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PubMed1999

Messenger ribonucleic acids encoding a serotonin receptor and a novel gene are induced in Sertoli cells by a secreted factor(s) from male rat meiotic germ cells.

Using Sertoli cell-germ cell cocultures and messenger RNA (mRNA) differential display, we have identified a complementary DNA of 355 nucleotides that is up-regulated in Sertoli cells by pachytene spermatocytes. The mRNA differential display pattern was confirmed by Northern blotting. Sequence analysis revealed a homology of 91% (nt) and 86% (aa) to a serotonin receptor. The mRNA encoding the serotonin receptor was detected in Sertoli cells after 18 h of coculture. Its induction did not require cell contact, as germ cell-conditioned medium also induced the mRNA. The germ cell factor(s) inducing the serotonin receptor mRNA is more than 10 kDa, survives freezing and thawing, and is heat sensitive. A high dose of serotonin (10 microM) or the serotonin receptor agonists (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl and quipazine induce the serotonin receptor mRNA in Sertoli cells after 24 h. The antagonists, ketanserin and spiperone, inhibit the serotonin-mediated mRNA induction but fail to inhibit the germ cell-mediated induction, suggesting that the germ cell factor(s) up-regulates the serotonin receptor by a distinct pathway. A second clone of 380 nucleotides, induced in Sertoli cells by pachytene spermatocytes or germ cell-conditioned medium, did not show homology to database sequences. The germ cell factor(s) inducing the second clone is larger than 10 kDa, but is inactivated by freezing/thawing and boiling. The induction of a serotonin receptor mRNA and a second novel mRNA in Sertoli cells by pachytene spermatocytes demonstrates that meiotic germ cells induce mRNA encoding an important receptor in Sertoli cells.

Amino Acid Sequence↗
PubMed1986

Incipient germ cell tumor in Sertoli-cell-only syndrome testis, accompanied with retroperitoneal teratocarcinoma and widespread metastases.

Incipient germ cell tumor in Sertoli-cell-only syndrome testis was examined in an autopsy case of retroperitoneal teratocarcinoma with widespread metastases. Although both testes of a 28-year-old man had clinically been small and free from tumor mass to palpation, histopathological examinations revealed a malignancy in the right testis with the appearance of Sertoli-cell-only syndrome. The left testis showed solely the histology of Sertoli-cell-only syndrome. The testicular malignancy consisted of undifferentiated, atypical germ cells mainly confined within approximately one-tenth of seminiferous tubules, and only one small cartilage nodule. Some tubules showed intratubular growth pattern suggestive of seminoma. A few syncytiotrophoblast-like giant cells occurred in the tubules. These findings seem to furnish substantial evidence to the concept that atypical germ cells are the origin of testicular germ cell tumors of different types.

Adult↗
PubMed1990

Prepubertal rat Sertoli cells secrete a mitogenic factor(s) that stimulates germ and somatic cell proliferation.

Sertoli cells were isolated from prepubertal 6- and 12-day-old rats. The Sertoli cell-conditioned media (SCCM-6 and SCCM-12) can markedly stimulate the proliferation of somatic cells and quiescent rat prespermatogonia in a dose-dependent and an age-related manner. SCCM-12 stimulated cell proliferation of BALB/c 3T3 fibroblasts up to 7-fold over control values, but did not stimulate to the same degree the germ cell mitotic activity. SCCM-6 stimulated proliferation of prespermatogonia up to 5-10-fold over controls. The mitogenic factor(s) in SCCM-6 appears to be more specific to prespermatogonia than to somatic cells which is consistent with the in vivo stimulation of mitosis in germ cells 5-6 days after birth and with the action of 'mitosis inducing substance'. The mitogenic factor(s) appears to be protein with a molecular weight over 8000 and sensitive to heat and trypsin treatment. These results suggest that the different mitogenicity of prepubertal rat SCCM on germ and somatic cells may be due to secretion of multiple mitogens by Sertoli cells in an age-dependent manner.

Animals↗
PubMed1999

Sertoli cell expression of galectin-1 and -3 and accessible binding sites in normal human testis and Sertoli cell only-syndrome.

Galectins are vertebrate lectins interacting with beta-galactosides and derivates thereof such as blood group A, B and H determinants. The expression of gelectin-1 and -3 and galectin-specific binding sites by human Sertoli cells was analyzed in normal human testis and Sertoli cell only-syndrome (SCOS). Staining intensity was scored semiquantitatively on a 4-grade scale. Sertoli cells in normal testes displayed a moderate cytoplasmic and weak nuclear staining for galectin-1-specific binding sites. Galectin-3-specific binding sites were expressed in Sertoli cells less intensely than accessible ligands for galectin-1 (mean score 2.25 for galectin-1 and 1.50 for galectin-3). Germ cells were only weakly reactive. Tubular walls were negative for both classes of galectin-specific binding sites. In SCOS, galectin-1 binding was moderate to strong and more pronounced than galectin-3 binding by Sertoli cells (mean scores 4.00 and 2.25). Tubular walls were negative for galectin-staining. The ratio for galectin-1-/galectin-3-specific binding (staining score ratio) was 1.50 form normal testis and 1.78 for SCOS disclosing a relative increase of galectin-3 binding sites in the latter. Staining with galectin-1- and -3-specific antisera showed a strong cytoplasmic galectin-1 immunoreactivity in Sertoli cells of normal and SCOS testis (score 4.00 for both). Anti-galectin-3 did not stain Sertoli cells or germ cells in normal testis. Only Leydig cells were labeled (score 3.00). In SCOS a weak to moderate nuclear staining of Sertoli cells was noted (score 2.00). Galectin-3 expression and galectin-1-specific binding sites were found to be increased in Sertoli cells of SCOS. This modulation of reactivity can have implications for Sertoli cell interactions with galectin-reactive extracellular matrix components like laminin and for anti-apoptotic effects.

Adult↗
PubMed1995

Isolation and culture of Sertoli cells from the testes of adult Siberian hamsters: analysis of proteins synthesized and secreted by Sertoli cells cultured from hamsters raised in a long or a short photoperiod.

In the present study, we describe two procedures for isolation and culture of Sertoli cells from the testes of adult Siberian hamsters. In procedure I, collagenase and pancreatin were used for differential enzymatic digestion of the tissue at 32 degrees C, and effects of several attachment factors were examined. Sertoli cells isolated from adult hamsters were not affected by addition of Na selenite, epidermal growth factor, insulin, and transferrin, with or without 5% fetal bovine serum, to Dulbecco's modified Eagle's medium + Ham's F-12. Significant increase in the yield and plating efficiency of Sertoli cells was achieved by developing a novel procedure (procedure II) for Sertoli cell isolation. In this procedure, testicular tissue was exposed to only one enzyme (collagenase I) for two consecutive digestions at 37 degrees C, and the total period of enzymatic exposure was reduced relative to that of procedure I. Another objective of this study was to compare the functions of Sertoli cells isolated from spermatogenetically active and inactive adult testes. Isolated Sertoli cells from 60 +/- 5-day-old hamsters raised in long day conditions (LD, 16L:8D) or in short day conditions (SD, 6L:18D) were stimulated with FSH and testosterone in the presence of 35S-methionine for 24 h on Day 4 of culture. The medium was concentrated, and equal amounts of radioactive proteins from LD and SD hamster Sertoli cell cultures were analyzed by two-dimensional PAGE. Compared to Sertoli cells derived from SD hamsters, Sertoli cells from LD animals produced greater amounts of two secretory proteins and a smaller amount of one.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

A rat Sertoli cell factor similar to basic fibroblast growth factor increases c-fos messenger ribonucleic acid in cultured Sertoli cells.

We have shown that the cultured Sertoli cell from the immature rat contains a fibroblast growth factor (FGF)-like factor. It behaves as a cationic peptide, is a potent competence factor for BALB/c3T3 mouse embryo fibroblasts, and displays a high affinity for heparin. Both bovine basic FGF and Sertoli cell FGF-like factor rapidly increase c-fos mRNA in cultured Sertoli cells. FSH, serum, and phorbol esters individually stimulate c-fos in cultured Sertoli cells whereas platelet-derived growth factor, epidermal growth factor, and insulin-like growth factor-I have little affect. However, unlike FSH, basic FGF does not stimulate an increase in cAMP and unlike either serum or phorbol esters, basic FGF does not stimulate phosphoinositol turnover or intracellular calcium changes. When Sertoli cell protein kinase C activity is suppressed by preexposure to phorbol ester, basic FGF continues to be a potent stimulator of c-fos, indicating that the calcium/phospholipid pathway is not involved in FGF induction. Basic FGF and FSH also increase jun-B mRNA levels in cultured Sertoli cells. In response to FGF, jun-B is more transiently increased than c-fos. In contrast, in response to FSH, jun-B persists longer than c-fos. These results indicate that cultured Sertoli cells contain a FGF-like factor that increases c-fos mRNA via a mechanism not involving cAMP and the calcium/phospholipid pathways. The different responsiveness of c-fos and jun-B to FSH and basic FGF may explain differences in the ultimate actions of these two ligands.

Animals↗
PubMed1992

Production of the cysteine proteinase inhibitor cystatin C by rat Sertoli cells.

The Sertoli cells of the rat testis produce cystatin C, a cysteine proteinase inhibitor. Primary culture of Sertoli cells secreted both unglycosylated and glycosylated forms of rat cystatin C. Despite the low concentration of cystatin C in rete testis fluid, equilibrium dissociation constants (Ki) for the interaction between cystatin C and lysosomal cathepsins indicate that this molecule could be involved in the local regulation of testicular cysteine proteinase activity which may be necessary for spermatogenesis and spermiogenesis.

Amino Acid Sequence↗
PubMed1998

Abnormalities in functional development of the Sertoli cells in rats treated neonatally with diethylstilbestrol: a possible role for estrogens in Sertoli cell development.

Diethylstilbestrol (DES) was administered neonatally (Days 2-12; 10 microg on alternate days) to rats, and developmental changes in Sertoli cell function were evaluated at 18, 25, and 35 days of age and compared to those observed in rats administered a GnRH antagonist (GnRHa; Days 2 and 5; 10 mg/kg) or a vehicle (controls). DES and GnRHa treatments resulted in similar reductions in both Sertoli cell numbers (40% for DES, 48% for GnRHa) and suppression of testicular growth at 18 and 25 days, though by 35 days the suppression was more pronounced (p < 0.001) in DES-treated animals. Plasma FSH levels were suppressed markedly at 18 and 25 days, but not at 35 days, in GnRHa-treated rats, whereas in DES-treated rats the FSH levels were suppressed significantly only at 35 days. Both treatments suppressed plasma levels of inhibin B, though this was more pronounced (p < 0.05) in DES- than in GnRHa-treated rats. In controls, Sertoli cell immunoexpression of inhibin alpha, sulfated glycoprotein-1 (SGP-1), and androgen receptor (AR) increased in intensity and changed to an adult, stage-dependent pattern by 25 days. In GnRHa-treated rats these changes were reduced in intensity but were similar to those in controls at 35 days. In DES-treated rats, the increase in intensity and stage-dependent pattern of immunoexpression of inhibin alpha, SGP-1, and AR were virtually absent at 25 days but were present by 35 days. Germ cell volume per Sertoli cell was reduced in GnRHa- and DES-treated rats compared with controls at 18 and 25 days but was significantly greater (p < 0. 001) in DES- than in GnRHa-treated rats at 35 days. The proportion of apoptotic to viable germ cells was increased (p < 0.01) in GnRHa- and DES-treated rats compared with controls at 18 and 25 days; but at 35 days, values in GnRHa-treated rats had declined to control values whereas those for DES-treated rats remained 10-fold elevated (p < 0.001). In adulthood, testis weight and daily sperm production were reduced by 43% and 44%, respectively, in GnRHa-treated rats, but spermatogenesis was grossly normal. Comparable changes were observed in approximately 25% of DES-treated rats, but the majority exhibited > 60% reduction in testis weight with many Sertoli cell-only tubules and very low daily sperm production. Taken together, these data are interpreted as providing evidence for direct modulation of Sertoli cell (maturational) development by DES.

Aging↗
PubMed1997

Immunohistochemical analysis of androgen effects on androgen receptor expression in developing Leydig and Sertoli cells.

Leydig and Sertoli cells are both targets of androgen action in the testis. Androgen exerts contrasting effects on the two cell types partially inhibiting steroidogenesis in adult Leydig cell and stimulating adult Sertoli cell functions required to support spermatogenesis. The developmental changes in the messenger RNA (mRNA) levels of androgen receptor (AR) also differ between Leydig and Sertoli cells, with Leydig cell AR mRNA being highest on day 35 postpartum, whereas Sertoli cell AR mRNA levels are highest on day 90. The purpose of the present study was to determine if the concentrations of AR in Leydig and Sertoli cells are differentially regulated during development using quantitative immunostaining. AR protein levels were measured in rat testes after hormonal treatments at three developmental stages: on days 21, 35, and 90 postpartum. At each age, five groups of animals were treated for 4 days with: 1) vehicle; 2) LHRH antagonist (NalGlu, 0.3 mg/kg BW.day) to suppress endogenous levels of androgen that accompany inhibition of LH and FSH secretion; 3) NalGlu + LH (0.2 mg/kg BW.day); 4) NalGlu + testosterone (T, at 7.5 mg/kg BW.day); and 5) NalGlu + MENT (a potent synthetic androgen, 7 alpha-methyl-19-nortestosterone, 0.7 mg/kg BW.day). AR protein was visualized by immunohistochemistry and measured by computer-assisted image analysis in Leydig and Sertoli cells using frozen sections of tests. After NalGlu treatment, AR levels in Leydig cells declined sharply to 42% and 31% of vehicle control (P < 0.01) in the 21 and 35 days postpartum age groups, respectively, but in 90-day-old rats there was no change. AR levels were partially maintained by exogenous LH, and completely maintained by exogenous androgen treatments in Leydig cells from 21- and 35-day-old rats, whereas in Leydig cells from 90-day-old rats, AR levels were unaffected in all treatment groups. In contrast, after NalGlu treatment, the AR concentration in Sertoli cells from 90-day-old rats were reduced to 32% of control (P < 0.01). Moreover, in Sertoli cells from 90-day-old rats, AR levels were partially maintained by LH and completely maintained by androgens. A similar trend was observed on day 35. On day 21, however, AR levels in immature Sertoli cells were unaffected in all treatment groups. These results indicate that androgen maximally stimulates AR levels in immature Leydig cells but is without significant effect in adult Leydig cells. In contrast, AR levels in Sertoli cells are more sensitive to androgen regulation in adult compared with immature animals. These findings indicate that there are distinct mechanisms controlling AR concentrations in Leydig and Sertoli cells during the development of the testis.

Androgens↗
PubMed1997

Recognition of phosphatidylserine on the surface of apoptotic spermatogenic cells and subsequent phagocytosis by Sertoli cells of the rat.

In a primary co-culture of spermatogenic and Sertoli cells of the rat, many spermatogenic cells die by apoptosis and are subsequently engulfed by Sertoli cells. We investigated the mechanism of this phagocytosis reaction. Testicular cells from 20-day-old rats were cultured, and spermatogenic cells and Sertoli cells were separated. When the recovered spermatogenic cells were maintained without Sertoli cells, the viability of the cells decreased and they became more susceptible to phagocytosis by Sertoli cells. Phagocytosis was severely impaired when liposomes containing acidic phospholipids, such as phosphatidylserine, phosphatidylinositol, and cardiolipin, were included in the reaction, whereas those consisting of neutral phospholipids showed little effect. Such anionic liposomes were more efficiently engulfed by Sertoli cells than were the other neutral liposomes. Also, the number of spermatogenic cells that exposed phosphatidylserine to the surface increased when cells were maintained in single culture. The results indicate that upon induction of spermatogenic cell apoptosis, phosphatidylserine and probably other acidic phospholipids, which are normally localized in the inner leaflet of the plasma membrane, translocate to the outer leaflet and serve as a signal for phagocytosis by Sertoli cells.

Animals↗
PubMed1998

Immunodetection of cell adhesion molecules in rat Sertoli cell cultures.

PROBLEM: The presence of cell adhesion molecules (CAMs) in Sertoli cells has not been explored extensively. The expression of CAMs involved in cell-matrix and cell-to-cell interactions in Sertoli cell cultures was examined. METHOD OF STUDY: Immunohistochemical and Western blot techniques were applied to rat Sertoli cell cultures using specific antibodies to alpha 3, alpha 5, and alpha 6 integrin subunits; NCAM; and cadherins. RESULTS: Expression of alpha 3 and alpha 6 integrin subunits (mainly laminin receptors) and lack of expression of alpha 5 integrin subunit (fibronectin receptor) was observed in Sertoli cells by immunohistochemistry. These cells also expressed neural CAM (NCAM) and N-cadherin. By Western blot analysis, Sertoli cell extracts reacted with antibodies to alpha 3 integrin subunit revealed a band approximately 130 kDa, whereas no expression of alpha 5 integrin subunit was detected. Cell extracts incubated with antibodies to pan cadherin exhibited a band approximately 120 kDa, whereas bands of 180, 140, and 120 kDa were observed with antibodies to NCAM. CONCLUSION: New data about the expression of receptors for extracellular matrix proteins (alpha 3 and alpha 6 integrin subunits) as well as cell-to-cell adhesion molecules (NCAM and cadherins) are reported in rat Sertoli cell cultures.

Animals↗
PubMed2005

Atypical development of Sertoli cells and impairment of spermatogenesis in the hypogonadal (hpg) mouse.

Testes of hypogonadal (hpg) mice show arrested postnatal development due to congenital deficiencies of gonadotrophin-releasing hormone (GnRH) and gonadotrophin synthesis and secretion. Follicle-stimulating hormone (FSH), androgen or oestrogen treatment restore qualitatively normal spermatogenesis in hpg testes. Understanding the cellular and molecular changes accompanying hormone-induced spermatogenesis in hpg mice requires detailed morphological analyses of the germ cells and Sertoli cells in the untreated hpg testis. We compared seminiferous epithelial cytology in adult hpg, immature and adult wild-type mice using unbiased optical disector-based stereology, immunolocalization of Sertoli cell microtubules (MT), espin (a component of the blood-testis barrier), markers of Sertoli cell maturity (p27(kip1) and WT-1), and electron microscopy. Hpg testes had marked reductions in weight, seminiferous cord volume and length, and severe spermatogenic impairment with germ cells per testis < 1% of adult wild-type testes. Sertoli cell nuclei expressed WT-1 in hpg testes, but often were centrally located, similar to 9-14-day-old wild-type testes, and they expressed p27(kip1), indicating that hpg Sertoli cells were post-mitotic. Hpg testes had significantly (P < 0.05) reduced Sertoli cells per testis (0.56 million) compared with 10-day wild-type (1.15 million) and adult wild-type testes (2.06 million). Immunofluorescence labelling of normal adult Sertoli cells showed supranuclear MT columns and basally located espin, but these features were absent in 10-day-old and hpg Sertoli cells. Hpg Sertoli cells showed pleomorphic nuclear ultrastructure with mature-type nucleoli, similar to normal adult-type Sertoli cells, but hpg Sertoli cells exhibited incomplete tight junctions that lacked ectoplasmic specializations. We conclude that in hpg mice, chronic gonadotrophin insufficiency restrains Sertoli cell proliferation and maturation, forming pseudo-adult-type Sertoli cells that are incapable of supporting germ cell proliferation and maturation.

Animals↗
PubMed1988

Pachytene spermatocyte proteins influence Sertoli cell function.

Isolated Sertoli cells were cultured on MatrigelTM-coated Millipore filters in bicameral chambers. The Sertoli cells form confluent epithelial sheets that, by virtue of the Sertoli cell tight junctions, form transepithelial permeability barriers between the apical and basal domains of the cells. These Sertoli cells secrete metabolically labeled proteins in a polarized manner. Three peptides, P1 (pI = 4.5-5.0, MW = 70,000), P2 (pI = 4.5-5.0, MW = 50,000), and P3 (pI = 4.0-4.7, MW = 34,000) are secreted apically from the epithelial sheets of Sertoli cells and are not found in basal secretions from the same Sertoli cells. Pachytene spermatocyte-conditioned medium contains proteins released from the germ cells that are uniquely different from the Sertoli cell-secreted proteins. Addition of the pachytene spermatocyte-conditioned medium to the apical reservoir of the bicameral chambers over an epithelial sheet of Sertoli cells stimulated the synthesis and secretion of total protein, transferrin, and specifically induced peptides S1 and S2 from Sertoli cells. As controls, conditioned medium from 3T3 fibroblasts and round spermatids did not stimulate the Sertoli cells. Hence, the ability of pachytene spermatocyte proteins to induce specific Sertoli cell secretion indicates that the pachytene spermatocytes are able to influence their surrounding milieu, and provides further support to the concept of a paracrine interaction between germ cells and Sertoli cells during spermatogenesis.

Animals↗
PubMed2002

Glutathione S-transferase alpha expressed in porcine Sertoli cells is under the control of follicle-stimulating hormone and testosterone.

Glutathione S-transferases (GSTs) are a family of detoxification isoenzymes present in different tissues including the testis and that conjugate many toxic substrates to glutathione. Among these substrates are carcinogens, mutagens and products of oxidative processes. In the present report we show that GSTalpha is expressed in somatic testicular Leydig cells and Sertoli cells. GSTalpha expression in Sertoli cells is under the hormonal control of FSH, testosterone, and estradiol. In Leydig cells, immunoreactive GSTalpha was present at the neonatal, pubertal, and adult periods. In Sertoli cells, GSTalpha was predominant in pubertal and adult testes (but not in neonatal testes), suggesting that its expression is controlled by gonadotropins. The regulatory action and the mechanisms of action of FSH and testosterone on GSTalpha mRNA and protein levels were studied by using a model of primary cultures of porcine testicular Sertoli cells. FSH increased GSTalpha mRNA levels in a dose-dependent manner (ED50 = 18.5 nm/ml) with a maximal effect observed after 48 h of exposure (a 3-fold increase; P < 0.001). In addition, FSH increased GSTalpha protein, which was detected as a doublet of 28 kDa. Treatment with testosterone enhanced GSTalpha mRNA levels in a dose-dependent (ED50 = 1.4 ng/ml) and time-dependent manner with a maximal effect delayed at 8 h of exposure (a 2-fold increase; P < 0.001). Similarly, Sertoli cell treatment with testosterone metabolites, dihydrotestosterone (DHT) and estradiol, led to an increase in GSTalpha mRNA levels. Because stimulatory effects of FSH and androgens were also observed on GSTalpha protein, we therefore had to determine whether the different hormones were affecting GSTalpha gene transcriptional activity, or GSTalpha mRNA stability, or both. FSH and 8-Br-cAMP (but not testosterone) increased the stability of GSTalpha mRNA. The effects of FSH and testosterone on GSTalpha protein were additive, confirming that both hormones act through distinct mechanisms on the expression of the enzyme. Taken together, the present observations indicate that Sertoli cell GSTalpha is targeted by FSH, testosterone, and its metabolites, and they reinforce the concept that Sertoli cells exert a protective role and are under endocrine control to ward against toxic agents in the context of Sertoli-germ cell interactions during spermatogenesis.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Basic fibroblast growth factor is a testicular germ cell product which may regulate Sertoli cell function.

Previously, a proteinacious factor secreted by a mixture of rat testicular spermatocytes and round spermatids was shown to stimulate transferrin mRNA and protein levels in Sertoli cells. To identify the germ cell-secreted proteins which affect Sertoli cell functions, concentrated germ cell-conditioned medium was fractionated by reverse-phase HPLC. The fraction which eluted at 35% acetonitrile increased transferrin secretion in Sertoli cell cultures 2.4-fold above the basal level. Both the active fraction and a protein extract from cultured germ cells were positive for basic fibroblast growth factor (bFGF) as determined by Western blot analysis and immunoprecipitation. The apparent molecular sizes of the immunoreactive proteins were 30, 27, and 24 kilodaltons (kDa). By immunohistochemistry, bFGF was shown to be present in pachytene spermatocytes and Leydig cells. The bFGF receptor was also examined by immunohistochemistry and found to be present in Leydig cells, round and elongated spermatids, and Sertoli cells. The presence of receptors was more pronounced in stages I-VIII. Western blot analysis confirmed that the receptors were expressed in isolated round spermatids, elongated spermatids, and Sertoli cells. Two major receptor species with apparent molecular sizes of 120 and 145 kDa were detected in the rat testis. Germ cells contained both of these receptors, but Sertoli cells possessed only the 120-kDa receptor. From these experiments, it is evident that bFGF is a germ cell product which may regulate Sertoli cell function. The expression of bFGF and its receptor may be an important component of germ cell-Sertoli cell and/or germ cell-germ cell communication during spermatogenesis.

Animals↗
PubMed2005

Effect of antispermatogenic agents on cell marker enzymes of rat Sertoli cells in vitro.

OBJECTIVE: To examine the role of Sertoli cells in the antispermatogenic action of two nonsteroidal male contraceptive compounds (CDRI-84/35 and gossypol) by evaluating their effect on some key parameters of Sertoli cell function in vitro. METHODS: Primary cultures of Sertoli cell were established from 18-day-old rat testis and treated on day 5 with different concentrations (1.0, 0.1, 0.01, and 0.001 mM) of either CDRI-84/35 or gossypol in vitro. Lactate (secretion), along with beta-glucuronidase, gamma-glutamyl transpeptidase, lactate dehydrogenase (LDH) and aromatase activities, was measured in these cells to examine the functions targeted by antispermatogenic agents in Sertoli cells. RESULTS: CDRI-84/35 significantly affected Sertoli cell parameters (stimulation in most of the cases) that are important for germ cell development like lactate secretion, LDH activity, aromatase activity (estradiol secretion) and so on. Gossypol in comparison to CDRI-84/35 had a more severe effect on Sertoli cells with complete inhibition of enzyme activities at higher concentrations. CONCLUSION: It is probable that the antispermatogenic action of CDRI-84/35 and gossypol is routed through Sertoli cells by disruption of important cell functions that support spermatogenesis in vivo. However, the two compounds appear to have different course of action in Sertoli cells, ultimately leading to spermatogenic failure.

Animals↗
PubMed1985

Stimulation of ribonucleic acid and deoxyribonucleic acid synthesis in spermatogenic cells by their coculture with Sertoli cells.

Germ cells were isolated from 30-day-old rat testes and purified by a Percoll gradient. Germ cells were cultured for 24 h to eliminate contaminant cells by utilizing the property of many nongerm cells of attaching to plastic surfaces. Germ cells were subsequently seeded on top of 7-day-old Sertoli cell monolayers. RNA and DNA synthetic activities were estimated by the rate of incorporation of [3H]uridine and [3H] thymidine into trichloroacetic acid-precipitable material. Germ cells increased RNA synthesis from 54,053 +/- 22,824 to 168,019 +/- 57,137 dpm/2 h X 10(6) cells (mean +/- SD) between 4 and 24 h of coculture, respectively (P less than 0.01), while they decreased this activity from 32,150 +/- 6,800 to 6,014 +/- 5,243 dpm when they were cultured alone for the same periods. Coculture of germ cells with either a cell derived from the adult bovine aortic endothelial cells (ABAE), or rat fibroblasts in primary culture prevented the fall in RNA synthesis but did not stimulate it. Addition of high concentrations of lactate to the culture medium did not have any affect in germ cells cultured alone but produced a slight stimulation in germ cells cocultured with ABAE cells, which was much smaller than the effect observed during coculture with Sertoli cells. Germ cells in coculture with Sertoli cells also increased their DNA synthesis from 2,186 +/- 765 at 4 h to 9,679 +/- 4,057 dpm [3H]thymidine/2 h X 10(6) cells at 24 h (P less than 0.05). When Sertoli cells were treated with FSH (1 microgram/ml), the synthesis of both RNA and DNA of germ cells cocultured with these cells was significantly higher than for germ cells cocultured with nontreated Sertoli cells. The system of coculture of Sertoli cells and germ cells consisted of the reassociation of the two previously isolated cells in a coculture, followed by their separation which permitted the effect of the coculture on the individual cell type to be assessed. By using this technique is was possible to obtain evidence suggesting that a chemical messenger other than lactate might be involved in the stimulatory effect of Sertoli cells on germ cells and that FSH increased this putative chemical messenger.

Animals↗
PubMedDate not supplied

Development and characterization of a prepubertal rat Sertoli cell line, 93RS2.

Sertoli cells in the seminiferous epithelium provide both structural and nutritional support to germ cells during spermatogenesis. Primary Sertoli cells in culture are an effective tool for the in vitro study of Sertoli cell function; however, primary cultures are inherently variable, time consuming to prepare, expensive, and wasteful of animals. We therefore developed a Sertoli cell line, called 93RS2, by immortalizing primary Sertoli cells derived from prepubertal rats with SV40 tsA255. This cell line proliferates at the permissive temperature (32 degrees C) and has enhanced expression of a differentiated Sertoli cell phenotype at the nonpermissive temperature (40-41 degrees C). Cytogenetic analysis demonstrated that 93RS2 has 42 chromosomes per cell, the same as a normal rat. mRNA analysis showed that this cell line, when cultured at a nonpermissive temperature, exhibited increased expression of transferrin in the presence of testosterone and enhanced expression of sulfated glycoprotein-2. A tumorigenicity assay showed that 93RS2 cells were temperature-dependent for growth in soft agar and were capable of forming tumors in nude mice. In conclusion, this rat 93RS2 cell line should be useful for the study of Sertoli cell function.

Animals↗