is a potent chemotherapeutic drug used against a variety of cancers. It acts through interaction with polymerases and topoisomerase II and free radical production. Doxorubicin activity is not specific to cancer cells and can also damage healthy cells, especially those undergoing rapid proliferation, such as spermatogonia. In previous studies our group showed that etoposide, another topoisomarese II poison, causes irreversible damage to Sertoli cells. Thus, the aim of this study was to address the effects of doxorubicin on Sertoli cell morphology and function and on the seminiferous epithelium cycle when administered to prepubertal rats. METHODS: Prepubertal rats received the dose of 5 mg/Kg of doxorubicin, which was fractioned in two doses: 3 mg/Kg at 15dpp and 2 mg/Kg at 22dpp. The testes were collected at 40, 64 and 127dpp, fixed in Bouin's liquid and submitted to transferrin immunolabeling for Sertoli cell function analysis. Sertoli cell morphology and the frequency of the stages of the seminiferous epithelium cycle were analyzed in PAS + H-stained sections. RESULTS: The rats treated with doxorubicin showed reduction of transferrin labeling in the seminiferous epithelium at 40 and 64dpp, suggesting that Sertoli cell function is altered in these rats. All doxorubicin-treated rats showed sloughing and morphological alterations of Sertoli cells. The frequency of the stages of the seminiferous epithelium cycle was also affected in all doxorubicin-treated rats.Conclusions and DiscussionThese data show that doxorubicin administration during prepuberty causes functional and morphological late damage to Sertoli cells; such damage is secondary to the germ cell primary injury and contributed to enhance the spermatogenic harm caused by this drug. However, additional studies are required to clarify if there is also a direct effect of doxorubicin on Sertoli cells producing a primary damage on these cells.     

Feedback Regulation of the Proliferation of the Undifferentiated Spermatogonia In the Chinese Hamster By the Differentiating Spermatogonia

In the seminiferous epithelium the differentiating spermatogonia proliferate following a very strict synchronous pattern, and undergo the S phase during parts of particular epithelial stages. the undifferentiated spermatogonia do not divide synchronously and display maximum proliferative activity in stages XI-III. Hence the S-phase-specific cytotoxic agent Ara-C kills different proportions of these two cell types dependent on the epithelial stage. We have studied the effect of several combinations of degrees of cell loss to both compartments on proliferation of the undifferentiated spermatogonia. It was found that when the differentiating spermatogonia are removed, the proliferation of the undifferentiated spermatogonia is not inhibited at epithelial stage III, as seen in controls. However, when the undifferentiated spermatogonia were already arrested in G₁, removal of the differentiating spermatogonia did not evoke proliferation again. When the population of undifferentiated spermatogonia was reduced in an area where the differentiating spermatogonia were left intact, the inhibition of the proliferation of undifferentiated spermatogonia took place around stage III as usual. It is concluded that in the normal adult seminiferous epithelium, the length of the period of active proliferation of the undifferentiated spermatogonia is regulated by negative feedback from the differentiating spermatogonia.