Depletion of the spermatogonia from the seminiferous epithelium of the rhesus monkey after X irradiation

In unirradiated testes large differences were found in the total number of spermatogonia among different monkeys, but the number of spermatogonia in the right and the left testes of the same monkey appeared to be rather similar. During the first 11 days after irradiation with 0.5 to 4.0 Gy of X rays the number of Apale spermatogonia (Ap) decreased to about 13% of the control level, while the number of Adark spermatogonia (Ad) did not change significantly. A significant decrease in the number of Ad spermatogonia was seen at Day 14 together with a significant increase in the number of Ap spermatogonia. It was concluded that the resting Ad spermatogonia are activated into proliferating Ap spermatogonia. After Day 16 the number of both Ap and Ad spermatogonia decreased to low levels. Apparently the new Ap spermatogonia were formed by lethally irradiated Ad spermatogonia and degenerated while attempting to divide. The activation of the Ad spermatogonia was found to take place throughout the cycle of the seminiferous epithelium. Serum FSH, LH, and testosterone levels were measured before and after irradiation. Serum FSH levels already had increased during the first week after irradiation to 160% of the control level. Serum LH levels increased between 18 and 25 days after irradiation. Serum testosterone levels did not change at all. The results found in the rhesus monkey are in line with those found in humans, but due to the presence of Ad spermatogonia they differ from those obtained in non-primates.     

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 the white-lipped peccary (Tayassu pecari)

This study aimed to characterize the stages of the seminiferous epithelium cycle by the tubular morphology method, and to determine the number of differentiated spermatogonia generations in the adult white-lipped peccary. Twenty adult white-lipped peccaries, obtained from commercial slaughterhouse, were used. Fragments of the testicular parenchyma were fixed in 3% glutaraldehyde and embedded into a methacrylate resin. The number of germ and Sertoli cells was estimated by the analysis of cell populations in 50 transversal sections of seminiferous tubules in different stages of the cycle. The tubular morphology method allowed the identification of cellular associations characteristic of the eight stages of the seminiferous epithelium cycle in white-lipped peccaries. The results showed the presence of six generations of differentiated spermatogonia in white-lipped peccaries, and that the cell composition of the eight stages of the seminiferous epithelium cycle in this species is very similar to that described for collared peccaries.     

Seminiferous tubule involution in elderly men

The observation of different types of seminiferous tubules (from tubules with normal spermatogenesis to sclerosed tubules) in aging human testes points to the progressive stages of tubular involution in elderly men. The tubules with hypospermatogonesis (reduced number of elongated spermatids) show numerous morphological anomalies in the germ cells, including multinucleated cells. Abnormal germ cells degenerate, causing Steroli cell vacuolation. These vacuoles correspond to dilations of the extracellular spaces resulting from the premature exfoliation of germ cells. Degenerating cells that are phagocytized by Sertoli cells lead to an accumulation of lipid droplets in the Sertoli cell cytoplasm. The loss of germ cells begins with spermatids, but progressively affects the preceding germ cell types, and tubules with maturation arrested at the level of spermatocytes or spermatogonia are observed. Simultaneously, an enlargement of the tunica propria occurs. This leads to the formation of sclerosed tubules, some of which display a low seminiferous epithelium consisting of a few cells--including lipid-loaded Sertoli cells and both Ap and Ad spermatogonia--and others, showing complete sclerosis, are devoid of seminiferous epithelium. The development of tubular involution is similar to that reported after experimental ischemia, which also seems to cause nonspecific effects on the testis such as multinucleate cells, vacuoles, and increased lipids in Sertoli cells.     

Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF

To study self-renewal and differentiation of spermatogonial stem cells, we have transplanted undifferentiated testicular germ cells of the GFP transgenic mice into seminiferous tubules of mutant mice with male sterility, such as those dysfunctioned at Steel (Sl) locus encoding the c-kit ligand or Dominant white spotting (W) locus encoding the receptor c-kit. In the seminiferous tubules of Sl/Sl(d) or Sl(17H)/Sl(17H) mice, transplanted donor germ cells proliferated and formed colonies of undifferentiated c-kit (-) spermatogonia, but were unable to differentiate further. However, these undifferentiated but proliferating spermatogonia, retransplanted into Sl (+) seminiferous tubules of W mutant, resumed differentiation, indicating that the transplanted donor germ cells contained spermatogonial stem cells and that stimulation of c-kit receptor by its ligand was necessary for maintenance of differentiated type A spermatogonia but not for proliferation of undifferentiated type A spermatogonia. Furthermore, we have demonstrated that their transplantation efficiency in the seminiferous tubules of Sl(17H)/Sl(17H) mice depended upon the stem cell niche on the basement membrane of the recipient seminiferous tubules and was increased by elimination of the endogenous spermatogonia of mutant mice from the niche by treating them with busulfan.     

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.     

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.     

Distribution of beta 1 integrin subunit in rat seminiferous epithelium.

We have studied the presence and distribution of beta 1 integrins in the seminiferous epithelium of prepubertal and adult rats. Our immunofluorescence data show that in the adult the antibody recognizes specific areas localized around the heads of elongating and maturing spermatids and above spermatogonia at stages I-VII. The following were found to be negative: a) areas adjacent to spermatogonia at stages IX-XIV and adjacent to spermatocytes and to round spermatids; b) spermiated spermatozoa. In the prepubertal rat, positive tubules are first apparent around Day 17 of age. Immunofluorescence and immunoprecipitation studies show that Sertoli cell monolayers from 3-wk-old rats express beta integrins in vitro.     

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)     

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.     

Loss of sperm in juvenile spermatogonial depletion (jsd) mutant mice is ascribed to a defect of intratubular environment to support germ cell differentiation.

C57BL/6(B6)-jsd/jsd mice are sterile due to the defective spermatogenesis in the testes. To know the cause of the deficient spermatogenesis in B6-jsd/jsd mice, we examined whether the problem is within or outside the seminiferous tubules by transplanting tubules from cryptorchid testes of B6- +/+ mice into B6-jsd/jsd testes or tubules from B6-jsd/jsd mice into testes of (WB x C57BL/6)F1-W/Wv (hereafter, WBB6F1-W/Wv) mice. Type A spermatogonia differentiated into spermatids in seminiferous tubules from cryptorchid testes transplanted into B6-jsd/jsd testes. In contrast, in B6-jsd/jsd tubules transplanted into WBB6F1-W/Wv testes, type A spermatogonia were stimulated to mitotic proliferation, but didn't proceed to any differentiated germ cells. The present results suggest that the cause of the deficient spermatogenesis in B6-jsd/jsd mice is a defect of intratubular environment to support germ cell differentiation.     

The sensitivity of quiescent and proliferating mouse spermatogonial stem cells to X irradiation.

The radiosensitivity of spermatogonial stem cells to X rays was determined in the various stages of the cycle of the seminiferous epithelium of the CBA mouse. The numbers of undifferentiated spermatogonia present 10 days after graded doses of X rays (0.5-8.0 Gy) were taken as a measure of stem cell survival. Dose-response relationships were generated for each stage of the epithelial cycle by counting spermatogonial numbers and also by using the repopulation index method. Spermatogonial stem cells were found to be most sensitive to X rays during quiescence (stages IV-VII) and most resistant during active proliferation (stages IX-II). The D0 for X rays varied from 1.0 Gy for quiescent spermatogonial stem cells to 2.4 Gy for actively proliferating stem cells. In most epithelial stages the dose-response curves showed no shoulder in the low-dose region.     

Long-term effects of irradiation before adulthood on reproductive function in the male rhesus monkey.

Today, many patients, who are often young, undergo total body irradiation (TBI) followed by bone marrow transplantation. This procedure can have serious consequences for fertility, but the long-term intratesticular effects of this treatment in primates have not yet been studied. Testes and epididymides of rhesus monkeys that received doses of 4-8.5 Gy of TBI at 2-4 yr of age were studied 3-8 yr after irradiation. In all irradiated monkeys, at least some seminiferous tubule cross-sections lacked germ cells, indicating extensive stem cell killing that was not completely repaired by enhanced stem cell renewal, even after many years. Testes totally devoid of germ cells were only found in monkeys receiving doses of 8 Gy or higher and in both monkeys that received two fractions of 6 Gy each. By correlating the percentage of repopulated tubules (repopulation index) with testicular weight, it could be deduced that considerable numbers of proliferating immature Sertoli cells were killed by the irradiation. Because of their finite period of proliferation, Sertoli cell numbers did not recover, and potential adult testis size decreased from approximately 23 to 13 g. Most testes showed some dilated seminiferous tubules, indicating obstructed flow of the tubular fluid at some time after irradiation. Also, in 8 of the 29 irradiated monkeys, aberrant, densely packed Sertoli cells were found. The irradiation did not induce stable chromosomal translocations in spermatogonial stem cells. No apparent changes were seen in the epididymides of the irradiated monkeys, and the size of the epididymis adjusted itself to the size of the testis. In the irradiated monkeys, testosterone and estradiol levels were normal, whereas FSH levels were higher and inhibin levels lower when testicular weight and spermatogenic repopulation were low. It is concluded that irradiation before adulthood has considerable long-term effects on the testis. Potential testis size is reduced, repopulation of the seminiferous epithelium is generally not complete, and aberrant Sertoli cells and dilated tubules are formed. The latter two phenomena may have further consequences at still longer intervals after irradiation.     

Arrest of spermatogonial differentiation in jsd/jsd, Sl17H/Sl17H, and cryptorchid mice.

The nature of the spermatogenic arrest in cryptorchid C57Bl mice and in jsd/jsd and Sl17H/Sl17H mutant mice was identified by studying whole mounts of seminiferous tubules. In all three types of mice, virtually only A spermatogonia were found, topographically arranged in clones of 1 to 16 (rarely more) cells. These clonal sizes are typical for undifferentiated spermatogonia. The proportion of these cells lying in chains of more than 2 cells (50-70%) was comparable to that seen in epithelial stages VII-VIII in the normal epithelium. It is concluded that in all three types of mice, spermatogenesis is arrested at the point where the undifferentiated A spermatogonia, specifically A(al) spermatogonia, differentiate into the first generation of the differentiating-type spermatogonia, the A1 spermatogonia. The remaining A spermatogonia were proliferating, but no accumulation of spermatogonia was present, as spermatogonial apoptosis also took place. Spermatogonial clones of all sizes were seen to undergo apoptosis, but there were relatively many large apoptotic clones, indicating that the clones became more vulnerable when they became larger. In contrast to what is seen in the normal epithelium, odd-numbered clones, not composed of 2(n) cells, were present, as well as clumps of 2 or more spermatogonial nuclei in the same cytoplasm, in all three types of mice. This indicates a lack of integrity of spermatogonial clones, also observed in other situations with a relative paucity of cells on the basal membrane. It is concluded that the differentiation of the undifferentiated spermatogonia, affected in all three types of mice as well as in vitamin A-deficient animals, is a rather vulnerable point in the spermatogenic developmental pathway.     

Expression of calbindin-D28k in developing and growing chick testes.

Calbindin, a 28-kDa vitamin D-dependent calcium-binding protein was localized immunohistochemically in developing and growing chick testes. The protein first appeared in the germinal epithelium of developing testes of the eight-day-old embryo and remained therein throughout development. Calbindin was not present in the germinal epithelium after hatching. Calbindin was next detected in the spermatogonia and spermatocytes of one-week-old and growing chick testes. Calbindin-positive spermatogonia and spermatocytes gradually increased in number and staining intensity as the seminiferous tubules further developed. A few interstitial Leydig cells were positive for calbindin from five-week-old and older chicks. Comparison of the time-course of appearance and increase in calbindin content in spermatogonia and spermatocytes with spermatogenesis in chickens suggests that calbindin may be involved in the mitotic process in spermatogonia and spermatocytes.     

Expression of calbindin-D 28k in developing and growing chick testes

Summary Calbindin, a 28-kDa vitamin D-dependent calcium-binding protein was localized immunohistochemically in developing and growing chick testes. The protein first appeared in the germinal epithelium of developing testes of the eight-day-old embryo and remained therein throughout development. Calbindin was not present in the germinal epithelium after hatching. Calbindin was next detected in the spermatogonia and spermatocytes of one-week-old and growing chick testes. Calbindin-positive spermatogonia and spermatocytes gradually increased in number and staining intensity as the seminiferous tubules further developed. A few interstitial Leydig cells were positive for calbindin from five-week-old and older chicks. Comparison of the time-course of appearance and increase in calbindin content in spermatogonia and spermatocytes with spermatogenesis in chickens suggests that calbindin may be involved in the mitotic process in spermatogonia and spermatocytes.     

Renewal and proliferation of spermatogonia during spermatogenesis in the Japanese quail,Coturnix coturnix japonica

Summary Four different types of spermatogonia were identified in the seminiferous tubules of the Japanese quail: a dark type A (A_d), 2 pale A type (A_p1 and A_p2), and a type B. A model is proposed describing the process of spermatogonial development in the quail. The A_d spermatogonia are considered to be the stem cells. Each divides to produce a new A_d spermatogonium and a A_p1 spermatogonium during Stage IX of the cycle of the seminiferous epithelium. An A_p1 spermatogonium produces two A_p2 spermatogonia during Stage II of the cycle, A_p2 spermatogonia produce four type B spermatogonia during Stage VI of the cycle, and type B spermatogonia produce eight primary spermatocytes during Stage III of the cycle. Consequently, 32 spermatids can result from each division of an A_d spermatogonium. Spermatogonial development in the quail differs from the process described in mammals in that there are fewer mitotic divisions and they are all synchronized with the cycle of the seminiferous epithelium. It is suggested that the fewer mitotic divisions explain why a smaller area of the seminiferous tubule is occupied by a cellular association in the quail than in mammals like the rat, ram and bull. The duration of spermatogenesis from the division of the A_d spermatogonia to sperm release from the seminiferous epithelium was estimated to be 12.77 days.     

Morphologic study of the testes from spontaneous unilateral and bilateral abdominal cryptorchid boars

Macroscopical and histological characteristics were examined in both testes from three healthy boars, three boars with unilateral abdominal cryptorchidism on the right side, and three boars with bilateral abdominal cryptorchidism. Abdominal cryptorchidism, unilateral and bilateral, provoked a significant decrease of the weight and volume of the ectopic testes. The scrotal testis of the unilateral cryptorchid boars showed an increase in its volume and weight. Cryptorchidism also induced abnormalities in the histological structure of seminiferous tubules, lamina propria, and interstitial tissue of the abdominal testes. The number of seminiferous tubules decreased; the seminiferous epithelium was constituted by few spermatogonia with an atypical pattern and by abnormal Sertoli cells. The lamina propria showed a variable degree of thickening and collagenization. The interstitial tissue was very developed but displayed a decrease in the Leydig cell population. These abnormalities were more critical in bilateral cryptorchidism than in unilateral cryptorchidism. The scrotal testis of the unilateral cryptorchid boars showed normal appearance, but a decrease of the number of seminiferous tubules was observed. Moreover, the seminiferous tubules showed impaired spermatid maturation. The alterations observed in the abdominal testes of the unilateral and bilateral cryptorchid boars were attributed to defective proliferation and differentiation of Sertoli cells and Leydig cells. The anomalies in the scrotal testis of the unilateral cryptorchid boars were due to disturbances in the Sertoli cell activity.