The effects of postnatal FSH substitution on Sertoli cell number and the sperm production capacity of the adult boar

The role of FSH for Sertoli cell establishment and sperm production in the boar is not definitely known. In order to elucidate its function FSH was substituted postnatally in male pigs and the resulting effects on testicular histological traits and sperm production capacity were investigated when the boars had reached maturity. Six male piglets received pFSH from 18 to 48 days postnatally. Another six piglets instead received saline and served as controls. Blood samples were drawn to measure FSH, LH, testosterone and estradiol. After 28 weeks, the boars were trained to mount a dummy so that the spermatogenic capacity was tested by increasing the frequency of semen collection at the age of 30 weeks. Libido (latency time) and ejaculate criteria (volume, motility, morphological abnormalities) were determined. Thereafter the boars were killed and their testes analyzed for morphology, number of Sertoli cells, germ cells and Leydig cells as well as the ratio between mitosis and apoptosis in the tubules.FSH concentrations were twofold due to FSH application when compared to the controls. LH was low during the first 2 weeks of FSH treatment. Thereafter concentrations increased in three of the six treated animals but not in controls. Testosterone increased slightly over the application period both in the controls and the treated piglets. Estradiol levels were similar in both groups. Increased ejaculation frequency reduced sperm concentrations and sperm motility in all boars and the percentage of morphologically abnormal sperm increased. Ejaculate volumes and the time of latency were not significantly altered. No differences were observed between the controls and the FSH treated boars. The testicular parameters of both FSH- and control boars were identical for morphology, number of spermatogenic and somatic cells as well as mitosis–apoptosis equilibrium. The data demonstrated that a prolonged postnatal period of FSH concentrations does not influence the sperm production of the adult boar.     

Heat shock protein 27 expression in the human testis showing normal and abnormal spermatogenesis

Heat shock proteins (HSPs) are molecular chaperones involved in protein folding, assembly and transport, and which play critical roles in the regulation of cell growth, survival and differentiation. We set out to test the hypothesis that HSP27 protein is expressed in the human testes and its expression varies with the state of spermatogenesis. HSP27 expression was examined in 30 human testicular biopsy specimens (normal spermatogenesis, maturation arrest and Sertoli cell only syndrome, 10 cases each) using immunofluorescent methods. The biopsies were obtained from patients undergoing investigations for infertility. The seminiferous epithelium of the human testes showing normal spermatogenesis had a cell type-specific expression of HSP27. HSP27 expression was strong in the cytoplasm of the Sertoli cells, spermatogonia, and Leydig cells. Alternatively, the expression was moderate in the spermatocytes, weak in the spermatids and absent in the spermatozoa. In testes showing maturation arrest, HSP27 expression was strong in the Sertoli cells, weak in the spermatogonia, and spermatocytes. It was absent in the spermatids and Leydig cells. In Sertoli cell only syndrome, HSP27 expression was strong in the Sertoli cells and absent in the Leydig cells. We report for the first time the expression patterns of HSP27 in the human testes and show differential expression during normal spermatogenesis, indicating a possible role in this process. The altered expression of this protein in testes showing abnormal spermatogenesis may be related to the pathogenesis of male infertility.     

Immunolocalization of proliferating cell nuclear antigen (PCNA) in cynomolgus monkey (Macaca fascicularis) testes during postnatal development

The age-related distribution of proliferating cell nuclear antigen (PCNA) in the testes of cynomolgus monkeys (Macaca fascicularis) during postnatal development was detected using light-microscopic immunohistochemistry. In neonatal testes, some PCNA-positive spermatogonia, Sertoli cells, peritubular cells, and Leydig cells were detected. In early infantile testes, only a few of these cell types were positive. In late infantile testes, the numbers of positive cells were greater than in the earlier developmental stages. In pubertal testes, the numbers of positive spermatogonia, spermatocytes, Sertoli cells, peritubular cells, and Leydig cells were considerably higher. In adult testes, a larger percentage of spermatogonia and spermatocytes was positive, and peritubular cells and Leydig cells were occasionally positive; secondary spermatocytes, spermatids, and Sertoli cells were not positive. We concluded that immunolocalization of PCNA can serve as a tool for studying proliferation status in developing testes of cynomolgus monkeys. A relatively low proliferative activity in early infantile testes and a remarkable increase of proliferative activity in pubertal testes correlate with the fluctuations of steroidogenic functions during postnatal development in cynomolgus monkeys.     

A role for kit receptor signaling in Leydig cell steroidogenesis

Kit and its ligand, Kitl, function in hematopoiesis, melanogenesis, and gametogenesis. In the testis, Kitl is expressed by Sertoli cells and Kit is expressed by spermatogonia and Leydig cells. Kit functions are mediated by receptor autophosphorylation and subsequent association with signaling molecules, including phosphoinositide (PI) 3-kinase. We previously characterized the reproductive consequences of blocking Kit-mediated PI 3-kinase activation in KitY(719F)/Kit(Y719F) knockin mutant male mice. Only gametogenesis was affected in these mice, and males are sterile because of a block in spermatogenesis during the spermatogonial stages. In the present study, we investigated effects of the Kit(Y719F) mutation on Leydig cell development and steroidogenic function. Although the seminiferous tubules in testes of mutant animals are depleted of germ cells, the testes contain normal numbers of Leydig cells and the Leydig cells in these animals appear to have undergone normal differentiation. Evaluation of steroidogenesis in mutant animals indicates that testosterone levels are not significantly reduced in the periphery but that LH levels are increased 5-fold, implying an impairment of steroidogenesis in the mutant animals. Therefore, a role for Kit signaling in steroidogenesis in Leydig cells was sought in vitro. Purified Leydig cells from C57Bl6/J male mice were incubated with Kitl, and testosterone production was measured. Kitl-stimulated testosterone production was 2-fold higher than that in untreated controls. The Kitl-mediated testosterone biosynthesis in Leydig cells is PI 3-kinase dependent. In vitro, Leydig cells from mutant mice were steroidogenically more competent in response to LH than were normal Leydig cells. In contrast, Kitl-mediated testosterone production in these cells was comparable to that in normal cells. Because LH levels in mutant males are elevated and LH is known to stimulate testosterone biosynthesis, we proposed a model in which serum testosterone levels are controlled by elevated LH secretion. Leydig cells of mutant males, unable to respond effectively to Kitl stimulation, initially produce lower levels of testosterone, reducing testosterone negative feedback on the hypothalamic-pituitary axis. The consequent secretion of additional LH, under this hypothesis, causes a restoration of normal levels of serum testosterone. Kitl, acting via PI 3-kinase, is a paracrine regulator of Leydig cell steroidogenic function in vivo.     

Immunolocalization of inhibin and activin alpha and betaB subunits and expression of corresponding messenger RNAs in the human adult testis

Inhibin B is a testicular peptide hormone that regulates FSH secretion in a negative feedback loop. Inhibin B is a dimer of an alpha and a beta(B) subunit. In adult testes, the cellular site of production is still controversial, and it was hypothesized that germ cells contribute to inhibin B production. To determine which cell types in the testes may produce inhibin B, the immunohistochemical localization of the two subunits of inhibin B were examined in adult testicular biopsies with normal spermatogenesis, spermatogenic arrest, or Sertoli cell only (SCO) tubules. Moreover, using in situ hybridization with mRNA probes, the mRNA expression patterns of inhibin alpha and inhibin/activin beta(B) subunits have been investigated. In all testes, Sertoli cells and Leydig cells showed positive immunostaining for inhibin alpha subunit and expressed inhibin alpha subunit mRNA. Using inhibin beta(B) subunit immunoserum on testes with normal spermatogenesis and with spermatogenic arrest, intense labeling was located in germ cells from pachytene spermatocytes to round spermatids but not in Sertoli cells. Inhibin beta(B) subunit mRNA expression was intense in germ cells from spermatogonia to round spermatids and in Sertoli cells in these testes. In testes with SCO, high inhibin beta(B) subunit mRNA labeling density was observed in both Sertoli cells and Leydig cells, whereas beta(B) subunit immunostaining was negative for Sertoli cells and faintly positive for Leydig cells. These results agree with the recent opinion that inhibin B in adult men is possibly a joint product of Sertoli cells and germ cells.     

Photoperiodic variations of somatic and germ cell populations in the Soay ram testis

Seasonal changes in the Leydig, Sertoli and stem cells (undifferentiated A0 and cyclic A1) spermatogonia and the daily spermatid production were analysed in the testes of adult Soay rams exposed to short days (8L:16D) or long days (16L:8D) for 12 weeks. The total numbers of Leydig, Sertoli and stem cells (A0 + A1) were not affected by the treatments, but the size of the Leydig and the Sertoli cells, the efficiency of spermatogenesis (i.e. the number of male gametes produced by an A1 spermatogonium) and the daily sperm production were all significantly reduced in the rams exposed to long days. There was a positive correlation between the concentration of FSH and testosterone and many of the cytological changes consistent with a causal role for these hormones in mediating the effects of photoperiod on the testicular function in the ram.     

Trout sertoli and leydig cells: Isolation,separation, and culture

Trout testes at various stages of maturation were dissociated by perfusion at 12°C with collagenase plus pronase and then with collagenase alone, followed by slight shaking overnight in 1% bovine albumin. This step provided a suspension of isolated somatic and germ cells, clusters of interstitial cells, and either intact spermatogenetic cysts (meiotic testes) or clusters of Sertoli cells (other testes). Most of the spermatozoa were removed from the testis cell suspension by centrifugation in Percoll (density 1.065 g/ml). Sertoli and Leydig cells were prepared by a two-step separation method: (1) the testis cell suspension was separated by sedimentation at unit gravity into “isolated cell” and “cell cluster” populations; (2) these populations were fractionated by isopyknic centrifugation in Percoll gradients. In terms of somatic cell composition, a nearly pure Sertoli cell (clusters) population was obtained between 1.017 and 1.033 g/ml and a Leydig cell (clusters) enriched population of between 1.033 and 1.048 g/ml (testes resuming spermatogenesis) or 1.048 and 1.062 g/ml (other testes). These various cell populations were cultured in modified Leibovitz L15 medium for 10–15 days. When seeded, the Sertoli cells had a normal ultrastructure that remained unchanged for at least 10 days, and the steroidogenic activity of Leydig cells could be stimulated by salmon gonadotropin. Leydig cells remained 3β-HSD positive and produced progesterone and 17α, 20β-OH progesterone for at least 11 days. This study points out that viable and differentiated trout somatic testicular cells can be prepared and cultured for several days.     

Fas-Fas ligand system as a possible mediator of spermatogenic cell apoptosis in human maturation-arrested testes

To elucidate the mechanism of maturation arrest, known as one of the male infertility, we addressed whether germ cell apoptosis occurs during maturation arrest, and if so, whether Fas and Fas ligand expressions are involved in the apoptosis. By electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), typical apoptotic features were frequently found around the spermatocytic stage in maturation arrest, compared to that in normal testes. When paraffin-embedded sections reacted with anti-Fas antiserum, staining for Fas was found in the plasma membranes of spermatocytes in the maturation-arrested testes, while no positive spermatogenic cells were seen in the normal testes. On the other hand, positive immunostaining for Fas ligand was restricted to Sertoli cells in the maturation-arrested testes as well as in the normal testes, although the intensity of staining for Fas ligand in normal testicular Sertoli cells was much weaker than that of maturation-arrested ones. Thus, these findings demonstrate that "maturation arrest" is characterized by frequent apoptosis of spermatocytes, and that Fas and Fas ligand staining are associated with a high frequency of apoptosis.     

Nestin, a neuroectodermal stem cell marker molecule, is expressed in Leydig cells of the human testis and in some specific cell types from human testicular tumours

The intermediate filament protein nestin is predominantly expressed in some stem/progenitor cells and appears to be a useful molecular tool to characterise tumours originating from precursor cells of neuroectodermal and mesenchymal lineages. Leydig cells originate in the adult testis by differentiation from stem cells and express a variety of neural and neuroendocrine markers. The possible expression of the neural stem cell marker nestin in Leydig cells and testicular tumour cells was determined by analysing the patterns of nestin expression in normal and pathological human testes by Western blot and immunohistochemical methods. In normal testis, nestin was found in some vascular endothelial cells, a subset of peritubular spindle-shaped cells and some Leydig cells; spermatogenic and Sertoli cells were unstained. In normal Leydig cells, nestin was distributed in the perinuclear cytoplasm and accumulated in the crystalloids of Reinke with ageing. In non-tumour pathologies (cryptorchidism, impaired spermatogenesis), the seminiferous tubules were immunonegative, whereas hyperplastic Leydig cells showed cytoplasmic immunolabelling. In testicular malignancies, nestin was localised in the Sertoli cells of the seminiferous tubules affected with intratubular germ cell neoplasia, in the hyperplastic Leydig cells associated with these tumours and in some components (mesenchymal and neuroepithelial cells) of teratomas; spermatocytic and non-spermatocytic seminomas were unstained. Some vascular endothelial cells were immunolabelled in all tumour samples. Thus, nestin is expressed in a population of normal and hyperplastic Leydig cells and in Sertoli cells in the presence of intratubular germ-cell neoplasia. Nestin may be a good marker for identifying components of testicular teratomas.The two first authors participated equally in this workThis work was supported by a grant from the Fondo de Investigaciones Sanitarias (FIS 02/3003 to M.V.T. Lobo)     

Effects of acid sphingomyelinase deficiency on male germ cell development and programmed cell death

Deficiency of acid sphingomyelinase (ASM), an enzyme responsible for producing a pro-apoptotic second messenger ceramide, has previously been shown to promote the survival of fetal mouse oocytes in vivo and to protect oocytes from chemotherapy-induced apoptosis in vitro. Here we investigated the effects of ASM deficiency on testicular germ cell development and on the ability of germ cells to undergo apoptosis. At the age of 20 weeks, ASM knock-out (ASMKO) sperm concentrations were comparable with wild-type (WT) sperm concentrations, whereas sperm motility was seriously affected. ASMKO testes contained significantly elevated levels of sphingomyelin at the age of 8 weeks as detected by high-performance, thin-layer chromatography. Electron microscopy revealed that the testes started to accumulate pathological vesicles in Sertoli cells and in the interstitium at the age of 21 days. Irradiation of WT and ASMKO mice did not elevate intratesticular ceramide levels at 16 h after irradiation. In situ end labeling of apoptotic cells also showed a similar degree of cell death in both groups. After a 21-day recovery period, the numbers of primary spermatocytes and spermatogonia at G2 as well as spermatids were essentially the same in the WT and ASMKO testes, as detected by flow cytometry. In serum-free cultures both ASMKO and WT germ cells showed a significant increase in the level of ceramide, as well as massive apoptosis. In conclusion, ASM is required for maintenance of normal sphingomyelin levels in the testis and for normal sperm motility, but not for testicular ceramide production or for the ability of the germ cells to undergo apoptosis.     

Selective loss of Sertoli cell and germ cell function leads to a disruption in sertoli cell-germ cell communication during aging in the Brown Norway rat

We investigated the effects of aging on Sertoli cell-germ cell interactions from Brown Norway rats using the induction of four specific mRNAs as markers. The testes from aging (24 mo old) Brown Norway rats can be normal size or regressed. One marker, a von Ebner's-like protein, is expressed in coculture and "in vivo" in germ cells from normal testes of 6- and 24-mo-old rats but not in germ cells from regressed testes of 24-mo-old rats. A second germ cell marker, the Huntington disease protein, is expressed in all germ cells. Two Sertoli cell markers, a serotonin receptor and a novel gene, are induced in Sertoli cells by meiotic germ cells. The serotonin receptor mRNA is expressed in Sertoli cells from 20-day, 6-mo, and 24-mo normal testes but not in those from 24-mo regressed testes. The novel gene is induced in Sertoli cells from all testes. We conclude that Sertoli cells from aged regressed testes are unable to respond to selective signals from germ cells from young rats, and germ cells from regressed testes show a similar selective loss. Such disruptions in communication between Sertoli cells and germ cells likely contribute to germ cell loss during aging.     

Leydig cell hyperplasia in fetal mice treated transplacentally with ethinyl estradiol

Pregnant female mice were given ethinyl estradiol on days 11 through 17 of gestation. On day 18 the dams were killed and the male fetuses were examined for testicular differentiation. Three of 12 males from dams treated with the highest dose of ethinyl estradiol showed cryptorchid testes with uterine tubes. Light and electron microscopic evaluation of the testes, both cryptorchid and normal, demonstrated foci of hyperplasia of Leydig cells showing cytoplasmic and nuclear pleomorphism, increase in lipid droplets, and decrease in smooth endoplasmic reticulum and ribosomes when compared to testes from control fetal mice. Morphometric determinations of the testes indicated that the number of Leydig cells in a unit area (mm2) in the interstitial tissue showed a dose-response relationship to ethinyl estradiol in the normal testes. The number of Leydig cells in the testes exposed to the highest dose of estrogen showed a significant difference between cryptorchid and normal testes: the former had fewer Leydig cells than the latter. These morphological observations indicate that hyperplasia of Leydig cells of fetal mouse testis at term can be induced by transplacental treatment with ethinyl estradiol and suggest that a malignant transformation into a Leydig cell tumor is possible.     

Expression of anti-Müllerian hormone (AMH) in the equine testis

Anti-Müllerian hormone (AMH) induces regression of Müllerian ducts during male fetal development; in the human male, it is expressed in Sertoli cells during fetal development (and through puberty). The objective was to characterize expression of AMH in the fetal, neonatal, prepubertal, and adult equine testis, as well as in equine cryptorchid testes, in select testicular neoplasms, and in intersex gonads, based upon immunohistochemistry (IHC). Testes were removed from equine fetuses at 5.5, 10, and 11 months of gestation, at 12 months of age, and from adult stallions. In addition, cryptorchid testes, testis tumors (teratomas, seminomas, Sertoli cell tumors), and male intersex gonads were examined by IHC for expression of AMH using a goat polyclonal primary antibody (alpha-AMH) directed against a C-terminal peptide antigen from human AMH. Immunolabeling with alpha-AMH was localized to Sertoli cells within the developing seminiferous tubules of fetal, neonatal and prepubertal equine testes, with no expression detected in Sertoli cells from normal adult equine testes. Furthermore, expression was detected in cryptorchid testes (in animals up to 3-4 years of age) and in Sertoli cell tumors and male intersex gonads. In conclusion, AMH was strongly expressed by Sertoli cells in fetal, neonatal and prepubertal equine testes, but not in normal adult testes. That AMH was expressed in cryptorchid testes may provide a useful biomarker for detection of cryptorchid testes, as well as for immunohistochemical characterization of testicular tumors and intersex gonads in the horse.     

Regulation of pig Leydig cell aromatase activity by gonadotropins and Sertoli cells

The present work was done to investigate the cell localization of testicular aromatase activity and its regulation in immature pig testis using an in vitro model. Leydig cells and Sertoli cells were isolated from immature pig testes and cultured alone or together in the absence or presence of human chorionic gonadotropin (hCG) or porcine follicle-stimulating hormone (pFSH) for 2 days. At the end of incubation, the amounts of testosterone (T), estrone sulfate (E1S) and estradiol (E2) were measured. Then the cells were incubated for 4 h in the presence of saturating concentrations of delta 4-androstenedione (3 microM) and the amounts of E1S and E2 were measured again (aromatase activity). The ability of Sertoli cells to produce estrogens was very low and neither hCG nor pFSH had any significant effect. hCG stimulated, in a dose-dependent manner, the secretion of T and E1S by Leydig cells cultured alone as well as the aromatase activity of these cells. The main estrogen produced by Leydig cells was E1S. pFSH also stimulated the above parameters of Leydig cell function; this may have been due to the contamination of this hormone with luteinizing hormone (LH). Coculture of Leydig cells with Sertoli cells without gonadotropins had very small effects on T and E1S production and on aromatase activity. However, treatment of coculture with increasing concentrations of hCG had a dramatic effect on Leydig cell functions. For each hCG concentration, the amounts of T and E1S secreted, as well as the aromatase activity of the coculture, were 2- to 3-fold higher than those of Leydig cells cultured alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sertoli cell development and function in an animal model of testicular dysgenesis syndrome

Pregnancy exposure to di(n-butyl) phthalate (DBP) in rats induces a testicular dysgenesislike syndrome (TDS) in male offspring. Earlier studies suggested altered Sertoli cell development/maturation may result, especially in testes that become cryptorchid. This study quantitatively assessed Sertoli cell numerical and functional development in DBP-exposed rats and compared (unilaterally) cryptorchid and scrotal testes. Pregnant rats were gavaged with 500 mg/kg/day DBP or corn oil from embryonic (E) Days 13.5 to 21.5. Male offspring were sampled on E21.5 or Postnatal Day 6, 10, 15, 25, or 90. Sertoli cell number in DBP-exposed males was reduced by approximately 50% at E21.5 but recovered to normal by Days 25-90, accompanied by significant changes in plasma inhibin B and testosterone levels. Sertoli cell maturational development in DBP-exposed males, assessed using five protein markers (anti-müllerian hormone, cytokeratin, androgen receptor, CDKN1B, and Nestin), was largely normal, with some evidence of delayed maturation. However, in adulthood, Sertoli cells (SC) in areas lacking germ cells (Sertoli cell-only [SCO] tubules) often exhibited immature features, especially in cryptorchid testes. Sertoli cells in DBP-exposed animals supported fewer germ cells during puberty, but this normalized in scrotal testes by adulthood. Scrotal and especially cryptorchid testes from DBP-exposed animals exhibited abnormalities (SCO tubules, focal dysgenetic areas) at all postnatal ages. Cryptorchid testes from DBP-exposed animals exhibited more Sertoli cell abnormalities at Day 25 compared with scrotal testes, perhaps indicating more severe underlying Sertoli cell malfunction in these testes. Our findings support the concept of altered Sertoli cell development in TDS, especially in cryptorchid testes, but show that maturational defects in Sertoli cells in adulthood most commonly reflect secondary dedifferentiation in absence of germ cells.     

Immunoexpression of androgen receptors and aromatase in testes of patient with Klinefelter's syndrome

Klinefelter's syndrome (47, XXY) is the most common chromosome aneuploidy in men and is usually characterized by underdeveloped testes and sterility. The aim of the present study was to detect cellular distribution of androgen receptors (AR) and aromatase in testes of patient with KS. The tissue sections were processed for morphological and immunohistochemical staining. Additionally, levels of FSH, LH, PRL, estradiol, and testosterone were measured in the plasma. Morphological analysis revealed a complete absence of spermatogenesis. No germ cells were present in seminiferous tubules. In some tubules, nests of apparently degenerating Sertoli cells were found. In the interstitium, Leydig cell hyperplasia was observed. Using immunohistochemistry, nuclear AR staining was detected in Sertoli cells and peritubular cells, whereas in Leydig cells the staining was exclusively cytoplasmic. The immunostaining of aromatase was detected in the cytoplasm of Sertoli cells and Leydig cells. Increased levels of gonadotropins and decreased level of testosterone concomitantly with the cytoplasmic localization of AR in Leydig cells might contribute to the impaired testicular function in patient with KS.