Differential localization of inhibin subunit proteins in the ovine testis during fetal gonadal development

Inhibins and activins are dimeric proteins that are involved in cell proliferation, apoptosis, and differentiation in a number of systems and have previously been detected in fetal testes of many species. This study used immunohistochemistry to examine the localization of inhibin alpha-, betaA-, and betaB- subunits during ovine testicular development from days 40-135 of gestation. Localization of inhibin betaA- and betaB-subunit messenger RNAs was confirmed by in situ hybridization. The results showed that there was differential localization of inhibin alpha-, betaA-, and betaB-subunits to specific cells in the ovine fetal testis from 40 days of gestation. All three inhibin subunits were present in Sertoli cells throughout gestation, whereas the rete epithelium and gonocytes did not express inhibin alpha-subunit. These data suggest that the fetal Sertoli cells have the capacity to produce all forms of inhibins and activins, i.e. inhibin A and B, and activins A, AB, and B, whereas the rete testis epithelial cells can only synthesize activin A. In the interstitium, the fetal Leydig cells expressed all three inhibin subunits, but this was restricted to the period between 40 and 90 days of gestation. Thereafter, inhibin alpha-subunit immunoreactivity was not observed in fetal Leydig cells, which suggests that only activin ligands are produced by Leydig cells during late gestation. Collectively, the data demonstrate that fetal ovine testes have the potential to produce the full repertoire of inhibins and activins from very early in testicular differentiation. The distinct and restricted localization of the various subunits to specific cells suggests that specific dimeric proteins have particular roles in the development and function of the fetal testis.     

Ontogeny of estrogen receptor-beta expression in rat testis

The recently discovered estrogen receptor-beta (ERbeta) is expressed in rodent and human testes. To obtain insight in the physiological role of ERbeta we have investigated the cell type-specific expression pattern of ERbeta messenger RNA (mRNA) and protein in the testis of rats of various ages by in situ hybridization and immunohistochemistry. In fetal testes of rats 16 days postcoitum and testes of 4-day-old animals, fetal germ cells (gonocytes) reveal the ERbeta mRNA in their cytoplasm and the ERbeta protein in their nucleus. In testes of 11- and 15-day-old rats, ERbeta mRNA and protein were detected in Sertoli cells and type A spermatogonia. No signal was found in other types of germ cells. In the adult testes, expression of ERbeta mRNA as well as ERbeta protein was found in pachytene spermatocytes from epithelial stages VII-XIV and in round spermatids from stages I-VIII. Low ERbeta expression was observed in all type A spermatogonia, including undifferentiated A spermatogonia, whereas no expression was found in In and type B spermatogonia and early spermatocytes. At all ages, Sertoli cells showed a weak hybridization signal as well as weak immunoreactivity for ERbeta. In adult testes, no ERbeta mRNA or protein was detected in the interstitial tissue, indicating that Leydig cells and peritubular myoid cells do not express ERbeta. The expression of ERbeta in fetal and late male germ cells as well as in Sertoli cells suggests that estrogens directly affect germ cells during testicular development and spermatogenesis.     

Wilms' tumor suppressor gene (WT1) as a target gene of SRY function in a mouse ES cell line transfected with SRY

With the aim of identifying the gene(s) located downstream from SRY, we transfected an ES cell line with XX karyotype, TMA-18, with a Sry DNA construct and established cell lines, TS18-1 and TS18-2, where the transfected Sry was expressed in the functional linear mRNA form. Among the five potential SRY-target genes examined, i.e., MIS, SF1, P450arom, Sox9 and WT1, only the expression of WT1 was induced de novo by the unscheduled expression of Sry in the transfected cell lines. No clear indication of Sry-induced enhancement of Sox9 expression was obtained in the present series of experiments. Function of a yet unidentified gene(s) located on the Y chromosome might be needed for the up-regulation of Sox 9 expression which takes place during the development of male gonads. Quantitative RT-PCR analysis of the patterns of WT1 expression in developing fetal gonads revealed that although both male and female fetal gonads express WT1, male gonads invariably expressed WT1 mRNA at higher levels than female ones after the Sry expression. Immunohistochemical analysis of the male fetal gonads between 10.5 and 13.5 dpc demonstrated the presence of strong WT1 immunoreactivity in Sertoli cells of the primordial testes. Suggestions were made in the past indicating that both SF1 and WT1 proteins might be active in a common pathway upstream from Sry. Our results showed that WT1 is located downstream, rather than upstream from Sry and behaves independently from SF1. Analysis using an appropriate in vitro system will be essential to understand the molecular mechanisms of SRY action within cells.     

Induction of Ets-1 in endothelial cells during reendothelialization after denuding injury

The requirement of Y-chromosome activity for the differentiation of somatic cells and germ cells was studied in the fetal gonads of X/XSxra mouse embryos where the activity of the Sxra fragment of the Y chromosome is influenced by the inactivation and reactivation of the X chromosome. In the interstitial somatic cells, random inactivation of the X and the XSxra chromosomes took place which was revealed by the mosaic expression of an X-linked lacZ transgene. The Sertoli cells, however, displayed a preferentially active XSxra chromosome and the presence of Sxra-active Sertoli cells was associated with the morphogenesis of testicular tubules in the sex-reversed gonads. The activity of the Y-chromosome fragment is therefore necessary for the differentiation of the Sertoli cells which may direct the development of the testis. The expression pattern of the X-linked transgene in X/XSxra germ cells suggests that both the X and the XSxra chromosomes are active. This finding suggests that the presence of Sxra has no impact on the reactivation of the X chromosome in the germ cells and that the X chromosome can be reactivated even though the germ cells are found in the testicular environment. Our results are consistent with the concept that the activity of genes on the XSxra fragment is essential for the differentiation of Sertoli cells and the morphogenesis of the testis, but not for premeiotic differentiation of germ cells in sex-reversed mice.     

Relaxin-like factor expression as a marker of differentiation in the mouse testis and ovary

Expression of the relaxin-like factor (RLF) was studied at the messenger RNA (mRNA) and protein levels in the testes and ovaries of the mouse, as well as through testicular development and differentiation in the mouse testis. In situ hybridization or RT-PCR, and immunohistochemistry using a polyclonal antibody raised against a recombinant protein, provided mutually confirmatory results for a high expression of RLF in the Leydig cells of the adult testis and at a much lower level of expression in the luteal cells of the ovary through the cycle, pregnancy, and in lactation. Analysis of protein and mRNA expression, through postnatal testicular development, indicated moderate RLF expression also in the fetal population of Leydig cells, even in the hpg mutant mouse, lacking an active pituitary-gonadal axis. Prepubertal Leydig cells, however, exhibit only very low-level RLF gene expression, this phenotype persisting in the adult hpg mouse. In summary, fetal Leydig cells express RLF in an LH/human CG-independent fashion, whereas LH/human CG is essential to induce RLF expression in the adult-type Leydig cell. In cultured adult Leydig cells or in the mouse tumor MA-10 cell line, RLF mRNA is expressed in a constitutive fashion. RLF thus seems to be a useful marker of Leydig cell differentiation status.     

Expression of inhibin alpha-subunit in horse testis

Inhibin is believed to play roles in the pituitary secretion of FSH and in the paracrine regulation of testicular function. Although it has been generally accepted that inhibin is produced in Sertoli cells, there was a recent evidence for the localization of inhibin in Leydig cells of primates, rat and sheep. However, there is no report on the expression of inhibin in the adult horse testis. Therefore, using immunohistochemistry, western blotting and in situ hybridization techniques, the present study examined inhibin alpha-subunit (Ih-alpha) expression in the adult horse testis. For the detection of Ih-alpha protein, we used anti-porcine Ih-alpha antibody in immunohistochemistry and western blotting. Furthermore, digoxigenin-labeled complementary RNA probes were prepared to detect intracellular messenger RNA (mRNA) of Ih-alpha. Immunostainings for Ih-alpha were found not only in Leydig cells but also in Sertoli cells. The intensity in Leydig cells was stronger than in Sertoli cells. Immunoreactivities for Ih-alpha were found at approximately 46 kDa, 56 kDa and 90 kDa in the homogenates from testicular interstitial tissues. The bands at 56 kDa and 90 kDa agree with previous report, but not at 46 kDa. Signals for mRNA of Ih-alpha by in situ hybridization were detected in Leydig cells and in the basal region of seminiferous epithelium including Sertoli cells. These results suggest that Ih-alpha is expressed in Leydig cells and Sertoli cells of horse testis, and the expression level should be higher in Leydig cells than Sertoli cells.     

Alteration of Sertoli cell differentiation in the presence of carcinoma in situ in human testes

PURPOSE: We investigated Sertoli cells in testicular biopsies with carcinoma in situ (CIS) in respect to cytokeratin expression to elucidate the status of Sertoli cell differentiation adjacent to CIS in human testes. Cytokeratin 18 intermediate filaments indicate a state of undifferentiation usually observed in Sertoli cells of prepubertal testes. MATERIALS AND METHODS: 29 testicular biopsies presenting CIS were investigated by means of immunohistochemistry, using a polyclonal antibody against placental-alkaline phosphatase to detect CIS cells and a monoclonal antibody against human cytokeratin 18 to show expression of cytokeratin 18 intermediate filaments in Sertoli cells. RESULTS: All tubules bearing CIS showed positive cytokeratin expression of Sertoli cells if tubules were devoid of normal germ cells. However, a total of 13 specimen revealed CIS cells together with normal germ cells. In the presence of CIS cells together with round or elongated spermatids, adjacent Sertoli cells did not express cytokeratin immunoreactivity. In the case of combined presence of CIS and spermatogonia and primary spermatocytes, Sertoli cells could be found either immunopositive or immunonegative, and were positive in tubules with CIS and spermatogonia only. CONCLUSIONS: Sertoli cells associated with CIS cells undergo a process of dedifferentiation, seen by the re-expression of cytokeratin intermediate filaments. We suggest that this dedifferentiation results in a loss of Sertoli cell function and leads to a cessation of spermatogenic activity.     

A substance secreted by rat Sertoli cells induces feminization of embryonic chick testes in vitro

Male and female gonads from 7- to 9-day-old chick embryos were cultured for 6 days in Sertoli cell-conditioned medium or in serum-free medium to investigate the possible effect of substances secreted by rat Sertoli cells on chick gonad development. Histological analysis showed that whereas all female gonads proceed through normal ovarian development in both culture media, most of male gonads showed clear feminization only when cultured in Sertoli cell-conditioned medium; male gonads cultured in serum-free medium developed as normal testes. Because the only substance detected in our conditioned medium with the potential to cause these effects was sex-specific antigen (Sxs), our results provide further evidence that Sxs antigen may play a role in sexual differentiation in birds, and probably in mammals.     

Sertoli cell-specific expression of rat androgen-binding protein in transgenic mice: effects on somatic cell lineages

The Sertoli cells of many species produce an androgen binding protein (ABP) which carries testicular androgens to the epididymis and is thought to play a role in sperm maturation. In the present report we analyzed the morphological modifications present in Leydig, Sertoli, and peritubular cells of the testis of young adult male mice transgenic for ABP gene, which overproduce ABP in testis. By in situ hybridization we demonstrated that ABP is specifically produced by Sertoli cells. Using light and electron microscopy, we detected scattered alterations of the seminiferous tubule cells which include cell degeneration and vacuolization. Leydig and Sertoli cells present morphological signs of hyperfunctioning compensatory mechanisms which include increased amounts of lipid droplets probably due to the existence of a stimulated steroid synthesis that in turn could be a consequence of the decreased unbound testosterone and/or a direct paracrine effect of ABP. Peritubular cells also present numerous signs of hyperstimulation.     

Differential regulation of leucine-rich primary response gene 1 (LRPR1) mRNA expression in rat testis and ovary

In immature rat Sertoli cells, leucine-rich primary response gene 1 (LRPR1) represents a follicle stimulating hormone (FSH)-responsive gene; the function of the encoded protein is not yet known. LRPR1 mRNA expression is up-regulated very rapidly and specifically by FSH, both in cultured Sertoli cells and in vivo in regulation in more detail, in testis and ovary of fetal, immature, and adult rats. In addition, we have studied the expression of FSH receptor (FSHR) mRNA in relation to LRPR1 mRNA expression. In rat testis, LRPR1 mRNA and FSHR mRNA followed a similar expression pattern, during postnatal development and also at different stages of the spermatogenic cycle in the adult rat. Furthermore, after short-term challenge of the FSH signal transduction pathway in intact immature rats by injection with a relatively high dose of FSH, an inverse relationship between LRPR1 mRNA (up-regulation) and FSHR mRNA expression (down-regulation) was observed. Similar studies in the ovary provided completely different results. LRPR1 mRNA in the postnatal ovary is present well before expression of FSHR mRNA can be first detected. In addition, incubation of ovaries of immature rats with FSH or dibutyryl cyclic AMP (dbcAMP) did not result in up-regulation of LRPR1 mRNA expression. During fetal development, the LRPR1 mRNA expression pattern involved many more tissues, in contrast to the relatively tissue-specific expression of LRPR1 mRNA in gonads of 21 day old and adult rats. Moreover, LRPR1 mRNA expression could be detected as early as 12.5 days post-coitum, whereas FSHR mRNA is absent at this stage of fetal development. We concluded that the pronounced regulation of LRPR1 by FSH observed in the immature rat testis does not occur in the ovary. Furthermore, in the ovary LRPR1 mRNA expression does not appear to be dependent on FSH action. Finally, the LRPR1 gene product may play a general role during fetal development.     

Sertoli cells of the mouse testis originate from the coelomic epithelium

During mouse development, the gonad begins to form shortly before 10. 5 days postcoitum (dpc) on the ventromedial side of the mesonephros. The XY gonad consists of germ cells and somatic cells. The origin of the germ cells is clearly established; however, the origin of the somatic cells, especially the epithelial supporting cell lineages, called Sertoli cells, is still unclear. Sertoli cells are the first somatic cell type to differentiate in the testis and are thought to express Sry, the male sex-determining gene, and to play a crucial role in directing testis development. Previous data have suggested that the somatic cells of the gonad may arise from the mesonephric tubules, the mesonephric mesenchyme, or the coelomic epithelium. Immunohistochemical staining of the gonad at 11.5 dpc showed that the basement membrane barrier under the coelomic epithelium is discontinuous, suggesting that cells in the coelomic epithelium at this stage might move inward. To test this possibility directly, cells of the coelomic epithelium were labeled using the fluorescent lipophilic dye, DiI. We show that when labeled at tail somite 15-17 stages, corresponding to 11.2-11.4 dpc, the coelomic epithelial cells of both sexes migrated into the gonad. In XY gonads, the migrating coelomic epithelial cells became Sertoli cells, as well as interstitial cells. This ability of the coelomic epithelium to give rise to Sertoli cells was developmentally regulated. When labeled at tail somite 18-20 stages, corresponding to 11.5-11.7 dpc, the coelomic epithelial cells no longer became Sertoli cells. Instead, cells that migrated into the gonad stayed outside testis cords, in the interstitium. Migration gradually decreased and ceased by tail somite 30 stage, corresponding to 12.5 dpc, after testis cords had formed and the basement membrane layer underlying the coelomic epithelium had thickened to form the tunica albuginea. In XX gonads, coelomic epithelial cells also migrated into the gonad, but there was no obvious fate restriction during the same developmental period. Taken together, our data show that the coelomic epithelium is a source of Sertoli cells as well as other somatic cells of the gonad in the developing mouse testis.     

Cellular localization of insulin-like growth factor II mRNA in the human fetus and the placenta: detection with a digoxigenin-labeled cRNA probe and immunocytochemistry

IGF-II plays a major role in the regulation of human fetal growth and development. However, more extensive information on the cellular sites of IGF-II synthesis in the fetus would provide more insight into its role in fetal organogenesis. Thus we have determined the sites of IGF-II synthesis in 18-26-wk gestation human fetal tissues using in situ hybridization with a digoxigenin-labeled cRNA probe to localize IGF-II mRNA in fetal liver, kidney, adrenal gland, cerebral cortex, costal cartilage, skeletal muscle, and lung, and in placental tissue. In human fetal tissues it has to date been impossible to clearly assign IGF-II mRNA to epithelial cells of entodermal origin. Besides their already known localization in cell matrix and a variety of mesodermal cell types, strong IGF-II mRNA-positive signals were detected in epithelial cells in the liver (hepatocytes), bronchial and bronchiolar epithelium, undifferentiated renal tubular epithelium, mature glomerular epithelium, pelvic urothelium, and adrenal epithelial cells of the zona persistens. To identify the cellular location of immunoreactive IGF-II, we also performed immunocytochemical studies in tissues of the same fetuses. Every tissue studied except the cerebral cortex contained immunoreactive cells; however, immunostaining was generally weaker than in situ hybridization signals. Our data show that the distribution of IGF-II in human fetal tissue is much more widespread than hitherto thought. A digoxigenin-labeled detection system for IGF-II is more capable of detecting the cellular expression pattern of IGF-II than radioactive probes and is suitable for analysis of routinely prepared paraffin-embedded material.