Reduced testicular steroidogenesis in tumor necrosis factor-alpha knockout mice

We previously demonstrated that the expression of Mullerian inhibiting substance (MIS) in Sertoli cells is downregulated by tumor necrosis factor alpha (TNF-alpha), which is secreted by meiotic germ cells, in mouse testes. Several studies have reported that MIS that is secreted by Sertoli cells inhibits steroidogenesis and, thus, the synthesis of testosterone in testicular Leydig cells. Here, we demonstrate that in TNF-alpha knockout testes, which show high levels of MIS, steroidogenesis is decreased compared to that in wild-type testes. The levels of testosterone and the mRNA levels of steroidogenesis-related genes were significantly lower after puberty in TNF-alpha knockout testes than in wild-type testes. Furthermore, the number of sperm was reduced in TNF-alpha knockout mice. Histological analysis revealed that spermatogenesis is also delayed in TNF-alpha knockout testes. In conclusion, TNF-alpha knockout mice show reduced testicular steroidogenesis, which is likely due to the high level of testicular MIS compared to that seen in wild-type mice.     

Müllerian inhibiting substance activity in bovine fetal, newborn and prepubertal testes

Mullerian Inhibiting Substance (MIS) activity in bovine fetal, newborn, and prepubertal testes was investigated. MIS was present in all fetuses studied during the 1st 6 weeks after birth. Activity was present but diminished at 8 weeks. Loss of activity appeared to coincide with the time of weaning. Electron microscopic studies of the neonate testes revealed highly active Sertoli cells with a morphology suggestive of a protein secreting cell, and implicate the Sertoli cell as the source of MIS. It is concluded that the neonate calf testes may be an excellent source of material for the isolation and biochemical characterization of MIS.     

Expression of Connexin 43 in normal canine testes and canine testicular tumors

In human testis, gap junctions containing connexin(Cx)43 are located within the seminiferous epithelium between Sertoli cells and between Sertoli and germ cells. Cx43 is known to play a role in the differentiation and proliferation of these cell types. It can further be associated with human seminoma development. The dog has been proposed as a model for studies of the male reproductive system, because of the frequent occurrence of testicular neoplasms. Thus, we investigated Cx43-mRNA and -protein expression in testes of normal prepubertal dogs, adult dogs, and in canine testicular tumors. Sertoli cells in prepubertal cords express Cx43 mRNA, but do synthesize only less Cx43 protein. Within the seminiferous tubules, Cx43 mRNA was detected in Sertoli cells, spermatogonia, and spermatocytes. Cx43 protein was mainly present in the basal compartment. In canine testicular tumors Cx43 mRNA was detectable in both seminoma and neoplastic Sertoli cells, whereas Cx43 protein was only found in neoplastic Sertoli cells. Our data indicate that Cx43 is regulated differentially in testicular tumors and that alterations of Cx43 expression may be involved in the pathogenesis of canine testicular malignancies. This study represents the first morphological work on the spatiotemporal expression pattern of Cx43 in normal and neoplastic canine testis.     

The critical period for mullerian duct regression in the dog embryo.

The embryonic period during which Mullerian duct regression and Mullerian Inhibiting Substance (MIS) secretion occur was determined in canine embryos removed from timed pregnancies (32, 36, 37, 39, 42, and 46 days gestation). Sex chromosomes of each embryo were identified in metaphase spreads prepared from fibroblast cultures. Testicular differentiation, defined by seminiferous tubule formation and the presence of Sertoli cells and Leydig cells, and the degree of Mullerian duct regression were determined by careful morphologic analysis of histologic sections of canine embryonic gonads (n = 20) and Mullerian ducts (n = 20). MIS was detected immunohistochemically in embryonic testes using avidin-biotin complex enhancement of a specific rabbit polyclonal anti-MIS antibody. Testicular differentiation was observed at 36 days gestation. The earliest evidence of Mullerian duct regression in male embryos was observed at 36 days gestation, and regression was completed by 46 days gestation. Positive staining for MIS was present in testes from 36 to 46 days (n = 9). Staining was absent in the undifferentiated testis (n = 1) at 32 days gestation and in ovaries at all ages tested (n = 10). Thus, MIS is normally present throughout the critical period for Mullerian duct regression in the embryonic male dog.     

Immortalization of germ cells and somatic testicular cells using the SV40 large T antigen

We report the immortalization, using the SV40 large T antigen, of all the cell types contributing to a developing seminiferous tubule in the mouse testis. Sixteen peritubular, 22 Leydig, 8 Sertoli, and 1 germ cell line have been established and cultured successfully for 90 generations in a period of 2.5 years. Immortalized peritubular cells were identified by their spindle-like appearance, their high expression of alkaline phosphatase, and their expression of the intermediary filament desmin. They also produce high amounts of collagen. Immortalized Leydig cells are easily identifiable by the accumulation of lipid droplets in their cytoplasm and the production of the enzyme 3-beta-hydroxysteroid dehydrogenase. Some Leydig cell lines also express LH receptors. The immortalized Sertoli cells are able to adopt their typical in vivo columnar appearance when cultured at high density. They exhibit a typical indented nucleus and cytoplasmic phagosomes. Some Sertoli cell lines also express FSH receptors. A germ cell line (GC-1spg) was established that corresponds to a stage between spermatogonia type B and primary spermatocyte, based on its characteristics in phase contrast and electron microscopy. This cell line expresses the testicular cytochrome ct and lactate dehydrogenase-C4 isozyme. These four immortalized cell types, when plated together, are able to reaggregate and form structures resembling two-dimensional spermatogenic tubules in vitro. When only the immortalized somatic cells are cocultured, the peritubular and Sertoli cells form cord-like structures in the presence of Leydig cells. Fresh pachytene spermatocytes cocultured with the immortalized somatic cells integrate within the cords and are able to survive for at least 7 days. The ability to perform coculture experiments with immortalized testicular cell lines represents an important advancement in our ability to study the nature of cell-cell and cell-matrix interactions during spermatogenesis and testis morphogenesis.     

The ontogeny of mullerian inhibiting substance in granulosa cells of the bovine ovarian follicle

Mullerian Inhibiting Substance (MIS) previously detected in the Sertoli cells of the calf testis has been localized in the granulosa cells of the ovarian Graafian follicle by using an immunoperoxidase technique and a monoclonal antibody (IG8) to MIS that almost completely blocks its biological activity. The immunoperoxidase technique (avidin-biotin complex method) demonstrated specific localization of MIS in the cytoplasm of the ovarian granulosa cells in the bovine Graafian follicles over a wide age span, i.e. one day, one week, three months, two-and-a-half years and five years. The presence of MIS in the ovary implies a function that is as yet unknown.     

Cell type-autonomous and non-autonomous requirements for Dmrt1 in postnatal testis differentiation

Genes containing the DM domain, a conserved DNA binding motif first found in Doublesex of Drosophila and mab-3 of Caenorhabditis elegans, regulate sexual differentiation in multiple phyla. The DM domain gene Dmrt1 is essential for testicular differentiation in vertebrates. In the mouse, Dmrt1 is expressed in pre-meiotic germ cells and in Sertoli cells, which provide essential support for spermatogenesis. Dmrt1 null mutant mice have severely dysgenic testes in which Sertoli cells and germ cells both fail to differentiate properly after birth. Here we use conditional gene targeting to identify the functions of Dmrt1 in each cell type. We find that Dmrt1 is required in Sertoli cells for their postnatal differentiation, and for germ line maintenance and for meiotic progression. Dmrt1 is required in germ cells for their radial migration to the periphery of the seminiferous tubule where the spermatogenic niche will form, for mitotic reactivation and for survival beyond the first postnatal week. Thus Dmrt1 activity is required autonomously in the Sertoli and germ cell lineages, and Dmrt1 activity in Sertoli cells is also required non-autonomously to maintain the germ line. These results demonstrate that Dmrt1 plays multiple roles in controlling the remodeling and differentiation of the juvenile testis.     

Dysplastic development of seminiferous tubules and interstitial tissue in rat hypogonadic (hgn/hgn) testes

The hypogonadic rat is characterized by male sterility, reduced female fertility, and renal hypoplasia controlled by a single recessive allele (hgn) on chromosome 10. Plasma testosterone is low and levels of gonadotropins are high in adult male hgn/hgn rats, indicating that the cause of hypogonadism lies within the testis itself. We found that the postnatal growth of the seminiferous tubules was severely affected. Here we describe the details of postnatal testicular pathogenesis of the hgn/ hgn rats. In these rats, gonadal sex determination and initial differentiation of each type of testicular cell occur, but proliferation, differentiation, and maturation of these cells during postnatal testicular development is severely affected. Postnatal pathological changes include reduced proliferation and apoptotic cell death of Sertoli cells, abnormal mitosis and cell death of gonocytes, reduced deposition of extracellular matrix proteins into the basal lamina, lack of the formation of an outer basal lamina, formation of multiple layers of undifferentiated peritubular cells, and the delayed appearance and islet conformation of adult-type Leydig cells. Apoptotic cell death of Sertoli cells and disappearance of FSH receptor mRNA expression indicate that this mutant rat is a useful model for Sertoli cell dysfunction. The abnormalities listed above might be caused by defective interactions between Sertoli cells and other types of testicular cells. Because the results presented here strongly indicate that a normal allele for hgn encodes a factor playing a critical role in testicular development, the determination of the gene responsible for hgn and the analysis of early alterations of gene expression caused by mutations in this gene would provide important information on the mechanisms of testicular development.     

Aromatase in the human testis

Low levels of testicular estrogen synthesis have been reported in a number of species, but the cellular localization has not been unequivocally established. To study aromatase in the human testis, we have combined immunocytochemistry with direct measurement of enzyme activity in the testicular 6μm cryosections. Thus, the functionality of the immunoreaction and its sensitivity can be assessed in quantitative terms. Testes were obtained from immediate autopsy from men aged 18–53 years, from surgery from two patients with prostatic cancer (67 and 74 years) and from two normal children aged 8 months and 3 years at autopsy. Benign testicular sex cord tumors were also examined from two unrelated patients aged 5 and 8 years with gynecomastia and diagnosed with Peutz-Jeghers syndrome. Our results consistently showed low to moderate staining intensity of immunoreactive aromatase in comparison to that of normal human placental cryosections. Immunoreactive aromatase was only present in the interstitial Leydig cells and absent from the Sertoli cells of all normal adult testes showing spermatogenesis. Aromatase activity correlated well with the intensity of the immunostain. However, there was no obvious relationship between the level of aromatase activity and increasing age. Generally higher levels were present in testes of young men (18–22 years). No immunostain in any cell type was detected in one 33-year-old patient with testicular cancer. In the testes of the two normal prepubertal boys, no immunostaining was observed. However, intensely stained Sertoli cells as well as high aromatase activity were observed in the testicular tumors of the patients with Peutz-Jeghers syndrome. Our results suggest that Leydig cells are the source of aromatase in normal men but that Sertoli cells may express this enzyme under abnormal conditions. The combined methods for measuring enzyme activity and immunoreactive aromatase are suitable for application to tissues expressing low levels of aromatase.     

Development of a conditionally immortalized testicular Sertoli cell line RTS3-3 from adult transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene

Transgenic mice and rats harboring temperature-sensitive simian virus 40 (tsSV40) large T-antigen gene are useful for establishing cell lines from tissues. We succeeded in establishing a conditionally immortalized testicular Sertoli cell line, RT3-3, from adult transgenic rats harboring the oncogene. The cells grew at permissive (33 degrees C) and intermediate (37 degrees C) temperatures but not at nonpermissive temperature (39 degrees C). Large T-antigen was expressed at 33 and 37 degrees C, whereas the expression level was gradually decreased at 39 degrees C, suggesting that the temperature-sensitive growth characteristics arise as a result of the function of tsSV40 large T-antigen. The cells showed biochemical features associate with normal Sertoli cells including expressions of mRNAs of sulfated glycoprotein-2 (SGP-2), transferrin (TF) and steel factor. Quantitative polymerase chain reaction revealed that nonpermissive temperature induced increase in the level of SGP-2. Moreover, levels of SGP-2 and/or TF were significantly elevated in the cells treatment with sodium butyrate and retinoic acid, inducers of cellular differentiation. To our knowledge, this is the first report of the establishment of a testicular Sertoli cell line from the transgenic rats. Thus, the conditionally immortalized cell line RTS3-3 with unique characteristics may serve as good experimental in vitro models for basic and applied biology of testicular Sertoli cells.     

Transforming growth factor-alpha and epidermal growth factor receptor gene expression and action during pubertal development of the seminiferous tubule.

The potential role of transforming growth factor-alpha (TGF-alpha) as a mediator of cell-cell interactions in the growth and development of the testis was examined. Developing rat testes were collected, and preparations of mesenchymal-derived peritubular cells and epithelial-like Sertoli cells were isolated from prepubertal, midpubertal, and late pubertal rat testes. The developmental expression of TGF-alpha and its receptor, the epidermal growth factor receptor (EGFR), in whole testis and isolated cell types was determined using a nuclease protection assay. TGF-alpha and EGFR gene expression were predominant early in testis development and decreased during pubertal development. TGF-alpha expression was greatest in prepubertal peritubular cells. Sertoli cell TGF-alpha expression remained relatively constant during development, with a slight decline at the later pubertal stages. EGFR gene expression was predominant in peritublar cells throughout development. A low level of EGFR expression was detected in Sertoli cells. Scatchard analysis confirmed the presence of high affinity receptors on peritubular cells; however, no functional receptors were detected on Sertoli cells from any stage of development examined. Interestingly, low-level EGFR gene expression was also detected in pachytene spermatocytes and round spermatids. TGF-alpha was found to stimulate [3H] thymidine incorporation into DNA and increase cellular proliferation of peritubular cells from each developmental stage, while having no effect on Sertoli cells. The in vivo physiological significance of TGF-alpha was evaluated in a line of transgenic mice which overexpress TGF-alpha in the mature testis. These transgenic animals had no abnormal testicular morphology or alterations in spermatogenesis. Observations demonstrate that gene expression of TGF-alpha and its receptor is high during early pubertal stages when somatic cell growth is predominant and low at late pubertal stages when somatic cell proliferation is reduced. TGF-alpha can act as an autocrine/paracrine mitogen for the mesenchymal-derived peritubular cell, while actions on the Sertoli cell population are not evident. The observation that spermatogenic cells express the EGFR gene, although the protein remains to be identified, implies that TGF-alpha may potentially mediate Sertoli-germinal cell interactions.     

Expression in vitro du gène de l'aromatase dans les cellules testiculaires du rat

The ability of the male gonad to convert androgens into estrogens is well known ; the microsomal enzymatic complex involved in this transformation is named aromatase and is composed of a specific cytochrome P450 aromatase (P450arom) and a ubiquitous reductase. Using a highly specific RT-PCR method we have measured the amount of P450arom mRNA in purified Leydig and Sertoli cells prepared from 20, 40 and 70–80 day-old rats. The amount of P450arom mRNA in the Leydig cells is independent of age (40 × 10⁻³ attomoles/μg of total RNA); in contrast, in the immature rat Sertoli cells, after 5 days of culture the amount of P450arom mRNA is 20-fold lower when compared to that of 20-day-old rat Sertoli cells (71 × 10⁻³ attomoles/μg of total RNA). Nevertheless, irrespective of the age, the addition of either FSH or dbcAMP for 6 h increases the level of P450arom mRNA in the rat Sertoli cell preparations. Therefore, we evidenced that during testicular maturation not only the Leydig cells but also the Sertoli cells of the rat have the capacity to express the gene for cytochrome P450 aromatase.     

Testicular cell differentiation in fetal mouse ovaries following transplantation into adult male mice

Testicular development is a complicated process involving differentiation and arrangement of several cell types. To analyze the process of testicular organization we examined the sequence of the appearance of testicular structures induced in fetal ovaries following transplantation. Fetal mouse ovaries on the twelfth day of gestation were transplanted beneath the kidney capsules of adult male mice. They continued to develop morphologically as ovaries until the eleventh day after transplantation, when seminiferous cord formation and testosterone production began in addition to follicle development (ovotestes). Between the eleventh and fourteenth day after transplantation, ovarian grafts frequently contained transitional structures consisting of Sertoli cells, pregranulosa cells, a third type of cells which show intermediate characteristics between Sertoli and pregranulosa cells, and oocytes enclosed by common basal lamina. Leydig cells or peritubular myoid cells were not found in the transitional area, whereas these cells were present around seminiferous cords composed only of Sertoli cells. Oocytes were absent or degenerated in the well-developed seminiferous cords. The present findings suggest that, in ovarian grafts, pregranulosa cells can differentiate into Sertoli cells, which are responsible for the organization of the seminiferous cords, degeneration of oocytes, and differentiation of other testicular somatic cell types.     

Intratubular germ cell neoplasia of unclassified type

Intratubular germ cell neoplasia of unclassified type (IGCNU) is the precursor lesion of adult testicular germ cell invasive tumors. Primordial germ cells (PGCs) are recognized as the cells of origin of testicular germ cell tumors (TGCTs) because of the genetic and phenotypic characteristics analyzed. The most important risk factors responsible for abnormal development of PGCs are environmental, including the testicular dysgenetic syndromes that generate a better microenvironmentfor survival of IGCNU cells, an abnormal relationship with Sertoli cells, and an abnormal hormonal exposure at the time of testicular differentiation in utero. Furthermore, a familial TGCT susceptibility gene (TGCT1), localized at Xq27, is associated with a higher risk for bilateral tumors and possibly cryptorchidism. The normal tetraploid pattern and the consequent genomic instability of germinal cell DNA are considered sufficient per se for neoplastic transformation. The altered expression of oncogenes and suppressor genes due to nonrandom chromosomal numerical aberrations are involved in the development of IGCNU. Some of these genes are considered responsible for bilaterality, while other genes characterize the similarity between IGCNU cells and PGCs or are involved in the neoplastic transformation, histotype differentiation, and invasivity. In spite of the monomorphic seminomatous appearance of cells in IGCNU, it is becoming increasingly evident that they hide an intrinsic heterogeneity capable of committing neoplastic cells to an embryonal and pluripotent development associated or not with a seminomatous phenotype.