Identification of antigen in rat spermatogenic cells interacting with an anti-human sperm monoclonal antibody

A monoclonal antibody (MAb) raised against human sperm protein, designated YWK-II, was used to determine the distribution of antigens in rat spermatozoa and rat testicular germ cells. By an indirect immunofluorescent method, the antibody localized over the rat spermatozoal head, except for the postacrosomal region. In paraffin sections of adult and immature rat testis, germ cells, at every developmental stage, and Sertoli cells stained, while interstitial cells and peritubular myoid cells remained unstained. When cocultures of Sertoli and germ cells were tested, only the germ cells stained intensely. Sertoli cells and peritubular myoid cells in cultures did not stain. In the epididymal sections, strong staining occurred with spermatozoa in the lumen and epididymal epithelial cells, with moderate staining in the myoid layers of epididymis. To determine the sperm antigen interacting with the YWK-II antibody, rat spermatozoa proteins were prepared and analyzed by an immunoblot technique. The monoclonal antibody interacted with a single protein, with an estimated molecular weight of 115,000, present in the cauda epididymal spermatozoa. Among the proteins of the caput epididymal spermatozoa, however, the antibody interacted with a major and a minor band with molecular weights of 115,000 and 88,000, respectively. On the other hand, with proteins prepared from the membrane fraction of adult and immature rat testis, the antibody reacted with two bands with estimated molecular weights of 88,000 and 115,000. In the lysate prepared from germ cells dissociated from Sertoli-germ cell cocultures, the antibody recognized only the 88,000 protein. The present results show that the YWK-II MAb interacts with two proteins with different molecular weights. The amount of the interacting proteins in spermatozoa varied with their location within the epididymis.     

β-hexosaminidase immunolocalization and α- and β-subunit gene expression in the rat testis and epididymis

β-hexosaminidase is an essential lysosomal enzyme whose absence in man results in a group of disorders, the G_M2 gangliosidoses. β-hexosaminidase activity is many times higher in the epididymis than in other tissues, is present in sperm, and is postulated to be required for mammalian fertilization. To better understand which cells are responsible for β-hexosaminidase expression and how it is regulated in the male reproductive system, we quantitated the mRNA expression of the α- and β-subunits of β-hexosaminidase and carried out immunocytochemical localization studies of the enzyme in the rat testis and epididymis. β-hexosaminidase α-subunit mRNA was abundant and differentially expressed in the adult rat testis and epididymis, at 13- and 2-fold brain levels, respectively. In contrast, β-subunit mRNA levels in the testis and epididymis were 0.3- and 5-fold brain levels. During testis development from 7–91 postnatal days of age, testis levels of α-subunit mRNA increased 10-fold and coincided with the appearance of spermatocytes and spermatids in the epithelium; in contrast, β-subunit mRNA was expressed at low levels throughout testis development. In isolated male germ cells, β-hexosaminidase α-subunit expression was most abundant in haploid round spermatids, whereas the β-subunit mRNA was not detected in germ cells. Within the epididymis both α- and β-subunit mRNA concentrations were highest in the corpus, with 1.5-fold and 9-fold initial segment values, respectively. Light microscopic immunocytochemistry revealed that β-hexosaminidase was localized to Sertoli cells and interstitial macrophages in the testis. In the epididymis, β-hexosaminidase staining was most intense in narrow cells in the initial segment, principal cells in the caput, and proximal corpus, and clear cells throughout the duct. Electron microscopic immunocytochemistry revealed that β-hexosaminidase was predominantly present in lysosomes in Sertoli and epididymal cells. The cellular and regional specificity of β-hexosaminidase immunolocalization suggest an important role for the enzyme in testicular and epididymal functions. Mol. Reprod. Dev. 46:227–242, 1997. © 1997 Wiley-Liss, Inc.     

Lysosomal enzymes in cells separated from rat testis

Fractions enriched with Sertoli cell (S), germ cells (G), and interstitial cells (I) were separated from rat testis after enzymic treatment and double filtration through nylon meshes. The fractions were analysed for protein content and for enzymic activity of 4 acid hydrolases known to be of lysosomal nature in other tissues. Acid phosphatase activity was preferentially recovered in Fraction G, the highest activity of beta-glucuronidase was found in Fraction I while the activity of aryl sulphatase and beta-N-acetyl-D-glucosaminidase was prominent in Fraction S. With the exception of acid phosphatase, the enzymes were mostly recovered in a subcellular fraction of whole testis homogenate separated between 600 and 27 000 g. The results may reflect the peculiar enzyme composition of the lysosomal apparatus of each cell type.     

Cyclic nucleotide phosphodiesterases in somatic and germ cells of mouse seminiferous tubules

The distribution of phosphodiesterase forms in somatic and germ cells, and their variations during testicular development and germ cell differentiation have been investigated. Seminiferous tubules from immature mice and Sertoli cells in culture possessed two enzyme activities which were comparable to forms described for different tissues and species: (a) a calcium-calmodulin-dependent enzyme with high affinity for guanosine 3',5'-(cyclic)-monophosphate (cGMP), and (b) a calcium-calmodulin-independent enzyme with high affinity for adenosine 3',5'-(cyclic)-monophosphate (cAMP) the activity of which increased in cultured Sertoli cells after treatment with FSH or dibutyryl cAMP. Seminiferous tubules from adult animals and germ cells at the meiotic and post-meiotic stage of differentiation possessed two enzyme forms that could be distinguished from those present in somatic cells of the seminiferous tubules: (a) a calcium-calmodulin-dependent form with high affinity for both cAMP and cGMP, similar to forms described in other tissues from different species, and (b) a calcium-calmodulin-independent phosphodiesterase with high affinity for cAMP and present only in post-meiotic cells, previously identified also in germ cells of the rat.     

Arylsulfatase A deficiency causes seminolipid accumulation and a lysosomal storage disorder in Sertoli cells

Sulfogalactosylglycerolipid (SGG) is the major sulfoglycolipid of male germ cells. During spermatogenesis, apoptosis occurs in >50% of total germ cells. Sertoli cells phagocytose these apoptotic germ cells and degrade their components using lysosomal enzymes. Here we demonstrated that SGG was a physiological substrate of Sertoli lysosomal arylsulfatase A (ARSA). SGG accumulated in Sertoli cells of Arsa(-/-) mice, and at 8 months of age, this buildup led to lysosomal swelling and other cellular abnormalities typical of a lysosomal storage disorder. This disorder likely compromised Sertoli cell functions, manifesting as impaired spermatogenesis and production of sperm with near-zero fertilizing ability in vitro. Fecundity of Arsa(-/-) males was thus reduced when they were older than 5 months. Sperm SGG is known for its roles in fertilization. Therefore, the minimal sperm fertilizing ability of 8-month-old Arsa(-/-) males may be explained by the 50% reduction of their sperm SGG levels, a result that was also observed in testicular germ cells. These unexpected decreases in SGG levels might be partly due to depletion of the backbone lipid palmitylpalmitoylglycerol that is generated from the SGG degradation pathway in Sertoli cells and normally recycled to new generations of primary spermatocytes for SGG synthesis.     

Enzyme histochemical studies on the pathological changes in human Sertoli cells

Two forms of human Sertoli cell disorders were characterized enzyme histochemically from the testicular biopsy material of infertile and subfertile patients. Sertoli cell asthenia: a slight injury of the Sertoli cell with exfoliation of individual germ cells; marked by the rarefaction of reaction zones of thiamine pyrophosphatase (TPPase) and a decrease in lactate dehydrogenase (LDH). Sertoli cell insufficiency: severe Sertoli cell damage with the formation of a "puff" and a heavy exfoliation of germ cells (dislocation of Sertoli cell nucleus and cytoplasm along with the related germ cells into the lumen of seminiferous tubule); marked by a heterogeneous activity pattern of TPPase, the disappearance of LDH, maintenance of a slightly weakened activity of alkaline phosphatase, and an increase of acid phosphatase. In the case of Sertoli-cell-only syndrome, the high prismatic Sertoli cells showed strong acid phosphatase activity with scattered weak TPPase reaction, whereas the flat or cube-like Sertoli cells exhibited weak acid phosphatase activity with only one small round reaction zone of TPPase in each cell. In addition, the frequency of the occurrence of Sertoli cell asthenia, Sertoli cell insufficiency, and Sertoli-cell-only syndrome is reported, and its correlation with the andrological diseases discussed.     

Myoinositol biosynthesis by Sertoli cells, and levels of myoinositol biosynthetic enzymes in testis and epididymis.

Levels of glucose-6-phosphate cyclase (myoinsitol-1-phosphate synthase, EC 5.5.1.4) and myoinositol-1-phosphate phosphatase (myoinositol-1-phosphatase, EC 3.1.3.25) were determined in extracts of testes from10-, 20-, and 30-day-old rats, and in extracts of Sertoli cells, germinal cells, and epididymides. The specific activity of the cyclase was approximately 1/10th that of the phosphatase in all extracts found to contain either enzyme. Among cells in the testis examined, Sertoli cells had highest levels of enzymes required for inositol biosynthesis from glucose, while spermatocytes and round spermatids did not have detectable activity. Spermatozoa from the epididymis also had no detectable cyclase or phosphatase activity. In contrast, extracts of washed epididymides contained exceedingly high specific activities of these enzymes. Primary cultures of Sertoli cells, maintained in a chemically defined medium without added inositol, released inositol into the medium during three successive 24-h periods. The amounts released were greater in cells stimulated by dibutyryl cyclic AMP. Results were interpreted to indicate that inositol in the fluid of seminiferous tubules most probably originates from Sertoli cells, which synthesize inositol from glucose. Additional inositol in the fluid of epididymal tubules could readily be provided by metabolism of glucose by epididymal epithelial cells     

Expression of claudin-1 and -11 in immature and mature pheasant (Phasianus colchicus) testes

The expression of claudin-1 and -11, tight junctions (TJs) proteins was examined in immature and adult pheasant (Phasianus colchicus) testes. Claudin-1 and -11 cDNA were highly similar to those of human, mice, and chicken. Claudin-1 mRNA and protein (21 kDa) levels in immature testes were higher than those of adult testis. In immature testes until 6 weeks of age, Claudin-1 was found at contacts between adjacent Sertoli cells and between Sertoli cells and germ cells. In adult testis, Claudin-1 was found in early spermatocytes migrating the blood testis barrier (BTB). Blood vessels were positive for claudin-1. Claudin-11 mRNA and protein (21 kDa) increased during adulthood development of testis. In immature testis, Claudin-11 was found in apicolateral contacts between adjacent Sertoli cells, indicating its involvement in cell adhesion in immature testis. In adult testis, strong wavy Claudin-11 immunoreactivity was parallel to basal lamina at the basal part of seminiferous epithelium, indicating that Claudin-11 at the inter-Sertoli TJs may act as a structural element of the BTB. Weak Claudin-1 and -11 immunoreactivity at contacts between Sertoli cells to elongating/elongated spermatids, meiotic germ cells, and basal lamina suggests that they also participate in the cell-cell and cell-extracellular matrix adhesion in pheasant testis. Testosterone increased claudin-11 mRNA in testis organ culture and Sertoli cell primary culture, suggesting positive regulation of claudin-11 gene by androgen in Sertoli cells of pheasant testis. This is the first report on the claudins expression at BTB in avian testis.     

Cathepsin A is expressed in a cell- and region-specific manner in the testis and epididymis and is not regulated by testicular or pituitary factors.

The epithelial cells of the testis are involved in the production, differentiation, and sustenance of sperm, and those of the epididymis play a major role in sperm maturation, protection, and storage. These tissues express various proteins that respond differently to androgens. Cathepsin A is a multifunctional lysosomal carboxypeptidase that also functions as a protective and an activator protein for neuraminidase and beta-galactosidase. In this study, cathepsin A was immunolocalized by light and electron microscopy using a polyclonal affinity-purified antibody on the testis and epididymis of normal, orchidectomized with or without testosterone supplementation, efferent duct-ligated, and hypophysectomized adult rats. In normal rats, cathepsin A expression was noted in lysosomes of Sertoli and Leydig cells but not in germ cells of the testis, as well as nonciliated cells of the efferent ducts. In the epididymis, a cell- and region-specific distribution of cathepsin A was noted. In experimentally treated animals, no changes were noted in the expression of cathepsin A. Immunolabeling of tissues examined at the electron microscopic level revealed that lysosomes were reactive. These data indicate cell- and region-specific expression of cathepsin A in cells of the testis and epididymis and also indicate that cathepsin A expression is not regulated by testicular or pituitary factors.     

Over expression of interleukin-1alpha,interleukin-1beta and interleukin-1 receptor antagonist in testicular tissues from sexually immature mice as compared to adult mice

The levels of IL-1alpha, IL-1beta and IL-1Ra were higher in homogenates of testicular tissue from sexually immature than those from mature mice. Immunohistochemical staining of testicular tissues from sexually immature and adult mice show that differentiated germ cells express higher levels of IL-1alpha compared to Sertoli cells and Leydig cells/interstitial cells. Peritubular cells of sexually immature and adult mice did not express IL-1alpha. Testicular tissue cells of adult mice showed high levels of expression of IL-1beta, mainly in the cytoplasm and nucleus of the spermatogonia and in spermatocytes. Sertoli cells and Leydig/interstitial cells were also highly stained for IL-1beta. However, peritubular cells did not express IL-1beta. On the other hand, testicular tissue cells from sexually immature mice, showed high levels of IL-1beta, mainly in spermatocytes. Spermatogonia showed low levels of IL-1beta expression. Also, high levels of IL-1beta expression were detected in Leydig/interstitial cells. Peritubular cells clearly showed IL-1beta expression. Testicular tissue cells from adult mice, showed IL-1Ra expression in spermatogonia, Sertoli and Leydig/interstitial cells. IL-1Ra expression was clearly present in the Golgi apparatus of spermatogonia and Sertoli cells. However, peritubular cells did not show IL-1Ra expression. Testicular tissue cells from sexually immature mice, also showed high levels of IL-1Ra expression mainly in the cytoplasm and nucleus of the spermatogonia and Sertoli cells. In addition, Leydig/interstitial cells and peritubular cells also expressed IL-1Ra. Our results demonstrate, for the first time, the expression of IL-1beta in germ and Sertoli cells, and IL-1Ra in Leydig/interstitial cells of testicular tissues from adult and sexually immature mice, under in vivo conditions. In addition, the relative elevated levels of the IL-1 system in the testis of immature mice compared to mature mice may indicate its involvement in the spermatogenesis.     

Changes in lipase and phosphatase activities of rat spermatozoa in transit from the caput to the cauda epididymidis.

Rat spermatozoa from the cauda epididymidis, freed from their cytoplasmic droplets and acrosomes, were found to have a lower lipid content and to incorporate [14C]glucose into their glycerides and glycerophosphatides at a lower rate than spermatozoa from the caput epididymidis. Against the background of the activities of some glycolytic enzymes which remained constant the activity of alkaline phosphatase decreased in spermatozoa migrating through the epididymis, whereas the activity of monoglyceride lipase increased. The corresponding enzyme activities of non-flagellate germ cells of the testis were measured for comparison. The triglyceride lipase of non-flagellate germ cells and of spermatozoa from both caput and cauda epididymidis was activated by cyclic 3':5'-AMP.     

Spontaneous germ cell death by apoptosis in epididymis of the adult bat Artibeus lituratus.

Many factors can lead cells to apoptosis during the various stages of cell life. This study was undertaken to characterize germ cell death in the epididymis of the adult Artibeus lituratus by histochemical and immunohistochemical techniques using light microscopy and transmission electron microscopy. The results showed that cells with a nuclear phenotype and ultrastructural characteristics of chromatin compaction were common in apoptosis. The Apoptag test confirmed that the suspected cells were apoptotic. It is suggested that immature germ cells, when released from the germinative epithelium, may be directed towards the epididymis instead of being disposed of in the testicle. Furthermore, intact immature cells can leave the testicle in the initial phases of apoptosis and complete this phenomenon in the epididymis.     

Isolation of sertoli cells from adult rat testes: an approach to ex vivo studies of Sertoli cell function

Much of what is known about the molecular regulation and function of adult Sertoli cells has been inferred from in vitro studies of immature Sertoli cells. However, adult and immature cells differ in significant ways and, moreover, many Sertoli cell functions are regulated by conditions that are difficult to replicate in vitro. Our objective was to develop a procedure to isolate Sertoli cells rapidly and in sufficient number and purity to make it possible to assess Sertoli cell function immediately after the isolation of the cells. The isolation procedure described herein takes less than 4 h and does not require culturing the cells. From a single 4-mo-old adult rat, we routinely obtain 7.0 +/- 0.4 x 10(6) Sertoli cells per testis, and from a 21-mo-old rat, 7.2 +/- 0.4 x 10(6) Sertoli cells per testis. The purity, determined by morphologic analyses of plastic-embedded cells or after staining for tyrosine-tubulin or vimentin, averaged 80%. The contaminants typically included germ cells (10%) and myoid cells (10%). The germ cell-expressed genes protamine-2 and hemiferrin were not detected in the Sertoli cell preparations by Northern blot analyses, but the Sertoli cell-expressed genes clusterin, cathepsin L, and transferrin were highly expressed. Transferrin mRNA levels were greater in Sertoli cells isolated from aged than from young adult rats, consistent with previous analyses of whole testes; and cathepsin L mRNA levels were far more highly expressed in Sertoli cells isolated from stages VI-VII than from other stages of the cycle of the seminiferous epithelium, also consistent with previous analyses of whole testes and isolated tubules. These studies indicate that the freshly isolated cells retain differentiated function, and thus it should be possible to assess the in vivo function of adult Sertoli cells by isolating the Sertoli cells and immediately assessing their function.     

Functional importance of palmitoyl protein thioesterase 1 (PPT1) expression by Sertoli cells in mediating cholesterol metabolism and maintenance of sperm quality

Sertoli cells are a type of nurse cell in the seminiferous epithelium that are crucial for sustaining spermatogenesis by extending nutritional and energy support to the developing germ cells. Dysfunction of Sertoli cells could cause disordered spermatogenesis and reduced fertility in males. In this study, we focused on the expression and function of palmitoyl protein thioesterase 1 (PPT1), a lysosomal depalmitoylating enzyme, in Sertoli cells. Here, we show that PPT1 expression in Sertoli cells is responsive to cholesterol treatment and that specific knockout of Ppt1 in Sertoli cells causes male subfertility associated with poor sperm quality and a high ratio of sperm deformity. Specifically, Ppt1 deficiency leads to poor cell variably accompanied with abnormal lysosome accumulation and increased cholesterol levels in Sertoli cells. Further, Ppt1 deficiency results in poor adhesion of developing germ cells to Sertoli cells in the seminiferous epithelium, which is likely to be responsible for the reduced male fertility as a consequence of declines in sperm count and motility as well as a high incidence of sperm head deformity. In summary, PPT1 affects sperm quality and male fertility through regulating lysosomal function and cholesterol metabolism in Sertoli cells.     

The histological changes in the testis, epididymis and prostate gland of the red-bellied tree squirrel, Callosciurus erythraeus, during the growth and reproductive cycle

The male reproductive glands of the red-bellied tree squirrel, Callosciurus erythraeus, in the infantile, and prepubertal males, as well as sexually functional, degenerating and redeveloping adults were studied histologically. In the infant, testes are characterized with solid seminiferous tubules filled with primordial germ cells and Sertoli cells. Interstitial cells are sparse. The prostate is composed of condensed cell cords grouped into lobules dispersed with interlobular tissues rich in fibroblasts. In the epididymis the highly convoluted tubule is lined with a simple cuboidal or columnar epithelium and thin smooth musculature without. In the prepubertal male, germ cells are engaged actively in mitosis. Primary spermatocytes are readily recognized. Leydig cells appear in groups in the interstitial tissue. In the prostate, cell cords become highly branched and collecting tubules make their appearance. The tubules in the epididymis are enlarged in diameter but their peripheral musculature becomes thinner. In functional males, meiosis is active and bundles of spermatozoa are scattered along the central lumen. Leydig cells have their cytoplasm highly enriched. The prostate is in the secretory phase. The tubule in the epididymis is filled with sperm. In the degenerating adult, meiosis is interrupted and necrotic germ cells are detached from germinal epithelium. In the prostate, secretory and collecting ducts are eventually reduced to condensed lobules separated by interlobular fibrous tissue. The tubule in the epididymis often fills with necrotic germ cells but no sperm. In the redeveloping adult, the histology of the testes, prostate and epididymis is similar to that of the prepubertal male. However, there is more fibrous tissue in the interlobular septa in the prostate gland and thick musculature at the periphery of the tubule in the epididymis.     

The Sertoli cell in vivo and in vitro

The Sertoli cell extends from the basement membrane of the seminiferous tubule towards its lumen; it sends cytoplasmic processes which envelop different generations of germ cells. The use of Sertoli cell culture began to develop in 1975. To reduce germ cell contamination immature animals are generally used as Sertoli cell donors. Sertoli cell mitosis essentially occurs in sexually immature testes in mammals; mitosis of these cells is observed in vitro during a limited period of time. Sertoli cells in vivo perform an impressive range of functions: structural support of the seminiferous epithelium, displacement of germ cells and release of sperm; formation of the Sertoli cell blood-testis barrier; secretion of factors and nutrition of germ cells; phagocytosis of degenerating germ cells and of germ cell materials. Some of the Sertoli cell functions can be studied in vitro. The recent development of Sertoli cell culture on permeable supports (with or without extracellular matrix) has resulted in progress in understanding the vectorial secretion of several Sertoli cell markers. In addition to FSH and testosterone, several other humoral factors are known to influence Sertoli cell function. Furthermore, myoid cells bordering the tubules as well as germ cells are capable of regulating Sertoli cell activity. Sertoli cells are the most widely used testicular cells for in vitro toxicology. The testis is highly vulnerable to xenobiotics and radiations, yet the number of studies undertaken in this field is insufficient and should be drastically increased.     

Overlapping functions of lysosomal acid phosphatase (LAP) and tartrate-resistant acid phosphatase (Acp5) revealed by doubly deficient mice.

To date, two lysosomal acid phosphatases are known to be expressed in cells of the monocyte/phagocyte lineage: the ubiquitously expressed lysosomal acid phosphatase (LAP) and the tartrate-resistant acid phosphatase-type 5 (Acp5). Deficiency of either acid phosphatase results in relatively mild phenotypes, suggesting that these enzymes may be capable of mutual complementation. This prompted us to generate LAP/Acp5 doubly deficient mice. LAP/Acp5 doubly deficient mice are viable and fertile but display marked alterations in soft and mineralised tissues. They are characterised by a progressive hepatosplenomegaly, gait disturbances and exaggerated foreshortening of long bones. Histologically, these animals are distinguished by an excessive lysosomal storage in macrophages of the liver, spleen, bone marrow, kidney and by altered growth plates. Microscopic analyses showed an accumulation of osteopontin adjacent to actively resorbing osteoclasts of Acp5- and LAP/Acp5-deficient mice. In osteoclasts of phosphatase-deficient mice, vacuoles were frequently found which contained fine filamentous material. The vacuoles in Acp5- and LAP/Acp5 doubly-deficient osteoclasts also contained crystallite-like features, as well as osteopontin, suggesting that Acp5 is important for processing of this protein. This is further supported by biochemical analyses that demonstrate strongly reduced dephosphorylation of osteopontin incubated with LAP/Acp5-deficient bone extracts. Fibroblasts derived from LAP/Acp5 deficient embryos were still able to dephosphorylate mannose 6-phosphate residues of endocytosed arylsulfatase A. We conclude that for several substrates LAP and Acp5 can substitute for each other and that these acid phosphatases are essential for processing of non-collagenous proteins, including osteopontin, by osteoclasts.     

During seminiferous tubule maturation testosterone and synergistic action of FSH with estradiol support germ cell survival while estradiol alone has pro-apoptotic effect

During establishment of spermatogenesis at the prepubertal age, an early germ cells apoptotic wave occurs likely aimed to remove abnormal germ cells and to maintain a proper cell number ratio between maturating germ cells and Sertoli cells. Here we assessed Sertoli and germ cell apoptosis in relation to morphological parameters of Sertoli cell maturation in neonatal rats under the influence of testosterone, estradiol and FSH given alone or in combinations. From postnatal day (PND) 5th to 15th male rats were daily injected with: 1) 2.5 mg of testosterone propionate (TP), or 2) 12.5 microg of 17beta-estradiol benzoate (EB), or 3) TP+EB, or 4) 7.5 IU of human purified FSH (hFSH), or 5) hFSH+EB or solvents (control-C). Autopsy was performed on PND 16th. Sertoli cell nuclei area and incidence of seminiferous tubule lumen formation (LF) were taken as markers of Sertoli cell maturation. Sertoli and germ cell apoptosis was assessed using TUNEL method. In comparison with C, the area of Sertoli cell nuclei was significantly reduced after EB (25.7+/-2.0 vs. 30.9+/-1.6 microm2 for C, p<0.001) and increased after hFSH+EB (33.1+/-2.3 microm2, p<0.05). Incidence of LF was completely arrested by steroid hormone treatments given separately, significantly inhibited after TP+EB (median: 0.0%, vs. 2.0% for C p<0.05) and significantly enhanced after hFSH+EB (median: 51.0%, p<0.001). hFSH alone did not influence LF. Incidence of TUNEL positive Sertoli cells significantly increased after EB (median: 2.9% vs. 0.5% for C, p<0.05) or TP+EB (median: 2.2%, p<0.01) and was not affected by other treatments. Incidence of TUNEL positive germ cells increased significantly after EB alone (median: 4.4% vs. 2.5%, for C, p<0.01 ) and was significantly decreased by hFSH+EB (median: 0.5%, p<0.01). CONCLUSIONS: 1) Administration of testosterone or estradiol to immature rats inhibits Sertoli cell maturation. 2) Estradiol stimulates Sertoli and germ cell apoptosis while testosterone has no effect. 3) Testosterone eliminates estradiol--induced germ cell apoptosis when both hormones act in concert. 4) FSH in concert with estradiol, but neither one of the hormone alone, accelerate Sertoli cell differentiation and effectively inhibit germ cell apoptosis. 5) During seminiferous tubule maturation testosterone and the synergistic action of FSH with estradiol support germ cell survival while estradiol alone has an inhibitory, pro-apoptotic effect.