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.     

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.     

Sertoli-germ cells interactions in the human testis.

In previous histoimmunochemical studies we reported that transferrin (TF) and insulin-like growth factor I (IGF-I) are present in the cytoplasm of the Sertoli cells of the adult human testis. Receptors for TF were found mainly in adluminal germ cells and type I receptors for IGF-I both in Sertoli and germ cells. Using electron microscopy, evidence of transfer of both TF and IGF-I from the Sertoli to the germ cells through a receptor-mediated endocytosis mechanism was also found. In this paper we report the results of the histoimmunochemical localization of alpha inhibin in the human fetal, prepubertal and adult testis. In 8- to 14-week-old fetal testes a positive immunostaining was found mainly in the interstitial cells, whereas no staining was found in the germ cords. In the prepubertal testis the immunostaining was present in the Sertoli cells but not in the interstitial cells. In the adult human testis the immunostaining was present not only in the Sertoli cells but also in the spermatocytes and in several Leydig cells. Using electron microscopy and immunogold labeling the presence of alpha inhibin immunoreactivity was found in the rough endoplasmic reticulum and in the Golgi cisternae of both Sertoli and Leydig cells. Moreover we found evidence of transfer of alpha inhibin from the Sertoli to the germ cells through receptor-mediated endocytosis.     

Testins are structurally related Sertoli cell proteins whose secretion is tightly coupled to the presence of germ cells

Previous studies from this laboratory have shown that Sertoli cell-enriched culture medium contained two immunologically and structurally related proteins designated CMB-22 and CMB-23 with Mr of 37,000 and 40,000, respectively. We have now demonstrated that both CMB-22 and CMB-23 are monomeric proteins with the following NH2-terminal amino acid sequences: CMB-22, NH2-TPDPSLDVEWNEWRTKHGKTYNMNEERLKR; CMB-23, NH2-XAPXPDPSLDVEXNEXRTK. These sequences are virtually identical except that CMB-23 has three extra NH2 terminus amino acids of X-A-P. Comparison of these sequences with those in the Protein Identification Resource revealed that they are unique proteins. CMB-22 and CMB-23 are highly concentrated in testes and their levels in this tissue increase with age. Studies using [35S]methionine incorporation and immunoprecipitation demonstrated that Sertoli cells synthesize and secrete these proteins in vitro. Because they seem not to have been isolated previously, are concentrated in and synthesized by the testes, and are structurally related, we propose that CMB-22 and CMB-23 be designated testin I and testin II, respectively. The distribution of these proteins in biological fluids were compared with those of testibumin and rat androgen binding protein (rABP), two other Sertoli cell proteins. The results suggest that testins, unlike testibumin and rABP, are not transported to the epididymis. Although the amount of testins secreted by Sertoli cells in vitro is similar to that of testibumin and rABP, the concentrations in testis and rete testis fluid are several orders of magnitude less than that of testibumin and rABP. These observations suggest that the secretion of these proteins in vivo might be suppressed by germ cells. The fact that 10 times more testins are secreted by tubules from immature rats than by those from adult rats and that there is an increase in the testicular content of testins following a single dose of busulfan, which depleted the germ cells from the seminiferous epithelium, supports this hypothesis. Thus, the secretion of testins by Sertoli cells appears to be tightly coupled to the presence of germ cells; there is an inverse relationship between the amount of testins in the testis and the number of germ cells. These results suggest that testins are unique testicular proteins that can be used to study Sertoli cell-germ cell interactions in the seminiferous epithelium.     

Fyn tyrosine kinase in Sertoli cells is involved in mouse spermatogenesis.

Fyn is a member of the Src family of non-receptor-type tyrosine kinases and plays an important role in signal transductions regulating cell proliferation and differentiation. Fyn immunoreactivity was localized in the Sertoli cells of mouse testes. Although fyn-deficient adult male mice were fertile, a significant reduction in testis weight and degenerated germ cells were observed at 3 and 4 wk of age. Electron microscopic examination revealed that fyn -/- testis has ultrastructural abnormalities in the specialized junctional structures of the Sertoli cells, the ectoplasmic specializations. Unusual vesicular structures were found in the actin filament layers of the ectoplasmic specializations of mutant mice. Immunohistochemical studies demonstrated that both Fyn and actin filaments were concentrated in the areas of ectoplasmic specializations. At these sites, a high level of phosphotyrosine was also immunostained in wild-type testes, whereas phosphotyrosine immunoreactivity was reduced in fyn -/- testes. Immunoblot analyses revealed that Fyn was mainly distributed within the Triton X-100-insoluble cytoskeletal fraction prepared from wild-type testes, suggesting that Fyn might be associated with cytoskeletal proteins such as actin filaments. These findings suggest that Fyn kinase functions at the ectoplasmic specializations of the Sertoli cells in the testes, regulating the dynamics of cytoskeletal proteins. Fyn-mediated signal transduction in the Sertoli cells may affect the survival and differentiation of germ cells at a specific stage during spermatogenesis.     

Regulation of testicular and Sertoli cell gamma-glutamyl transpeptidase by follicle-stimulating hormone

Hormonal deprivation achieved by hypophysectomy or gonadotropin-releasing hormone (GnRH)-antagonist treatment of immature rats resulted in markedly lower testicular gamma-glutamyl transpeptidase (GGT) activity than in the testes of age-matched controls. When begun 15 days after hypophysectomy, follicle-stimulating hormone (FSH) treatment significantly increased testicular GGT above that in testes from hypophysectomized controls in a time- and dose-dependent manner. In contrast, testosterone propionate had only a small effect. Testicular GGT was higher in adult hypophysectomized rats treated with FSH from the time of surgery than in untreated hypophysectomized rats; testosterone propionate treatment had no effect. GGT activity in Sertoli cells isolated from GnRH antagonist-treated or hypophysectomized immature rats was also lower than in cells from control rats. FSH treatment from the day of hypophysectomy resulted in Sertoli cell GGT values equivalent to those from intact controls. These data indicate that FSH regulates GGT activity in rat testis and Sertoli cells.     

Histochemical location of 17 beta-hydroxysteroid oxidoreductase in the adult and infantile human testis

17 beta-Hydroxysteroid oxidoreductase in the human testis was investigated histochemically using tissues obtained from seven patients with undescended testis or varicocele at the time of orchiopexy or high ligation of spermatic vein. Formazan precipitates were formed from nitro-blue tetrazolium in the tissue utilizing hydrogen released by oxidation of testosterone, which is catalyzed by the reductase function of 17 beta-hydroxysteroid oxidoreductase. The precipitates were formed specifically in the presence of 17 beta-hydroxy-C19-steroids under the conditions employed in the present study. In infantile testes, the precipitates were formed in cytoplasm of immature Sertoli cells, while in pubertal or adult testes, marked formazan precipitates were found in cytoplasm of both Sertoli and Leydig cells. The results indicate the presence of two distinct 17 beta-hydroxysteroid oxidoreductases in the human testis; one in Sertoli cells and detectable independent of age and the other only in functional Leydig cells.     

Isolation and culture of ram rete testis epithelial cells: structural and biochemical characteristics

Cultures of rete testis epithelial cell-enriched preparations from testes of adult rams have been investigated, and some of their properties have been determined. In monolayers, the cells form mosaic-like borders, and retain many ultrastructural features characteristic of rete epithelial cells in situ, including an indented nucleus with prominent heterochromatin clumps, short rod-shaped or round mitochondria that are easily distinguished from the elongated mitochondria of Sertoli cells, the presence of desmosomes, and few if any lipid droplets or vacuoles. Unlike Sertoli cell-enriched aggregates in culture, rete testis epithelial cell preparations do not form cytoplasmic extensions, and no associated germ cells are present. Rete cells in culture express cytokeratin and vimentin in the cytoskeleton, whereas Sertoli cells prepared from testes of adult rams contain vimentin but not cytokeratin. Both rete cells and Sertoli cells stain positively for laminin but not for fibronectin, Collagen Type I, or Collagen Type III. The rete cells synthesize and secrete several proteins into the culture medium, evident in gel electrophoresis patterns of radiolabeled proteins. This pattern is similar, but not identical, to that secreted by Sertoli cell-enriched preparations. Rete cells in culture in the presence of serum continue to undergo mitotic division, but Sertoli cells do not. A variety of criteria were employed to estimate the relative numbers of Sertoli cells present in the rete testis epithelial cell-enriched preparations from testes of adult rams, including morphological and ultrastructural differences between the two cell types, and the presence of desmosomal proteins and cytokeratin in rete cells but not in Sertoli cells. The relative number of fibroblast-like cells was determined by measuring the expression of fibronectin and Collagen Type I, and an immunocytochemical probe for the detection of Factor VIII was used to estimate the degree of contamination by vascular endothelial cells. Using these markers, we determined that the rete testis epithelial cell-enriched preparations were about 93% pure. Primary cultures under defined conditions contained relatively few Sertoli cells (0.4%), but were contaminated to a larger extent by fibroblast-like cells (approximately 4%) and by endothelial cells (about 3%). The possible functions of rete testis epithelial cells are discussed herein.     

Histological and histochemical studies on testis, epididymis and ductus defrens of the rooster (Gallus domesticus)]

Testis, epididymis and ductus deferens of the adult domestic fowl and male gonads of juvenile roosters have been studied by means of histochemical and histological methods. Testicular interstitial cells: According to enzyme-histochemical results oxidative energy production seems to be of minor importance. An extraordinarily high activity of lysosomal enzymes (acid phosphatase and esterases in the adult, esterases only in the immature) is observed. Positive reactions of 3beta-steroid-dehydrogenase and enzymes of the pentose phosphate cycle indicate steroid hormone production; the pathways of the steroid synthesis, however, are probably different in adult and immature testes. A remarkable LAP content of juvenile Leydig cells is a parameter of an increased protein metabolism. Areas of reserve cells: Focal accumulation of these cell types are observed in testis and epididymis of the immature and in the epididymis of the adult fowl. Reserve cells reveal distinct activities of LAP (prospective growth ability) and 3beta-HstDH (reserve capacity for steriod synthesis). All other enzymes studied react weakly, thus pointing to a generally low metabolic activity. Seminiferous tubules: The strong reaction of alkaline phosphatase in the peritubular cells may play a part in energy disposition for contractions. Sertoli cells of adult animals are rich in lysosomal enzymes and enzymes of the glycolytic chain but oxidoreductases react weaker than in mammalian Sertoli cells. This indicates that nutritive interactions between germ and Sertoli cells in birds are different from those in mammals: The basally orientated germ cells of birds contain strong activities of diaphorases, LDH, SDH, Cyto-Ox and seem to be metabolically rather indipendent from Sertoli cells.     

Germ cell transplantation in pigs.

Spermatogonial stem cells form the foundation of spermatogenesis, and their transplantation provides a unique opportunity to study spermatogenesis and may offer an alternative approach for animal transgenesis. This study was designed to extend the technique of spermatogonial transplantation to an economically important, large-animal model. Isolated immature pig testes were used to develop the intratesticular injection technique. Best results of intratubular germ cell transfer were obtained when a catheter was inserted into the rete testis under ultrasound guidance. The presence of infused dye or labeled cells was confirmed in the seminiferous tubules from 70 of 89 injected isolated testes. Infusion of 3-6 ml of dye solution or cell suspension could fill the rete and up to 50% of seminiferous tubules. The technique was subsequently applied in vivo. Donor cells included testis cells from 1- or 10-wk-old boars (from the recipients' contralateral testis or unrelated donors) and those from mice carrying a marker gene. Porcine testis cells were labeled with a fluorescent marker before transplantation. Testes were examined for the presence and localization of labeled donor cells immediately after transplantation or every week for 4 wk. Labeled porcine donor cells were found in numerous seminiferous tubules from 10 of 11 testes receiving pig cells. These results indicate that germ cell transplantation is feasible in immature pigs, and that porcine transplanted cells are retained in the recipient testis for at least 1 mo. This study represents a first step toward successful spermatogonial transplantation in a farm animal species.     

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.     

Sertoli cell proliferation in the adult testis--evidence from two fish species belonging to different orders

Germ cell survival and development critically depend on the cells' contact with Sertoli cells in the vertebrate testis. Fish and amphibians are different from mammals in that they show a cystic type of spermatogenesis in which a single germ cell clone is enclosed by and accompanied through the different stages of spermatogenesis by an accompanying group of Sertoli cells. We show that in maturing and adult testes from African catfish and Nile tilapia, Sertoli cell proliferation occurs primarily during spermatogonial proliferation, allowing the cyst-forming Sertoli cells to provide the increasing space required by the growing germ cell clone. In this regard, coincident with a dramatic increase in cyst volume and number of germ cells per cyst, in Nile tilapia, the number of Sertoli cells per cyst was strikingly increased from primary spermatogonia to spermatocyte cysts. In both African catfish and Nile tilapia, Sertoli cell proliferation is strongly reduced when germ cells have proceeded into meiosis, and stops in postmeiotic cysts. We conclude that Sertoli cell proliferation is the primary factor responsible for the increase in testis size and sperm production observed in teleost fish. In mammals, Sertoli cell proliferation in the adult testis is not observed under natural conditions. However, on the level of the individual spermatogenic cyst--similar to mammals--Sertoli cell proliferation ceases when germ cells have entered meiosis and when tight junctions are established between Sertoli cells. This suggests that fish are valid vertebrate models for studying Sertoli cell physiology.     

Immunohistochemical study of calmodulin in developing mouse testis

The purpose of this study was to determine the localization of calmodulin in the developing mouse testis by the indirect immunoperoxidase method. In addition, the amount of calmodulin in pachytene spermatocytes, spermatids, and residual bodies isolated from the mouse testis and epididymal spermatozoa was quantitated by the adenylate cyclase activation assay and by enzyme immunoassay. The relative levels of calmodulin in the developing mouse testis and in the isolated testicular germ cells were confirmed by western transfer staining. The level of immunoreactive calmodulin was very low in the testes from immature animals. In testes from the mature mouse, calmodulin was found to be localized in spermatocytes and spermatids, but was not found in spermatogonia, Sertoli cells, and interstitial cells. By contrast, immunochemical staining of tubulin was extremely intense in Sertoli cells. Biochemical determinations also showed that pachytene spermatocytes, round spermatids, spermatozoa, and residual bodies contained 14.9 micrograms, 15.8 micrograms, 2.3 micrograms and 5.2 micrograms of calmodulin per mg of protein, respectively. Both the immunochemical and the biochemical studies revealed that levels of calmodulin were high in the spermatocytes and in the round spermatids, as compared to the level in spermatozoa. This fact strongly suggests that the large amount of calmodulin in mammalian testes may be associated primarily with meiotic divisions and/or spermatogenesis.     

Expression of p57 in mouse and human testes

The expression of cyclin-dependent kinases inhibitors, p57kip2, was investigated during the postnatal development of mouse testis, and in adult human testis. Expression of p57kip2 mRNA was higher in immature than pubertal or adult mouse testes. In postnatal day 7 (PND7) testes, moderate p57kip2 immunoreactivity was found in spermatogonia, but signal was heterogeneous among the spermatogonia. In PND14 testes onward, strong immunoreactivity of p57kip2 was found in the nuclei of early spermatocytes but not in the late pachytene stage onward. In PND28 and PND50 testes, p57kip2 immunoreactivity was varying among the seminiferous tubules. There was no visible signal in late pachytene stage onward. In Leydig cells, heterogeneous immunoreactivity of p57kip2 was found in immature testis and the signal intensity was higher in adult testis than immature ones. In Sertoli cells, weak or negligible immunoreactivity of p57kip2 was found. In human seminiferous tubule, strong immunoreactivity of p57kip2 was found in the nucleus of early spermatocytes, but not in the late pachytene spermatocytes onward and Sertoli cells. These results suggest the possible role of p57kip2 in the regulation of early spermatogonial proliferation, meiotic progression of early spermatocytes and differentiation of Leydig cells in testis.     

Investigation of the fate of Sry-expressing cells using an in vivo Cre/loxP system

Sry (sex-determining region on Y chromosome) is expressed in the undifferentiated, bipotential genital ridges of mammalian XY fetuses. The expression of Sry initiates testis development, but the lineage of Sry-expressing cells is unclear. In this study, double-transgenic mice were analyzed using the Cre/loxP system. Cre under the control of the Sry promoter was expressed in the fetal gonads of transgenic mice similarly to endogenous Sry. The Sry/Cre-transgenic mice were crossed with CAG(cytomegalovirus immediate-early enhancer, chicken beta-actin promoter and fusion intron of chicken beta-actin and rabbit beta-globin)/loxP/CAT/loxP/LacZ-transgenic mice, in which the transgene expressed beta-galactosidase after a Cre-mediated recombination event. Sertoli cells, germ cells of testes and granulosa cells of ovaries of double-transgenic mice stained positive with X-gal. Cre expression was detected in germ cells and peritubular/Sertoli cells in adult testes. It is not clear whether beta-galactosidase expression in the Sertoli cells of the testes occurred as a result of Cre expression in the adult or in the fetal gonads. These analyses indicate that cells expressing Sry-inducing factors in female fetal gonads become granulosa cells.     

Structural and functional abnormality of ectopic testes in rats

Some males of a mutant strain of King-Holtzman rats exhibit an anomalous heritable defect manifested as either unilateral or bilateral ectopic testes. In the adult, these testes contain seemingly immature Sertoli and Leydig cells, seminiferous tubules greatly reduced in diameter, and exhibit arrested spermatogenesis. Thus, the affected testis is essentially sterile. An inability to produce normal amounts of testosterone and androstenedione by these gonads is probably a reflection of changes that have been effected in their Leydig cells. Thus, this study suggests that abnormal function of the Leydig and Sertoli cells and seminiferous tubule failure in these mutant animals result from the physiologically cryptorchid condition.     

Expression of inhibin α-subunit gene during mouse gametogenesis

Mammalian gametogenesis is regulated through complex interactions between germ and somatic cells. To investigate the mechanism underlying the differentiation of functional gametes, some genes specifically expressed during gametogenesis have been isolated and characterized. In a search for further examples of such genes, we have isolated from a newborn mouse testis cDNA library, a clone corresponding to mouse inhibin alpha-subunit. Although it is known that the inhibin alpha-subunit molecule is abundantly produced in ovarian follicle and in testicular Sertoli cells, the spatial and temporal patterns of expression of this gene remain to be elucidated. In this study, the patterns of expression of inhibin alpha-subunit mRNA during mouse gametogenesis were examined by RNA blot, cytoplasmic dot and in situ hybridization techniques. In the testis, the concentration of inhibin alpha-subunit mRNA increased from about 16 dpc (days post coitum), peaked at birth and then gradually decreased, paralleling testicular development. Inhibin alpha-subunit mRNA was localized in Sertoli cells of wild type as well as W/Wv testes. In adult testis, mRNA was restricted to the perinuclear cytoplasm of Sertoli cells. Inhibin alpha-subunit mRNA was expressed in follicle cells of adult ovary more abundantly than in adult testis. Analysis of expression during folliculogenesis showed that the accumulation of this mRNA began in preantrum follicles and the level of expression reached a maximum in Graafian follicles.     

Change of cyclin D2 mRNA expression during murine testis development detected by fragmented cDNA subtraction method

Using subtractive hybridization and polymerase chain reaction, we developed a differential cloning system, the fragmented cDNA subtraction method, that requires only small amounts of materials. The cloning system was used to isolate several cDNA fragments expressed more abundantly in the premeiotic day 3 post-natal mouse testis than in the adult mouse testis. The isolated cDNA fragments included cDNA encoding the murine cyclin D2. Northern blot and in situ hybridization analyses revealed that, during testis development, cyclin D2 expression was most abundant in the neonatal proliferating Sertoli cells. Those type A spermatogonia that were thought to divide mitotically also expressed cyclin D2 mRNA. Other spermatogenic cells, such as mitotically arrested gonocytes in neonatal testis and meiotically dividing germ cells in adult testis as well as adult Sertoli cells, were negative for the cyclin D2 signal. Adult W/W ^v mutant mice lacking germ cells expressed cyclin D2 mRNA in terminally differentiated Sertoli cells. Elimination of germ cells other than the undifferentiated type A spermatogonia by treating wild-type mice with an anti-c- kit monoclonal antibody did not result in the expression of cyclin D2 in Sertoli cells. These results demonstrate that there are lineage- and developmental-specific expression patterns of cyclin D2 mRNA during mouse testis development. At the same time, it is suggested that primitive type A spermatogonia affect the cyclin D2 expression of Sertoli cells.