Effect of the GnRH-agonist leuprolide on colonization of recipient testes by donor spermatogonial stem cells after transplantation in mice

Gonadotropin-releasing hormone (GnRH)-agonist or antagonist treatment supports recovery of spermatogenesis after irradiation damage in the rat and appears to be beneficial to colonization of recipient testes after spermatogonial transplantation from fertile donors to the testes of infertile recipients in rats and mice. In the present study, we quantified the effect of treatment of recipient mice with the GnRH-agonist leuprolide acetate on the extent of colonization by donor spermatogonial stem cells in the recipient testis. Testis cells from mice carrying transgenes, which produce beta-galactosidase in spermatogenic cells, were used as donor cells for transplantation to allow for quantification of donor spermatogenesis in the recipient testis by staining for enzyme activity. Donor cell colonization 3 months after transplantation was compared between recipients receiving leuprolide in different treatment protocols and untreated control mice. Two injections of leuprolide 4 weeks apart prior to transplantation with as little as 3.8 mg/kg resulted in a pronounced improvement in the number of donor-derived spermatogenic colonies as well as in the in the area of recipient seminiferous tubules occupied by donor cell spermatogenesis. Improved colonization efficiency by treatment with GnRH-agonist can make the technique of spermatogonial transplantation applicable to situations when only low numbers of donor cells are available.     

Single administration of di(n-butyl) phthalate delays spermatogenesis in prepubertal rats

Morphological alterations in seminiferous tubules caused by single administration of di(n-butyl) phthalate (DBP) in 3-week-old rats were investigated throughout the first wave of spermatogenesis. Single administration of DBP (500 mg/kg) showed progressive detachment and displacement of spermatogenic cells and disappearance of tubular lumen at 3 h after treatment, and then showed thin seminiferous epithelia and wide tubular lumen at day 1 (D1). At D1, quite significant numbers of apoptotic spermatogenic cells were detected, and then they gradually decreased in accordance with the passage of time. In contrast, the testes revealed lower weight gain, even after completion of first wave of spermatogenesis in the DBP-treated group, compared to the control. In order to clarify whether spermatogenic cells differentiate into mature spermatids in the DBP-treated rats, immunohistochemical staining for Hsc 70t, a specific marker for elongate spermatids, was carried out. As a result, the decrease in mature spermatids in the DBP-treated testes, compared to the control, was demonstrated. For example, at D20 (41-day-old) after treatment, the most advanced spermatids in the tubules from rats in the DBP-treated groups were steps 2-4, while those of the control were steps 12-13. Moreover, in some tubules, pachytene spermatocytes were the most advanced spermatogenic cell. At D30 (51-day-old) after treatment, maturation of spermatogenic cells in the DBP-treated rats proceeded further, and the most advanced spermatids in tubules were steps 8-9, while those of the control were steps 15-19. These results lead us to the postulation that a single administration of DBP to prepubertal rats delays maturation of spermatogenic cells, even after completion of first wave of spermatogenesis.     

Crystalloid formation in Leydig cells of rats (Rattus fuscipes)

Summary The ultrastructure of Leydig cells in a seasonally breeding rodent, Rattus fuscipes, was studied in the breeding and non-breeding season and compared with Leydig cell morphology after suppression of gonadotrophin secretion induced by hypophysectomy or chronic administration of testosterone. Serum luteinizing hormone (LH) and testosterone (T) were measured and in-vitro T production by testes was assessed by stimulation with human chorionic gonadotrophin (hCG). In non-breeding wild-trapped rats and rats with experimental suppression of gonadotrophins, the Leydig cells were atrophied and exhibited variable amounts of cytoplasmic lipid and crystalloid inclusions, the latter commonly dominating the cytoplasmic area. Compared with fertile rats, serum LH and hCG-stimulated T production of experimentally regressed rats was significantly reduced, confirming structural features indicative of Leydig cell inactivity. Atrophy of Leydig cell nuclei was accompanied by the formation of unusual intranuclear vesicles sometimes containing small crystalloids. Ultrastructural analysis suggested transfer of the vesicles to the cytoplasm where their unification gave rise to much larger crystalloid bodies. Crystalloids occurred when serum LH was depressed and with either full (T treatment) or arrested spermatogenesis (hypophysectomy) suggesting that their formation is governed by pituitary function and is not dependent upon the degree of spermatogenic activity.     

Expression and function of class B scavenger receptor type I on both apical and basolateral sides of the plasma membrane of polarized testicular Sertoli cells of the rat

Class B scavenger receptor type I (SR-BI), a multiligand membrane protein, exists in various organs and cell types. In the testis, SR-BI is expressed in two somatic cell types: Leydig cells and Sertoli cells. Unlike interstitially localized Leydig cells, Sertoli cells present within the seminiferous tubules keep contact with spermatogenic cells and form the tight junction to divide the seminiferous epithelium into the basal and adluminal compartments. In this study, the expression and function of SR-BI in rat Sertoli cells were examined with respect to dependency on the spermatogenic cycle, the plasma membrane polarity, and the pituitary hormone follicle-stimulating hormone (FSH). When the expression of SR-BI was histochemically examined with testis sections, both protein and mRNA were already present in Sertoli cells during the first-round spermatogenesis and continued to be detectable thereafter. The level of SR-BI mRNA expression in Sertoli cells was lower at spermatogenic stages I-VI than at other stages. SR-BI was present and functional (in mediating cellular incorporation of lipids of high density lipoprotein) at both the apical and basolateral surfaces of polarized Sertoli cells. Finally, SR-BI expression at both the protein and mRNA levels was stimulated by FSH in cultured Sertoli cells. These results indicate that SR-BI functions on both the apical and basolateral plasma membranes of Sertoli cells, and that SR-BI expression in Sertoli cells changes during the spermatogenic cycle and is stimulated, at least in cultures, by FSH.     

A Biological Model of Accelerated Senescence. 3. Histological Characteristics of Age-Related Changes of Spermatogenic Epithelium in SAM (Senescence-Accelerated Mouse) Mice

The data characterizing the age-related morphological changes in the spermatogenic epithelium of SAMP1 (senescence-accelerated prone) and SAMR1 (senescence-accelerated resistant) mice are presented. In many tubules, early spermatogenesis was accompanied by the formation of many morphologically abnormal germ cells on histological sections of the gonads of sexually immature (three–four weeks) mice of both strains. At this stage, destructive processes in the spermatogenic epithelium were more pronounced in SAMR1 mice. In sexually mature (two–three months) SAMP1 and SAMR1 mice, spermatogenesis as a whole proceeded normally. The first signs of regressive changes in the inner structure of most tubules (disintegration, detachment of spermatogenic epithelium from basal membrane) and morphology of germ cells (pycnosis, nuclear and cytoplasmic vacuolization) were found in SAMP1 mice at the age of six–seven months. In the older age groups (9–10 and 12–15 months), all types of spermatogenic cells were represented in both SAMP1 and SAMR1 mice, but most of these cells were atypical. Mitotic figures were recorded in a population of highly differentiated Sertoli cells.     

Stability of spermatogenic synchronization achieved by depletion and restoration of vitamin A in rats

Studies of synchronization of spermatogenesis following vitamin A deficiency have suggested that this may provide an in vivo model for the study of stage-dependent changes in hormonal action and protein secretion within the seminiferous epithelium. However, until now, no information on the stability or durability of this condition has been available. In this study, 200 seminiferous tubules from each of 40 rats (including controls) were classified according to their spermatogenic stage after withdrawal and replenishment of vitamin A. Following 15 wk withdrawal and subsequent replenishment of vitamin A, spermatogenesis was initiated in a synchronous fashion. This synchrony remained stable for more than 10 cycles of the seminiferous epithelium (2.5 spermatogenic cycles). In association with the extended period of vitamin A deficiency, a proportion of tubules (30%) showed morphological characteristics of either Sertoli cells only or Sertoli cells plus spermatogonia with occasional pachytene spermatocytes. During the 11-wk period of observation in this study, no significant change in proportions of damaged tubules were observed. Testicular testosterone concentrations, although elevated with respect to controls, showed no correlation with the stage of the cycle of the seminiferous epithelium observed, whereas pituitary and serum follicle-stimulating hormone levels were elevated, probably due to the number of damaged tubules observed. The persistence of synchrony in spermatogenesis following vitamin A treatment suggests that this model is applicable for studies of paracrine actions within the testis. However, the decreased ratio of synchrony observed with time may provide evidence that duration of the individual stages of the cycle of the seminiferous epithelium might be subject to temporal variation, leading to a progressive desynchronization of spermatogenesis in this model system.     

Hormone pretreatment enhances recovery of spermatogenesis in rats after neutron irradiation.

Previous studies showed that a 6-week pretreatment of rats with testosterone plus estradiol enhanced the recovery of spermatogenesis 9 weeks after gamma irradiation, resulting in a dose-modifying factor (DMF) of about 2. To test whether the effect of the hormone treatment was mediated through changes in oxygen tension, thiol levels or DNA repair, we irradiated the testes of rats with neutrons, which depend less on these factors than does low-LET radiation for their cytotoxic action. Control rats and rats treated with testosterone plus estradiol were irradiated with 0.7-2.7 Gy of cyclotron-generated high-energy neutrons. The recovery of spermatogenesis was assessed 9 weeks after irradiation by testis weights, sperm counts and the tubule repopulation indices. Greater recovery of spermatogenesis was observed for all end points, with a DMF of about 2 for rats treated with testosterone plus estradiol compared to the irradiated, cholesterol-treated rats. The equal protection factors for neutrons and gamma rays indicate that oxygen, thiols and repair of DNA damage are unlikely to be involved in the protective effect of the hormone treatment.     

Loss and recovery of androgen receptor protein expression in the adult rat testis following androgen withdrawal by ethane dimethanesulfonate

Androgens are especially important for the maintenance of spermatogenesis in adulthood and the experimental withdrawal of testosterone (T) production by ethane dimenthanesulfonate (EDS) is a valuable tool for studying androgen-dependent events of spermatogenesis. The aim of the present study was to investigate the specific changes in immunoexpression of androgen receptor (AR) in the testis in relation to degeneration and regeneration of Leydig cell (LC) population and seminiferous epithelium. Immunohistochemistry for AR and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) as well as TUNEL assay for apoptosis were performed on testicular sections of control and EDS-treated rats. Serum LH and T levels were measured by RIA. Our results revealed a total loss of AR immunoexpression from the nuclei of Sertoli (SCs), LCs and peritubular cells during the first week after EDS administration and that coincided with severe drop in T levels. Two weeks after EDS administration, the AR expression was recovered in these cells but normal stage-specificity in SCs was replaced by uniform intensity of AR immunostaining at all the stages of the spermatogenic cycle. The stage-specific pattern of androgen expression in SCs with a maximum at stages VII-VIII appeared 5 weeks after treatment. LC immunoreactivity for 3beta-HSD at different time points after EDS administration correlated with values of T concentration. The maximal germ cell apoptosis on day 7 was followed by total loss of elongated spermatids 2 weeks after EDS treatment. Regeneration of seminiferous epithelium 3 weeks after EDS administration and onwards occurred in tandem with the development of new LC population indicated by the appearance of 3beta-HSD-positive cells and gradual increase in T production. The specific changes in AR after EDS including their loss and recovery in Sertoli cells paralleled with degenerative and regenerative events in Leydig and germ cell populations, confirming close functional relationship between Sertoli, Leydig and germ cells.     

Effects and interactions of LH and LHRH agonist on testicular morphology and function in hypophysectomized rats

The effects of single or combined daily treatment with an LHRH agonist and low or high doses of LH upon the testes of adult hypophysectomized rats were studied for up to 2 weeks in which changes in testicular histology, particularly the interstitial tissue, were examined by morphometry and related to functional assessment of the Leydig cells in vivo and in vitro. Compared to saline-treated controls, LHRH agonist treatment did not alter testis volume or the composition of the seminiferous epithelium or any of the interstitial tissue components although serum testosterone and in-vitro testosterone production by isolated Leydig cells were significantly reduced. With 2 micrograms LH for treatment, testis volume was increased, spermatogenesis was qualitatively normal, total Leydig cell volume was increased, serum testosterone values were initially elevated but subsequently declined and in-vitro testosterone production was enhanced. Testis volume with 20 micrograms LH treatment was unchanged compared to saline treatment, the seminiferous epithelium exhibited severe disruption but total Leydig cell volume was greatly increased due to interstitial cell hyperplasia. This group showed elevated serum testosterone concentrations and major increases in testosterone production in vitro. Treatment with LHRH agonist with either dose of LH resulted in reduced testis volume, moderate to very severe focal spermatogenic disruption and increased total Leydig cell volume although serum testosterone values and in-vitro testosterone production were markedly reduced compared to control rats. It is concluded that, in the absence of the pituitary, LHRH agonist fails to disrupt spermatogenesis and the previously described antitesticular action of LHRH agonists in intact rats is therefore dependent upon the presence of LH, which alone or in combination with LHRH agonist, may focally disrupt spermatogenesis in hypophysectomized rats whereas the Leydig cells undergo hyperplasia. The findings show that impairment of spermatogenesis is accompanied by alterations of the interstitial tissue and suggest that communication between these two compartments is involved in the regulation of testicular function.     

Differential expression of N-Myc downstream regulated gene 2 (NDRG2) in the rat testis during postnatal development

N-Myc downstream regulated gene 2 (NDRG2) is expressed in the testis of adult animals and is involved in cell differentiation and development. However, little is known about the expression pattern of NDRG2 in the testis during postnatal development. Here, we show that NDRG2 is consistently expressed in Leydig cells in the rat testis during postnatal development. However, its expression has also been detected at a high frequency in spermatogenic cells of the seminiferous tubules in young rats but at a much lower frequency in adult rats. Furthermore, high levels of NDRG2 expression have been found in methoxyacetic-acid-induced apoptotic germ cells, particularly at stages X–XIII of the seminiferous epithelium cycle of adult rats. Interestingly, high levels of NDRG2 expression have also been observed in spontaneously apoptotic germ cells in the seminiferous tubules of young and adult rats. Thus, the expression of NDRG2 in germ cells seems to alter during spermatogenesis. These findings suggest that NDRG2 regulates testicular development and spermatogenesis in rats and is involved in the physiological and pathological apoptosis of germ cells. Wu-Gang Hou, Yong Zhao, and Lan Shen contributed equally to this study. This study was supported by the Natural Science Foundation of China (2006: no. 30600340; 2007: no. 30771138; 2008: no. 30871309).     

Androgenic gland cell structure and spermatogenesis during the molt cycle and correlation to morphotypic differentiation in the giant freshwater prawn, Macrobrachium rosenbergii

The freshwater prawn Macrobrachium rosenbergii shows three male morphotypes: blue-claw males (final stage having high mating activity), orange-claw males (transitional stage showing rapid somatic growth), and small males (primary stage showing sneak copulation). This morphotypic differentiation is considered to be controlled by androgenic gland hormone, which is probably a peptide hormone. However, its physiological roles are not fully understood. In the present study, we examined the correlation of androgenic gland cell structure to spermatogenic activity and morphotypic differentiation histologically in M. rosenbergii. spermatogenic activity showed close correlation to the molt cycle in orange-claw males and small males. spermatogonia increased in number in the late premolt stage, becoming spermatocytes in the postmolt stage, and spermatocytes differentiated into spermatozoa in the intermolt and early premolt stages. Ultrastructure of the androgenic gland was additionally compared among the molt stages, but, distinct histological changes were not observed in relation to spermatogenesis during the molt cycle. On the other hand, among the three morphotypes, the androgenic gland was largest in the blue-claw males, containing developed rough endoplasmic reticulum in the cytoplasm. These results suggest that, during spermatogenesis which is related to the molt cycle, the androgenic gland hormone is at rather constant levels and plays a role in maintaining spermatogenesis rather than directly regulating the onset of a specific spermatogenesis stage and that, during the morphotypic differentiation, the androgenic gland is most active in the blue-claw males and plays a role in regulating the observed high mating activity in M. rosenbergii.     

Touch imprint cytology in biopsy of the infertile testis.

OBJECTIVE: To identify different cell types in the testis by using touch imprint cytology and to compare the cytologic findings to the histopathologic diagnosis in infertile men. STUDY DESIGN: This prospective study used touch imprint preparations and included 20 infertile men. The biopsy material obtained was stained with toluidine blue, May-Grünwald-Giemsa stain and Papanicolaou stain. The cytologic results for oligospermic, normospermic and azospermic men were compared to the specific histopathologic diagnosis. The proportion of spermatogenic versus Sertoli cells was calculated. The scores were compared between three groups based on the results of the histologic biopsy: normal spermatogenesis, hypospermatogenesis and incomplete spermatogenic arrest. RESULTS: The mean ratio of the spermatogenic cells versus Sertoli cells was statistically significantly different in the three groups (P < .01). The mean ratio of spermatogenic cells to Sertoli cells was higher in cases with normal spermatogenesis than in cases with hypospermatogenesis and incomplete spermatogenic arrest, revealing a statistical difference (P<.01). This ratio was not statistically significantly different between the hypospermatogenesis and incomplete spermatogenic arrest groups. CONCLUSION: A cytologic demonstration of germinal cells by using touch imprint preparations may be a guide for histologic diagnosis.     

Histology of the normal and retained equine testis

Abdominal, inguinal and scrotal testes of horses were examined grossly and by light microscopy. An average of 1.5, 2.3 and 4.6 layers of spermatogenic cells, and mean seminiferous tubule diameters of approximately 66.2, 83.6 and 146.6 micron in the abdominal, inguinal and scrotal testes, respectively, were recorded. The interstitial spaces and the number of interstitial cells (of Leydig) seemed to be increased while spermatogenesis appeared to be arrested in the retained testes. Early spermatocytes were the most mature stages of the spermatogenic cells in the retained testes. An extensive vacuolation of spermatogenic cells was evident in these testes. The changes may result due to a high temperature of the abdominal environment in concert with the altered production of androgens.     

Gamma-ray exposure accelerates spermatogenesis of medaka fish,Oryzias latipes

To examine the spermatogenesis (and spermiogenesis) cell population kinetics after gamma-irradiation, the frequency and fate of BrdU-labeled pre-meiotic spermatogenic cells (spermatogonia and pre-leptotene spermatocytes) and spermatogonial stem cells (SSCs) of the medaka fish (Oryzias latipes) were examined immunohistochemically and by BrdU-labeling. After 4.75 Gy of gamma-irradiation, a statistically significant decrease in the frequency of BrdU-labeled cells was detected in the SSCs, but not in pre-meiotic spermatogenic cells. The time necessary for differentiation of surviving pre-meiotic spermatogenic cells without delay of germ cell development was shortened. More than 90% of surviving pre-meiotic spermatogenic cells differentiated into haploid cells within 5 days after irradiation, followed by a temporal spermatozoa exhaust in the testis. Next, spermatogenesis began in the surviving SSCs. However, the outcome was abnormal spermatozoa, indicating that accelerated maturation process led to morphological abnormalities. Moreover, 35% of the morphologically normal spermatozoa were dead at day 6. Based on these results, we suggest a reset system; after irradiation most surviving spermatogenic cells, except for the SSCs, are prematurely eliminated from the testis by spermatogenesis (and spermiogenesis) acceleration, and subsequent spermatogenesis begins with the surviving SSCs, a possible safeguard against male germ cell mutagenesis.     

Diethylstilbestrol induces rat spermatogenic cell apoptosis in vivo through increased expression of spermatogenic cell Fas/FasL system

The significant role that estrogens play in spermatogenesis has opened up an exciting area of research in male reproductive biology. The realization that estrogens are essential for proper maintenance of spermatogenesis, as well as growing evidence pointing to the deleterious effects of estrogen-like chemicals on male reproductive health, has made it imperative to dissect the role estrogens play in the male. Using a model estrogen, diethylstilbestrol (DES), to induce spermatogenic cell apoptosis in vivo in the male rat, we provide a new insight into an estrogen-dependent regulation of the Fas-FasL system specifically in spermatogenic cells. We show a distinct increase in Fas-FasL expression in spermatogenic cells upon exposure to diethylstilbestrol. This increase is confined to the spermatid population, which correlates with increased apoptosis seen in the haploid cells. Testosterone supplementation is able to prevent DES-induced Fas-FasL up-regulation and apoptosis in the spermatogenic cells. DES-induced germ cell apoptosis does not occur in Fas-deficient lpr mice. One other important finding is that spermatogenic cells are type II cells, as the increase in Fas-FasL expression in the spermatogenic cells is followed by the cleavage of caspase-8 to its active form, following which Bax translocates to the mitochondria and precipitates the release of cytochrome c that is accompanied by a drop in mitochondrial potential. Subsequent to this, activation of caspase-9 occurs that in turn activates caspase-3 leading to the cleavage of poly(ADP-ribose) polymerase. Taken together, the data indicate that estrogen-like chemicals can precipitate apoptotic death in spermatogenic cells by increasing the expression of spermatogenic cell Fas-FasL, thus initiating apoptosis in the same lineage of cells through the activation of the apoptotic pathway chosen by type II cells.     

Two states of active spermatogenesis switch between reproductive and non-reproductive seasons in the testes of the medaka, Oryzias latipes

Seasonal change in spermatogenesis was examined in the restricted spermatogonium‐type testes of a teleost, Oryzias latipes. Histological observation revealed that the number of each stage of germ cells during most of the non‐reproductive season, from October to January (O–J period) was nearly half of that during the reproductive season, from May to July (M–J period), except for type B spermatogonia (B‐gonia), which was actually equal. As a result, the ratio of primary spermatocytes (P‐cytes) to B‐gonia was remarkably small in the O–J period. Despite the differences between both time periods, the proliferative activity of type A spermatogonia (A‐gonia), B‐gonia, or P‐cytes was at a similar level in both periods. Moreover, in cultured testes treated with bromodeoxyuridine as a cell‐lineage tracer, P‐cytes differentiated to spermatids in 11–15 days in both M–J and O–J periods. These indicate that spermatogenesis is active in each period at a different state. In the spermatogenic testis, A‐gonial proliferation was maintained by human follicle stimulating hormone/luteinizing hormone in culture. Whereas cell death of B‐gonia and/or P‐cytes gradually increased in the M–J period in spite of those cells being constant in population sizes. In transition to the O–J period, A‐gonia and P‐cytes first decreased, which was accompanied by a decrease in proliferative activity of A‐gonia and relative increase of dead cells from B‐gonia and/or P‐cytes against live P‐cytes. These suggest that A‐gonial proliferation and cell death of B‐gonia and/or P‐cytes that is induced coordinately with B‐gonial differentiation are critical for the spermatogenic control.     

Evaluation of a peripherally selective antiandrogen (Casodex) as a tool for studying the relationship between testosterone and spermatogenesis in the rat.

The endocrine profile and the effects on spermatogenesis of the new antiandrogen, Casodex [2RS)-4-cyano-3-(4-fluorophenylsulphonyl)-2-anilide, CAS) were evaluated in the adult rat. In the first experiment rats were administered CAS at daily doses of 10, 20 and 40 mg/kg for 14 days. For comparison groups receiving flutamide (FL, 10 mg/kg) and ethane dimethane sulphonate (EDS) were included. Unlike FL, administration of CAS (10 and 20 mg/kg) did not significantly raise serum concentrations of gonadotropic hormones and testosterone. With 40 mg/kg CAS gonadotropin secretion, but not testosterone levels, were elevated on day 15. Administration of CAS lowered the weight of the seminal vesicles and coagulating glands comparable to the administration of the Leydig cell toxin EDS. In contrast to FL a significant loss of germ cells in stage VII of spermatogenesis was observed with CAS. In a second experiment the ability of FL and CAS to block testicular androgen action was compared in rats with reduced testicular androgen production induced by a gonadotropin-releasing hormone antagonist. Both antiandrogens markedly enhanced spermatogenic involution as revealed by quantitative flow cytometric analysis of germ cell numbers. The study demonstrates that (a) CAS is a peripherally selective antiandrogen and (b) CAS might provide a feasible approach to study androgen dependence of spermatogenesis in the presence of normal FSH levels.     

Suppression of spermatogenesis for cell transplantation in adult mice

Summary Spermatogenesis occurs within the testis of adult males by a complex and very well organized process. Breakthroughs in techniques such as cryopreservation and culture of spermatogenic cells and the maturation of these cells in exogenous testes after transplantation renewed the interest in this process. Transplantation of spermatogenic cells from a donor to a recipient animal needs a preparatory step that consists in the elimination of the endogenous population of spermatogenic cells. The most common method used to empty the seminiferous tubules is the treatment with busulfan (1,4-butanediol dimethanesulfonate). Busulfan partially eliminates stem cells because of its alkylating nature, but a residual component of stem cells survives the treatment and competes in the regeneration of the testis with transplanted cells. Estradiol has also been used as an agent that causes a delay in the process of spermatogenesis by altering its hormonal stimulation, although it does not affect the spermatogonia population. Therefore, we have tested different treatments with busulfan, estradiol benzoate, and also an agonist of the chorionic gonadotrophin-releasing hormone, leuprolide acetate, for the inhibition of endogenous spermatogenesis. We have found that a combination of estradiol, busulfan, and leuprolide can destroy the population of endogenous spermatogenic cells without altering Sertoli cells and maintains the optimal environment needed to allow the development of transplanted cells.     

The origin of the synchronization of the seminiferous epithelium in vitamin A-deficient rats after vitamin A replacement

In seminiferous tubules of vitamin A-deficient rats, the remaining spermatogonia were A spermatogonia. These cells were topographically arranged as single and paired cells and clones of 4, 8, or more cells. The bromodeoxy-uridine-labeling index and the mitotic index of these cells were found to be 9% and 1%, respectively, indicating that these cells were slowly proliferating. Administration of vitamin A (retinol-acetate) resulted in a reinitiation of spermatogenesis in such a way that the epithelium became stage-synchronized. The rate of development of the spermatogenic cells between 7 and 21 days after vitamin A replacement was found to be similar to that in normal rats. At 24-30 h after administration of vitamin A, a 4- to 6-fold increase in the labeling index was found. In contrast, after 2 days, the labeling index was low, while the mitotic index was elevated (10%). A high labeling index was found again after 3 days. Assuming that during the first 7 days after vitamin A replacement the rate of development of the spermatogenic cells also was normal, it could be deduced that the spermatogonia labeled 24-30 h after vitamin A administration were A1 spermatogonia. These cells would then divide into A2 spermatogonia after about 2 days, which in turn would traverse their S phase after about 3 days. Hence, spermatogenesis in vitamin A-deficient rats would be arrested shortly before the S phase of the A1 spermatogonia. After administration of vitamin A, the spermatogonia synchronously start the series of six divisions leading to the formation of spermatocytes and, ultimately, they develop into mature spermatids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear matrix in developing rat spermatogenic cells.

The nonchromatin structure or nuclear matrix in developing spermatogenic cells of the rat was studied using a biochemical fractionation in concert with resinless section electron microscopy. Observations demonstrated that the nuclear matrix of spermatogenic cells consisted of a three-dimensional network of filaments of variable thicknesses. In spermatogonia and spermatocytes the nuclear matrix consisted of relatively thin filaments, while that of round spermatids consisted of a thicker interconnecting network of filament. In elongating spermatids, the interior of the nuclear matrix consisted of a network of dense filaments bounded by a peripheral lamina. The protein composition of the nuclear matrix in spermatogenic cells was examined by high-resolution two-dimensional gel electrophoresis and correlated with morphological changes characteristic of each stage. The results showed that the proteins of nuclear matrix changed in a cell stage-specific manner. These stage-specific changes corresponded to the major transitions of chromatin structure and function during spermatogenesis. Furthermore, immunocytochemical and immunoblotting analysis of DNA topoisomerase II (topo II) revealed that this enzyme exhibited stage-specific variations and was associated with the nuclear matrix. These results suggest that the nuclear matrix in spermatogenic cells may be involved in mediating DNA modifications and maintaining nuclear organization during spermatogenesis. Mol. Reprod. Dev. 59:314-321, 2001.