Ad Spermatogonia Research Articles

Sertoli Cell Number Correlates with Serum Inhibin B in Infant Cryptorchid Boys

Postnatal maturation of Sertoli cells is crucial for male fertility. The aim of this study was to assess the association between the Sertoli cell number per tubule cross-section (SC/T), the serum level of the Sertoli cell-produced inhibin B, and the A-dark spermatogonia number per tubule (Ad/T) in cryptorchid boys. Forty infant cryptorchid boys aged 4-35 months (median: 13 months) were included in the study. During orchiopexy, blood samples for serum inhibin B, luteinizing hormone (LH), and follicle stimulating hormone (FSH) and testicular biopsies were obtained. Histological sections were evaluated by quantitative immunohistochemical and immunofluorescence analysis including VASA and SOX9 (Sertoli cell marker) in order to measure the tubular germ cell number (G/T), Ad/T, and SC/T. The SC/T correlated negatively with age (p < 0.0002) and positively with G/T, Ad/T, inhibin B, FSH, and LH (all p < 0.01). Inhibin B correlated with LH (p < 0.0001), but not with FSH (p = 0.2077). The SC/T:G/T ratio positively correlated with age (p < 0.0001). Boys with Ad spermatogonia at surgery had a higher number of Sertoli cells compared to boys without Ad spermatogonia. In conclusion, a correlation between Sertoli cell number and inhibin B was proven, indicating that inhibin B possibly reflects the function of Sertoli cells in infant cryptorchid boys.

PRDM Histone Methyltransferase mRNA Levels Increase in Response to Curative Hormone Treatment for Cryptorchidism-Dependent Male Infertility.

There is a correlation between cryptorchidism and an increased risk of testicular cancer and infertility. During orchidopexy, testicular biopsies are performed to confirm the presence of type A dark (Ad) spermatogonia, which are a marker for low infertility risk (LIR). The Ad spermatogonia are absent in high infertility risk (HIR) patients, who are treated with a gonadotropin-releasing hormone agonist (GnRHa) to significantly lower the risk of infertility. Despite its prevalence, little is known about the molecular events involved in cryptorchidism. Previously, we compared the transcriptomes of LIR versus HIR patients treated with and without hormones. Here, we interpreted data regarding members of the positive regulatory domain-containing (PRDM) family; some of which encoded histone methyltransferases that are important for reproduction. We found there were lower levels of PRDM1, PRDM6, PRDM9, PRDM13, and PRDM14 mRNA in the testes of HIR patients compared with LIR patients, and that PRDM7, PRDM9, PRDM12, and PRDM16 were significantly induced after GnRHa treatment. Furthermore, we observed PRDM9 protein staining in the cytoplasm of germ cells in the testes from LIR and HIR patients, indicating that the mRNA and protein levels corresponded. This result indicated that the curative hormonal therapy for cryptorchidism involved conserved chromatin modification enzymes.

Curative GnRHa treatment has an unexpected repressive effect on Sertoli cell specific genes

BackgroundFollicle stimulating hormone and testosterone stimulate Sertoli cells to support germ cell function and differentiation. During mini-puberty, when gonadotropin (GnRH) stimulates increases in plasma luteinizing hormone (LH) and testosterone levels, gonocytes are transformed into Ad spermatogonia. In cryptorchidism, impaired gonadotropin secretion during mini-puberty results in insufficient LH and testosterone secretion, impaired gonocyte transition to Ad spermatogonia, and perturbed Sertoli cell proliferation. Treatment with a gonadotropin-releasing hormone agonist (GnRHa/Buserelin) induced gonocytes to differentiate into Ad spermatogonia and rescued fertility. The present study evaluated the impact of low LH secretion on Sertoli cell function by comparing differential gene expression data between testes with low LH that lacked Ad spermatogonia (Ad-) and testes that completed mini-puberty (Ad+). Furthermore, we analyzed changes in the transcription of selected Sertoli cell specific genes in response to GnRHa treatment.ResultsAd- testes showed reduced expression of nine out of 40 selected Sertoli cell specific genes compared to Ad+ testes. GnRHa treatment repressed most of the Sertoli cell specific genes, including the inhibins, but it increased the expression of genes that regulate apoptosis (FASLG) and proliferation (GDNF).ConclusionsImpaired-minipuberty with decreased LH and testosterone levels affected Ad and Sertoli cell development through positive and negative regulation of morphoregulatory and apoptotic genes. GnRHa treatment had a repressive effect on most Sertoli cell specific genes, which suggested that Sertoli cells underwent a cellular rearrangement. We propose that gonadotropin-dependent increases in FASLG and GDNF expression drove Sertoli cell proliferation and germ cell self-renewal and supported the transition of gonocytes to Ad spermatogonia, independent of inhibins.

DMRTC2, PAX7, BRACHYURY/T and TERT Are Implicated in Male Germ Cell Development Following Curative Hormone Treatment for Cryptorchidism-Induced Infertility.

Defective mini-puberty results in insufficient testosterone secretion that impairs the differentiation of gonocytes into dark-type (Ad) spermatogonia. The differentiation of gonocytes into Ad spermatogonia can be induced by administration of the gonadotropin-releasing hormone agonist, GnRHa (Buserelin, INN)). Nothing is known about the mechanism that underlies successful GnRHa treatment in the germ cells. Using RNA-sequencing of testicular biopsies, we recently examined RNA profiles of testes with and without GnRHa treatment. Here, we focused on the expression patterns of known gene markers for gonocytes and spermatogonia, and found that DMRTC2, PAX7, BRACHYURY/T, and TERT were associated with defective mini-puberty and were responsive to GnRHa. These results indicate novel testosterone-dependent genes and provide valuable insight into the transcriptional response to both defective mini-puberty and curative GnRHa treatment, which prevents infertility in man with one or both undescended (cryptorchid) testes.

Hormonal Aspects of the Pathogenesis and Treatment of Cryptorchidism.

A normal functioning hypothalamic-pituitary-testicular axis is required for normal testicular descent. The percentage of cases that result from a disturbance in this axis remains controversial. Much has yet to be learnt about cryptorchidism, but is seems that the existence of A dark spermatogonia (Ad spermatogonia) is essential for later fertility. Bilateral cryptorchid patients have a high risk of later infertility, even though they undergo early surgery for cryptorchidism. It is possible today to distinguish-to a certain extent-between three different groups of cryptorchid patients based on testicular histology, gonadotropins, and inhibin B at the time of early surgery: Group 1, patients suspected of prepubertal transient hypothalamic-pituitary-testicular hypofunction and a high risk of later infertility; Group 2, patients with hypergonadotropic hypogonadism and a primary testicular dysfunction; and Group 3, patients with normal histology and normal serum levels of inhibin B and gonadotropins at the time of early surgery and a low risk of later infertility. Given the potential adverse effects of hormonal treatment, attention should be directed toward small doses of adjuvant gonadotropin-releasing hormone (GnRH) treatment for those who might benefit the most, that is, bilateral cryptorchid boys at early surgery without evidence of normal maturation of gonocytes into Ad spermatogonia. Optimally, gonadotropin levels in such patients should be measured to ensure that levels are not compensatory elevated, thereby supporting the suspicion of hypothalamic-pituitary-testicular hypofunction. Studies of GnRH-supplementary treatment should include testicular biopsy at surgery and at follow-up in childhood as well as examinations of fertility potential in adulthood.

Prospective study of histological and endocrine parameters of gonadal function in boys with cryptorchidism

A transient increase in gonadotropins and testosterone during mini-puberty causes gonocytes to differentiate into Ad spermatogonia, which establish male germ cell memory and male-specific DNA methylation pathways. Over half of patients with unilateral cryptorchidism and the majority of patients with bilateral cryptorchidism display an abnormal spermiogram, which indicates that unilateral cryptorchidism is a bilateral disease; therefore, it represents a serious andrological problem. The aim of this study was to evaluate relationships between hormonal parameters and testicular biopsy findings in boys with cryptorchidism. Seventy-one boys (median age 15 months; range 7-65 months) who underwent orchidopexy (24% had bilateral cryptorchidism) were tested for serum LH, FSH, and inhibin B. With ipsilateral testis biopsy histology, we determined the tubular fertility index (TFI), Ad spermatogonia counts, and Ad/tubular index (Ad/T). We compared age groups (<18 vs. >18 months old); groups with and without Ad spermatogonia; groups with unilateral and bilateral cryptorchidism; and extreme groups with high infertility risk (HIR; n=12; TFI <0.2; Ad/T=0) and low infertility risk (LIR; n=9; TFI >0.9; Ad/T>0.02). Of the specimens, 38% had no Ad spermatogonia. Age was significantly negatively correlated with TFI and Ad/T, but positively correlated with FSH. Median LH values were significantly higher in LIR than in HIR groups. Unilateral and bilateral cryptorchidism showed similarTFI, Ad/T, and hormone concentrations. The areas under ROC curves for FSH, LH, and inhibin B(0.66,0.601, and 0.599, respectively) showed low diagnostic value for predicting HIR (no Ad spermatogonia). Our observation of lower plasma LH levels in the group with the most pronounced testicular pathology was the opposite of what we would have expected if testicular pathological changes were caused by a primary gonadal defect. Therefore, low plasma LH levels in the HIR group confirmed the notion that this group of patients with cryptorchidism had hypogonadotropic hypogonadism. The estimated incidence of defective mini-puberty in boys with cryptorchidism could be as high as 50%. Testicular biopsies from boys with cryptorchidism lacked Ad spermatogonia. Fertility parameters worsened with age. Significantly lower basal LH in the HIR group indicated hypogonadotropic hypogonadism. Serum hormone levels could not predict histological biopsy findings.

Incidence of High Infertility Risk among Unilateral Cryptorchid Boys

Background: Increasing evidence of progressive damage to germ cell development in boys with cryptorchidism suggests recommending surgery until one year of age. However, despite early and successful orchidopexy, cryptorchid boys with impaired mini-puberty will suffer from infertility. We reviewed changes in the timing of surgery during the past decade and the incidence of unilateral cryptorchid boys with defective mini-puberty. Methods: Medical registries were reviewed for all patients who were operated on for cryptorchidism at the main pediatric urological center of the country. The ages of surgery in cases of unilateral cryptorchidism were compared between the years 2000-2001 and 2012-2013. A high risk of infertility was considered when no Ad spermatogonia were found. Two groups were compared: group I - operated on until the age of 1.5 years and group II - older than 1.5 years. Results: The average age at operation decreased from 5.3 to 4.1 years. Forty-six biopsies in boys with unilateral cryptorchidism were made during orchidopexy on undescended testicles. Overall, 44% in group I and 50 % in group II (p > 0.05) had no Ad spermatogonia. Conclusions: The average age of operation for cryptorchidism has decreased, but remains far above the recommended age. The high prevalence of histologically proven risk of infertility underscores the necessity of more education regarding the importance of earlier surgery and the research on hormonal prevention of infertility.

216 SPERMATOGONIAL STEM CELL ACTIVITY IN CRYPTORCHID TESTES

INTRODUCTION AND OBJECTIVES: Male infertility is a serious complication of cryptorchidism. In this study, we focused on early stage spermatogenesis and spermatogonial stem cell (SSC) activity in undifferentiated spermatogonia in cryptorchid testes to elucidate the mechanism of infertility caused by cryptorchidism. METHODS: Histological findings and expression patterns of the stem cell marker, undifferentiated embryonic cell transcription factor 1 (UTF1), were examined in a unilateral cryptorchid rat model induced by flutamide and in cryptorchid patinents. We removed both unilateral descended testis (UDT) and contralateral descended testis (DT), respectively, from cryptorchid and normal rats (control) 18 days postcoitum (dpc) to 144 days postpartum (dpp). Human testicular tissues were also excised from 5 boys (8–20 months old) during orchiopexy between January 2003 and December 2008. Hematoxylin and eosin staining, real-time RT-PCR, western blot analysis, and immunohistochemical analysis with UTF1 were performed to assess early stage spermatogenesis and SSC activity. RESULTS: Histopathological analysis showed that gonocyte differentiation into early A spermatogonia occurred at 9 dpp; however, this transformation was disturbed in UDT. Quantitative study revealed that UTF1 expression decreased with testicular development, but did not disappear in adulthood. Immunohistochemical analysis showed that all gonocytes and some early type A spermatogonia expressed UTF1 in rat DT. Furthermore, the UTF1-negative early A spermatogonia/positive early A spermatogonia ratio was significantly higher in the UDT group (0.69 0.04) than in the control (0.46 0.10; p 0.037) and DT (0.44 0.05; p 0.022) groups, indicating decreased early A spermatogonia with SSC activity in cryptorchid testes. In humans, UTF1-negative Ad spermatogonia/positive Ad spermatogonia ratios in UDT were also significantly higher than in the DT (0.55 0.36, 0.16 0.07, respectively; p 0.006). CONCLUSIONS: In cryptorchid testes, the differentiation from gonocytes into early A spermatogonia, as well as the stem cell activity of early A spermatogonia, were altered during the early stage of spermatogenesis, suggesting that the loss of SSC activity in cryptorchid rats resulted in altered spermatogenesis, thus interfering with fertility.

Cryptorchidism-pathophysiology, treatment concept and long-term follow up results

Undescended testes (cryptorchidism), incomplete descent at birth of one or both testes affects 1-3% of boys and is the most common endocrine disease in childhood. If untreated, the undescended testis may develop progressive failure of spermatogenesis and has a higher incidence of carcinoma that may manifest in adolescence and adulthood. Endocrine and primary end organ failures are the two etiological factors most frequently held responsible for the increased incidence of infertility in unilateral and bilateral cryptorchidism. The cryptorchid testis has a typical histology showing depletion of germ cells and impaired maturation of gonocytes accompanied by intestinal fibrosis and Leydig cell atrophy. In 70% of males with isolated cryptorchidism, hypogonadotropic hypogonadism is the cause of undescended testes. The number of Ad spermatogonia that develop in infancy during the period of mini puberty (the stem cells for mature spermatozoa) is severely reduced . The ultimate aim of all therapy for cryptorchidism is to have both testes in the scrotum and to achieve normal fertility. Hormonal treatment is recommended for all patients prior to orchidopexy and those at high risk of infertility (no Ad spermatogonia). Treatment includes Kryptocur for inducing epididymo-testicular descent and Buserelin (LH-RHa) for prevention of infertility. If unsuccessful surgery should be performed before patients second birthday.

EGR4 Is a Master Gene Responsible for Fertility in Cryptorchidism

The purpose of early medical or surgical treatment of boys with undescended testes is to prevent the development of infertility. However, early and successful surgery cannot prevent infertility in cryptorchid boys who lack type A dark (Ad) spermatogonia. The aim of this study was to compare the gene expression pattern of patients with completed transformation of gonocytes into Ad spermatogonia, associated with low infertility risk, with patients that had failed to undergo this process and had a high infertility risk. Genes expressed in the 16 cryptorchid testes were estimated using Affymetrix whole-genome microarray and compared to the expression profiles from four contralateral gonads of boys with unilateral testicular agenesis. Whole-genome expression profiling showed that boys in the high infertility risk group according to testicular histology, showed decreased or lack of expression of most of the genes essential for hypothalamo-pituitary-testicular axis function relative to low or intermediate risk group as well as controls. In particular, EGR4, which is involved in regulating the secretion of luteinizing hormone, was virtually not expressed. Thus, we found multiple differences in gene expression between the high and low infertility risk groups, confirming the importance of an intact hypothalamo-pituitary testicular axis and EGR4 in fertility development.

Regional differences in maturation of germ cells of cryptorchid testes: role of environment

To investigate differences in maturation of germ cells in cryptorchid testes in three different regions. A total of 103 consecutive patients were operated for unilateral undescended testis in Vojvodina, from March 2006 until September 2007, and had a testicular biopsy performed. Germ cells were counted, and the presence of Ad spermatogonia was noted. Biopsies were compared to biopsies of similar patients from two different regions: Philadelphia, USA (130), and Liestal, Switzerland (55 patients). In Vojvodina, 84.5% of patients had Sertoli cells only, or some spermatogonia, but no Ad spermatogonia, and 15.5% had Ad spermatogonia. In Philadelphia, 59.3% of patients had poor testicular histology, and 40.7% had Ad spermatogonia. In Liestal, 61.8% of patients had no, or some, spermatogonia, but no Ad spermatogonia, and 38.2% had Ad spermatogonia. There was a difference (p = 0.000025) between the patients with normal testicular histology from Philadelphia and those from Vojvodina, as well as between the patients from Vojvodina and Liestal (p = 0.0027). The reduction in the number of germ cells in patients with cryptorchidism from Vojvodina is more pronounced than patients from either Switzerland or USA. This is a unique observation, since such a study has not been published yet.

Is the prohibition of hormonal treatment for cryptorchidism, as suggested by the Nordic consensus group, justifiable?

Recently, the Nordic consensus on the treatment of undescended testis prohibited the use of hormones because of possible long-term adverse effects on spermatogenesis (1). This conclusion was based on articles published by groups from Finland and Denmark analyzing testicular histology and apoptosis of the germ cells (1). Even if human chorionic gonadotropin (HCG) treatment induces increased apoptosis of the germ cells, it is still irrelevant for subsequent fertility outcome. The follow-up spermiogram performed in these patients showed no significant difference in fertility outcome between the treated and untreated groups. The second reason was the observation of decreased numbers of germ cells in cryptorchid boys aged 1–3 years who were previously unsuccessfully treated with HCG in comparison to the untreated group (1). These results, although statistically significant, are again irrelevant for the fertility outcome because the germ cell count in both groups was below 0.2 germ cells per tubular cross-section. If the germ cell count is <0.2, the majority of patients will develop infertility irrespective of whether they had only surgery or hormonal pretreatment in addition to orchidopexy. Abnormal contralateral testis is ideal for evaluating the effect of hormonal treatment because there is no effect from abnormal position and increased temperature to interfere with the results. In contrast to the concerns raised by the Nordic consensus group, the results of our recent study showed that hormonal treatment for undescended testis improved the histopathology of the contralateral testis without harming the germ cells (2). Furthermore, neoadjuvant gonadotropin relasing hormone (GNRH) treatment was found to improve the fertility index in prepubertal cryptorchidism (3). Maximum salvage of active germinal tissue is achieved by treating cryptorchidism before the end of the first year of life (3). We entirely agree with the Nordic consensus group that infertility is the primary concern for the treatment of boys with unilateral or bilateral undescended testes. This especially because analysis of contralateral descended testis in unilateral cryptorchidism demonstrated that cryptorchidism is a bilateral disease, and infertility in cryptorchidism is endocrinopathy of mini-puberty (4,5). It is important to realize that male fertility potential depends on the presence of Ad spermatogonia (5–7). Development of Ad spermatogonia from gonocytes, which takes place during first months of life, was shown to be testosterone dependent (8). However, early and seemingly successful orchidopexy does not improve fertility in a substantial number of cryptorchid males because it does not address the underlying pathophysiology of cryptorchidism, namely, the impaired transformation of gonocytes into Ad spermatogonia (6,7). Furthermore, successfully surgically treated patients at risk for infertility after 6 months of luteinizing hormonerelasing hormone analogue (LH-RHa) treatment had a lasting increase in the number of germ cells in their cryptorchid testis (9). The efficacy of the LH-RHa treatment was the best in boys younger than 7 years (9,10). Treatment with LH-RHa normalized sperm concentration in 86% of unilateral cryptorchid males, who were in the high-risk group for developing infertility (Fig. 1). All males in the untreated group (surgery only) were severely oligospermic, with 20% being azoospermic (10). This profoundly changes our current concept of cryptorchidism treatment. For the first time, it is possible to demonstrate that infertility caused by cryptorchidism, which is believed to be a congenital malformation, can be successfully corrected if adequately treated. During the last 35 years, histological studies have contributed the most to our understanding of the aetiology of cryptorchidism. Only the comparison of histology and hormonal levels exemplify hypogonadotropic hypogonadism in the majority of cryptorchid boys. Why the Nordic consensus group did not take the histopathologic findings into consideration remains unclear. Nonetheless, their recommendation as to when a testicular biopsy should be obtained is passe. A testicular biopsy is the only sure way of identifying those cryptorchid boys who need to be treated hormonally with LH-RHa following successful surgery;

The importance of both an early orchidopexy and germ cell maturation for fertility

Delayed orchidopexy for undescended testes has been associated with abnormal testicular histology, but the effect on later fertility is unknown. We aimed to establish the importance of the first postnatal maturational step-ie, the transformation of gonocytes into Ad (dark) spermatogonia on fertility. We matched histological findings of the testes from 31 patients who had undergone an early orchidopexy with their total number of sperm. If Ad spermatogonia were present, 17 (94%) of 18 (95% CI 72.7-99.9) of the men had a total sperm count of 40 x 10(6)/ejaculate or greater. By contrast, despite successful early surgery, if Ad spermatogonia were absent, 12 (92%) of 13, (64.0-99.8) patients had abnormal spermiograms. Thus, the transformation of gonocytes into Ad spermatogonia is crucial for male fertility.

Importance of Early Postnatal Germ Cell Maturation for Fertility of Cryptorchid Males

Patients/Method: Twenty-seven adults who underwent an orchidopexy before 2 years of age have recently had their spermiograms analyzed. Results: Sixty-three percent of them had a normal sperm count; their germ cells had undergone the second stage of maturation and adult dark (Ad) spermatogonia were present at the time of surgery 20–25 years earlier. In contrast, 37% of them were infertile despite a seemingly successful orchidopexy; they had impaired second-step germ cell maturation and Ad spermatogonia were absent at orchidopexy. Conclusions: Transformation of gonocytes into Ad spermatogonia is crucial for fertility. This underscores the importance of a testicular biopsy at orchidopexy in order to identify those who could benefit from early LH-RH treatment after orchidopexy.

The Chance for Fertility in Adolescent Boys After Corrective Surgery for Varicocele

We estimated the efficacy of a surgical procedure in adolescent patients with varicocele and left testicular atrophy. A total of 25 young men (mean age 23.6 years) had a complete andrological followup for 10 years after successful ligation of the spermatic vein (according to Ivanissevich) to correct left varicocele and testicular atrophy. Levels of follicle-stimulating hormone, luteinizing hormone and testosterone in the plasma were normal in all cases. Patients were characterized into 2 distinct groups: group A--11 patients with asthenoteratospermia and group B--14 with a normal spermiogram. There was a significant difference between groups A and B in the number of sperm per ejaculate (32 x 10(6) in A and 234.9 x 10(6) in B, p < 0.001). Preoperatively there was no difference between these 2 groups regarding testicular atrophy of the left compared to the right testis (87% in A and 86.4% in B) and the comparative testicular histology of biopsies since the number of Ad spermatogonia per tubule was considerably reduced in all patients (right side 0.68 +/- 0.71 and left side 0.38 +/- 0.39 in A, and right side 0.15 +/- 0.17 and left side 0.14 +/- 0.11 in B). In both groups there was no distinguishable difference in the degree of atrophy of seminiferous tubules or Leydig cells in both testes, or patient age at surgery (mean 13.3 years in A and 12.8 years in B). While the left testis of patients in group B caught up in size to the contralateral testis, the left testicular volume in patients in group A was only 80% of the right testicular volume (p < 0.03, the Fisher test). One patient from group B required a second surgical procedure because of a relapse. Persistent atrophy of the left testis in group A indicates that some patients benefit suboptimally from the conventional surgical procedure.

Regulation of spermatogonial proliferation.

In nonprimates the spermatogonial compartment can be subdivided into (morphologically) undifferentiated spermatogonia and differentiating spermatogonia. Each cycle of the seminiferous epithelium the proliferative activity of the undifferentiated spermatogonia is stimulated, probably by factors secreted by Sertoli cells. Subsequently, during a period of active proliferation many Aal spermatogonia are formed. In the normal situation around epithelial stage III, proliferation is inhibited by the differentiating spermatogonia by way of a negative feedback system, probably involving a spermatogonial chalone. Then most of the Aal spermatogonia formed differentiate into Al spermatogonia. For this differentiation vitamin A, or factors secreted by Sertoli cells under the influence of vitamin A, is/are necessary. In the normal situation there is no regulation of the density of the undifferentiated spermatogonia. Different tubular areas can contain widely varying numbers of stem cells and other undifferentiated spermatogonia and consequently can produce widely varying numbers of Al spermatogonia. Only in extreme circumstances, such as after irradiation, the stem cells change the ratio between self-renewing and differentiating divisions in favor of self-renewal. Furthermore, in this situation the proliferation of the undifferentiated spermatogonia is not inhibited at epithelial stage III because of the lack of differentiating spermatogonia. Density regulation does take place during the development of the differentiating spermatogonia. In the chinese hamster it appeared that despite the variation in the numbers of Al spermatogonia produced in different areas, the density of the preleptotene spermatocytes was very much the same. It was found that the even distribution of spermatocytes in the epithelium was achieved by a density-dependent degeneration of differentiating spermatogonia in such a way that many of the latter cells degenerated in high density areas and only few or none in low density areas. In primates the undifferentiated spermatogonia can be subdivided into Ap and Ad spermatogonia. Both Ap and Ad spermatogonia can be seen to be topographically arranged in clones of 1 or 2n cells in situations in which their density is low. The Ad spermatogonia do not proliferate, but after cell loss these cells were found to transform into Ap spermatogonia that start to proliferate. The Ap spermatogonia only divide once every epithelial cycle, renewing themselves and giving rise to B spermatogonia. In the monkey the number of Ap spermatogonia could be increased by FSH treatment. Hence, there may be a correlation between FSH levels and the numbers of Ap spermatogonia. Fu

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.

Regulation of the proliferation of spermatogonial stem cells.

The proliferating cells in the seminiferous epithelium can be subdivided into the (morphologically) undifferentiated and the differentiating spermatogonia. In turn the undifferentiated spermatogonia can be subdivided according to their topographical arrangement into clones consisting of one cell, the A-single (As) spermatogonia, two cells, the A-paired (Apr) spermatogonia or groups of 4, 8 or 16 cells, the A-aligned (Aal) spermatogonia. Most likely the As spermatogonia are the stem cells of spermatogesis. When these cells divide their daughter cells can either migrate away from each other and become two new stem cells or stay together as Apr spermatogonia. The Apr spermatogonia can divide further to form Aal spermatogonia. Although suggestions have been made for the existence of a class of long-cycling stem cells with a higher probability for self-renewal, all cell kinetic data are still compatible with a homogeneous population of stem cells, the proliferative activity of which varies during the cycle of the seminiferous epithelium. Radiobiological studies have revealed that the radiosensitivity of the stem cells varies with their proliferative activity and that there is no particular class of radioresistant stem cells. In both rat and Chinese hamster the cell cycle time of the undifferentiated spermatogonia appeared to be much longer than that of the differentiating spermatogonia. All types of undifferentiated spermatogonia have the same minimum cell cycle time. However, the As spermatogonia, the stem cells, generally divide after longer intervals. The proliferative activity of the undifferentiated spermatogonia varies during the cycle of the seminiferous epithelium. At a certain moment during the epithelial cycle the undifferentiated spermatogonia, including the stem cells, are stimulated to proliferate. After a period of active proliferation the Apr and Aal spermatogonia are arrested in G1 phase possibly by the production of a chalone by the differentiating spermatogonia. In both rat and Chinese hamster the As spermatogonia are less sensitive towards this inhibition and continue to proliferate for some time longer. In the normal epithelium the probability of self-renewal of the stem cells varies with their proliferative activity in such a way that is low during active proliferation. In the normal epithelium there appeared to be no regulatory mechanism to ensure an even density of the stem cells. A large variation in stem cell density was found between areas. However, after heavy cell loss, for example after irradiation, the probability of self-renewal was found to be close to 100%. In primates the undifferentiated spermatogonia are composed of the so-called Ap and Ad spermatogonia.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.