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PubMed2001

Gene trap analysis of germ cell signaling to Sertoli cells: NGF-TrkA mediated induction of Fra1 and Fos by post-meiotic germ cells.

Analysis of complex signalisation networks involving distinct cell types is required to understand most developmental processes. Differentiation of male germ cells in adult mammals involves such a cross-talk between Sertoli cells, the somatic component which supports and controls germinal differentiation, and germ cells at their successive maturation stages. We developed a gene trapping strategy to identify genes, which, in Sertoli cells, are either up- or down-regulated by signals emitted by the germinal component. A library of approximately 2,000 clones was constituted from colonies independently selected from the Sertoli line 15P-1 by growth in drug-containing medium after random integration of a promoter-less (beta)geo transgene (neo(r)-lacZ fusion), which will be expressed as a fusion transcript from a 'trapped' cellular promoter, different in each clone. A first screen conducted on 700 events identified six clones in which beta-galactosidase activity was increased and one in which it was repressed upon addition of germ cells. The targeted loci were identified by cloning and sequencing the genomic region 5' of the insert. One of them was identified as the gene encoding Fra1, a component of the AP1 transcription regulatory complex. Accumulation of Fra1 mRNA was induced, both in 15P-1 and in freshly explanted Sertoli cells, by addition of either round spermatids or nerve growth factor (NGF). The effect of NGF was mediated by the TrkA receptor and the ERK1-ERK2 kinase kinase pathway. Fos and Fra1 transcription were induced within the first hour after addition of the neurotrophin, but, unlike what is observed after serum induction in the same cells, a second wave of transcription of Fra1, but not of Fos, started 16 hours later and peaked at higher levels at about 20 hours. These results suggest that AP1 activation may be an important relay in the Sertoli-germ cell cross-talk, and validate the gene trapping approach as a tool for the identification of target genes in cell culture systems.

Animals↗

PubMed1995

Neonatal hypothyroidism causes delayed Sertoli cell maturation in rats treated with propylthiouracil: evidence that the Sertoli cell controls testis growth.

BACKGROUND: The testes of rats treated neonatally with propylthiouracil (PTU) grow to almost twice their normal size. The cause of testicular enlargement has been suggested to be the result of delayed maturation of Sertoli cells, allowing Sertoli cell division to occur beyond the 15th postnatal day, the commonly recognized cutoff date for Sertoli cell divisions. It has been shown that an increased population of Sertoli cells in postnatal development supports increased numbers of germ cells in adult animals. After examining developing rats treated neonatally with PTU, we hypothesized that an approximate 10-day delay in maturation was occurring and proceeded to test this hypothesis experimentally. Thus the purpose of this report was to determine if a 10-day delay in maturation could explain the increased numbers of Sertoli cells and increased testis size in PTU-treated animals. METHODS: Both control animals and animals treated neonatally with PTU N = 5/group were sacrificed at 15 and 25 days of age and prepared for electron microscopy. RESULTS: Micrographs show and morphometric ultrastructural analysis of numerous parameters demonstrated at the 95% probability level that Sertoli cells from 25-day-old PTU animals are not different in size and most constituents (volume and surface area) from 15-day-old control animals and are less mature than 25-day-old control animals. Mitosis of Sertoli cells was observed in PTU-treated animals in 25-day-old animals but not in age-matched controls. The number of Sertoli cells in 25-day-old PTU-treated animals is significantly increased over age-matched controls. Micrographs show the presence of immature Sertoli cell nuclei in 25-day-old animals receiving PTU as well as increased germ cell degeneration in this group. Sertoli cell tight junction formation is also delayed in PTU-treated animals as compared with controls. CONCLUSIONS: Together, the data show that delayed maturation of Sertoli cells occurs in treated animals that corresponds to a minimum of 10 developmental days. In the immature state, Sertoli cells continue to divide. Data presented herein and published data related to PTU treatment indicate that delayed maturation of the Sertoli cell results in delayed maturation and proliferation of other testicular cell types. From this and from published data, the hypothesis is presented that the Sertoli cell is responsible for the overall control of testis development.

Animals↗

PubMed1983

Chemical messengers between Sertoli cells and neighbouring cells.

The Sertoli cell has been shown to secrete a number of compounds, some of which are postulated to serve as chemical signals to the neighbouring cells. The evidence for such communication between Sertoli cells and Leydig cells is briefly summarized. More emphasis is put on Sertoli cell-germ cell interactions. The morphological observations do not support a direct transfer of material from cell to cell. Therefore, the observed dependency of germ cells on Sertoli cells and vice versa must be explained by diffusion of substances from one cell to the other. Examples of Sertoli cell secretory products whose production is dependent on the type of surrounding germ cells are ABP, plasminogen activator, a somatomedin-like compound, transferrin and a high-molecular weight aromatase inhibitor.

Androgen-Binding Protein↗

PubMed1984

The actions of insulin-like growth factors I and II on cultured Sertoli cells.

Cultured Sertoli cells prepared from young rats (13 days old) showed increased incorporation of [3H]thymidine into DNA, increased production of lactate, and increased incorporation of [3H]leucine into protein in response to micromolar concentrations of insulin and nanomolar concentrations of insulin-like growth factor II (IGF-II). The first of these responses was also seen with nanomolar concentrations of IGF-I. Receptor affinity labeling studies using Sertoli cell membranes and whole Sertoli cells showed that these cells possess abundant growth factor receptors of type I (mol wt, 350,000) that show high affinity for IGF-I, moderate affinity for IGF-II, and low affinity for insulin. Sertoli cell membranes also show abundant growth factor receptors of type II (mol wt, 230,000) that show high affinity for IGF-II, moderate affinity for IGF-I, and no detectable affinity for insulin. Moreover, the responses of the Sertoli cell to insulin were observed at concentrations of 100 nM or higher, whereas insulin receptors are known to be saturated by insulin at concentrations of 10 nM or less. It is, therefore, concluded that Sertoli cells possess receptors for IGF-I and that the responses observed to insulin may result from binding of these hormones to receptors for IGF-I.

Animals↗

PubMed1995

Evidence that basement membrane prevents apoptosis of Sertoli cells in vitro in the absence of known regulators of Sertoli cell function.

Although the role of basement membrane in the morphological and functional differentiation of Sertoli cells has been well characterized, very little is known about its involvement in Sertoli cell survival and maintenance throughout life. When cultured on laminin or Matrigel, 80-90% of Sertoli cells retained their viability. Sertoli cells prevented from attachment and basement membrane deposition by plating on plastic surfaces coated with polyhydroxyethylmethacrylate (poly-HEMA) exhibited a loss of viability by approximately 50% within 24 h. Addition of soluble laminin did not prevent the loss of viability of Sertoli cells, whereas soluble Matrigel enhanced the survival significantly when added at a concentration of 100 micrograms/ml or more. The addition of FSH, epidermal growth factor, testosterone, retinoic acid, or a mixture of insulin, transferrin, and selenium had no significant effect on the viability of Sertoli cells cultured on polyHEMA for up to 72 h. When all of these hormones and factors were added together, a significantly higher percentage of cell survival was observed at 24, 48, and 72 h, but the percent survival was significantly lower than that seen on either laminin or Matrigel. The nature of cell death occurring in the Sertoli cells plated on polyHEMA was determined by agarose gel analysis that revealed a ladder of approximately 200-base pair DNA multiple fragments. Flow cytometric analysis of propidium iodide-stained cells indicated that most of the cells were apoptotic. Freshly isolated Sertoli cells and adherent cells on basement membrane did not show internucleosomal DNA breakdown or an apoptotic peak in the flow cytometric analysis. These results suggest that basement membrane plays a crucial role in Sertoli cell survival in vitro when it is used as a solid substratum for culture, and in the absence of basement membrane, FSH and other regulators of Sertoli cell function cannot prevent Sertoli cell apoptosis.

Animals↗

PubMedDate not supplied

Inhibin B levels in plasma of the male rat from birth to adulthood: effect of experimental manipulation of Sertoli cell number.

Sertoli cells undergo important changes in their number and function at different ages in the rat and may be the primary source of circulating inhibin B. The aims of this study were 1) to establish the profile of inhibin B levels from birth to adulthood in normal rats and 2) to identify whether experimental manipulation of Sertoli cell numbers was able to alter this profile. Levels of inhibin B, measured by a specific two-site assay, increased fivefold in normal Wistar rats between day 3 and days 10-15, plateaued, and then declined in late puberty to reach adult levels which were approximately 60% of those observed on days 10-15. The increase in inhibin B levels in the neonatal period coincided with the period of Sertoli cell multiplication as indicated by incorporation of bromodeoxyuridine. Neonatal treatment of rats with a GnRH antagonist (GnRHa) reduced Sertoli cell number and adult testis weight by 48% and significantly reduced plasma levels of inhibin B at all ages through to adulthood. Induction of neonatal hypothyroidism in Sprague-Dawley rats by administration of propylthiouracil (PTU) up to day 25 of age increased final testis weight by 41% (indicative of increased Sertoli cell numbers) and resulted in elevation of plasma levels of inhibin B at all ages beyond 7 days of age. The degree of change in inhibin B levels in adult rats in the two experimental treatment groups was approximately proportional to the change in final testis weight. Plasma follicle-stimulating hormone (FSH) showed changes opposite to inhibin B, with levels being lowered in PTU-treated rats and elevated (beyond day 25) in GnRHa-treated animals. The present results suggest that final Sertoli cell number per testis exerts an important effect on the circulating level of inhibin B (and FSH) in the rat. These findings are compared to the emerging data for the human male.

Aging↗

PubMed2007

Densin is a novel cell membrane protein of Sertoli cells in the testis.

Cell-cell interactions between Sertoli cells and germ cells are crucial for the maturation of germ cells in spermatogenesis but the structural and functional aspects of the interactions remain to be fully elucidated. Densin is a junction protein suggested to play a role in establishment of specific cell-cell contacts in the post-synaptic densities of the brain and the slit diaphragm of the kidney podocyte. In the present study, densin was discovered to be expressed in the testis of the man and the mouse. Expression of densin at the gene and the protein level was studied by using RT-PCR and Western blotting analyses, and the localization of densin was explored with immunofluorescence staining. RT-PCR and Western blotting analyses showed that densin is expressed at the gene and the protein levels. Immunofluorescence staining localized the expression of densin to the cell membranes of Sertoli cells suggesting that densin may be an adherens junction protein between Sertoli cells and developing germ cells. Densin is a novel testicular protein expressed in the cell membranes of Sertoli cells. Its functional role remains to be assessed.

Adult↗

PubMed1990

[Cell junctions between Sertoli cells and the blood-testis barrier in goldfish testis].

Freeze-fracture and etching technique combined with thin sectioning and lanthanum impregnation has been used for the study of Sertoli cell junctions and the blood-testis barrier formation in goldfish testis with lobular organization. Some observations and results are first given in this paper. The results of experiments can be summarized as the following: 1). Sertoli cell junctions are compound junctions of tight junctions, desmosomes and gap junctions. Tight junctions usually appear as parallel or network like ridges on the P face and fine grooves on the E face at the freeze-etching replicas. Desmosomes and gap junctions often are located between or nearby the ridges of tight junctions. In addition, endoplasmic reticulum cristae near the junction area can also be observed. 2). The number, area and density of each individual junction vary with the development and differentiation stages of germinal cells in the cyst. 3). Tight junctions can be observed at any stage during germinal cell differentiation through the period of spermatogenesis and spermiogenesis. However, they appear morphologically different as type I and type II. 4). Lanthanum can partially penetrate into the intercellular spaces of spermatogonium and early primary spermatocyte but can't penetrate after the stage of late primary spermatocyte. 5). The blood-testis barrier formation starts at the stage of pachytene spermatocytes. The formation of the blood-testis barrier is the result of the development of the tight junction from type I to type II.

Animals↗

PubMed1999

Effects of spinal cord injury on spermatogenesis and the expression of messenger ribonucleic acid for Sertoli cell proteins in rat Sertoli cell-enriched testes.

The study was an examination of the effects of spinal cord injury (SCI) on spermatogenesis and Sertoli cell functions in adult rats with Sertoli cell-enriched (SCE) testes. The effects of SCI on the seminiferous epithelium were characterized by abnormalities in the remaining spermatogenic cells during the first month after SCI. Three days after SCI, serum testosterone levels were 80% lower, while serum FSH and LH levels were 25% and 50% higher, respectively, than those of sham control SCE rats. At this time, the levels of mRNA for androgen receptor (AR), FSH receptor (FSH-R), and androgen-binding protein (ABP) were normal whereas those for transferrin (Trf) had decreased by 40%. Thereafter, serum testosterone levels increased, but they remained lower than those of the sham control rats 28 days after SCI; and serum FSH and LH levels returned to normal. The levels of mRNA for AR, ABP, and Trf exhibited a biphasic increase 7 days after SCI and remained elevated 28 days after SCI. FSH-R mRNA levels were also elevated 90 days after SCI. Unexpectedly, active spermatogenesis, including qualitatively complete spermatogenesis, persisted in > 40% of the tubules 90 days after SCI. These results suggest that the stem cells and/or undifferentiated spermatogonia in SCE testes are less susceptible to the deleterious effects of SCI than the normal testes and that they were able to proliferate and differentiate after SCI. The presence of elevated levels of mRNA for Sertoli cell FSH-R and AR, as well as of that for the Sertoli cell proteins, in the SCE testes during the chronic stage of SCI suggests a modification of Sertoli cell physiology. Such changes in Sertoli cell functions may provide a beneficial environment for the proliferation of the stem cells and differentiation of postmeiotic cells, thus resulting in the persistence of spermatogenesis in these testes.

Androgen-Binding Protein↗

PubMed2006

Giant Sertoli cell nodule of the testis: distinction from other Sertoli cell lesions.

The case of a 33-year-old man with a clinically suspected testicular neoplasm is reported here. The radical orchidectomy specimen showed a sharply demarcated, firm, yellow-white 1-cm nodule beneath the tunica albuginea at the upper pole. Microscopical examination showed the encapsulated nodule to be composed of tubules lined by immature Sertoli cells with interspersed spermatogonia and an interwoven network of hyalinised basement membrane having foci of calcification. Immunohistochemical studies verified the fetal phenotype of the Sertoli cells and the non-neoplastic nature of the germ cell component. Except for the large size, the findings were identical to those of a Sertoli cell nodule-a typically microscopic, unencapsulated lesion commonly detected in cryptorchid testes. The term "giant Sertoli cell nodule" is used for this unique, hitherto undescribed lesion and its distinction from other Sertoli cell lesions of the testis is considered here.

Adult↗

PubMed1996

Insulin-like growth factor I inhibits aromatization induced by follice-stimulating hormone in rat sertoli cell culture.

Sertoli cells in the testis and granulosa cells in the ovary convert androgen to estrogen under the primary control of FSH. Insulin-like growth factor I (IGF-I) markedly augments FSH-stimulated estrogen production in the rat granulosa cell. In this study we examined the regulation of aromatase by FSH and characterized the effects of IGF-I on FSH-induced estrogen production by Sertoli cells cultured from the testes of 16-day-old rats. FSH stimulated aromatization of androstenedione in Sertoli cell culture and achieved maximal effectiveness by 12 h of treatment. Analysis of aromatase mRNA by reverse transcription-polymerase chain reaction indicated a marked induction by FSH within 3 h of treatment that was dependent on FSH concentration. IGF-I inhibited FSH-stimulated aromatization dose-dependently; inhibition was approximately 50% by 6 h of cotreatment (p < 0.01). IGF-I was ineffective if added more than 3 h after addition of FSH. Aromatase mRNA was reduced by IGF-I (37 +/- 12%, p < 0.01), coincident with the decrease in estrogen production. To further address the mechanism of IGF-I inhibition, potential interactions with the cAMP and protein kinase C (PKC) signaling pathways were examined. IGF-I inhibited aromatase activity induced by dibutyryl cAMP and inhibited FSH-stimulated estrogen production in the presence of 3-isobutyl-1-methylxanthine, suggesting that IGF-I action was independent of cAMP production. Phorbol-12-myristate-13-acetate (PMA) and IGF-I were additive in their inhibition of FSH. However, down-regulation of PKC prevented PMA inhibition of FSH but not inhibition by IGF-I. We conclude that IGF-I specifically inhibits FSH-induced aromatization in the Sertoli cell in marked contrast to the effects of IGF-I on rat granulosa cells. Although IGF-I and PMA both inhibit aromatase induction, the independence of the IGF-I effect from PKC down-regulation suggests that the initial action of IGF-I is independent of PKC. As IGF-I treatment similarly alters FSH stimulation of both estrogen production and aromatase mRNA, it is likely that the effect of IGF-I on estrogen production in the Sertoli cell is a result, at least in part, of a decrease in aromatase mRNA.

Animals↗

PubMed2001

Sertoli cell proliferations of the infantile testis: an intratubular form of Sertoli cell tumor?

We report on six boys with intratubular Sertoli cell proliferations (ISCPs), studied by routine histologic methods, electron microscopy, and immunohistochemistry of anti-müllerian hormone (AMH), inhibin alpha-subunit, 3beta-hydroxysteroid dehydrogenase (3beta-HSD), proliferative cellular nuclear antigen, and p53, and carefully followed for extended periods with periodic clinical examinations, testicular ultrasonographies, and determinations of serum levels of AMH and inhibin B. Peutz-Jeghers syndrome was found in four of six patients, and gynecomastia occurred in five of six patients. One boy had isosexual pseudoprecocity. ISCPs were observed as multiple foci of seminiferous tubules with large and proliferated Sertoli cells replacing germ cells and limited by the basement membrane. Mitotic figures, atypia, and/or interstitial invasion were not observed. Bilateral ISCPs were the only pathologic finding in three patients (patient nos. 1-3) and were associated with a microscopic tumor that resembled a large-cell calcifying Sertoli cell tumor (LCCSCT) in a fourth patient (patient no. 4). In the two remaining patients (patient nos. 5 and 6) ISCPs and LCCSCT were found in both testes. Ultrastructural examination showed large Sertoli cells, with round nuclei, sparse organelles, and some glycogen. Inhibin alpha-subunit immunolocalization was positive in the five patients in whom it was determined (patient nos. 2-6), AMH was positive in those ISCPs associated with tumors (patient nos. 4-6) and negative in isolated ISCPs (patient nos. 2 and 3); 3beta-HSD and PCNA were variable, and p53 was negative in all ISCPs. Patient nos. 1-4 have been followed for 2-19 years. One of them is currently entering puberty, the other two have already completed puberty and have testes of normal size, and the remaining one is an adult with clinically normal testes and sperm production. None of these patients had evidence of tumor development during follow-up as shown by serial ultrasonographies and serum levels of AMH and inhibin B. Patient nos. 5 and 6 who had bilateral ISCPs and LCCSCT were orchidectomized and evolved for 2-10 years after surgery without tumor recurrence. The prognostic significance of ISCPs, particularly when they are the only pathologic finding in a testicular biopsy, is a matter of controversy. Based on the long normal evolution, we recommend a conservative approach to therapy. The bilateral and multicentric character of ISCPs and their association with Sertoli tumors and Peutz-Jeghers syndrome suggest that they represent either proliferative lesions with tumorigenic potential or the intraepithelial stage in the evolution of some testicular Sertoli cell tumors.

3-Hydroxysteroid Dehydrogenases↗

PubMed1999

Cell death patterns of the rat spermatogonial cell progeny induced by sertoli cell geometric changes and Fas (CD95) agonist.

Spermatogonial-Sertoli cell co-cultures, prepared from sexually immature rats (7-10 days old) and maintained for experimental purposes for a maximum period of time of eight days, were used to determine whether Sertoli cell geometry can influence spermatogonial cell growth, viability and differentiation. We have found that when Sertoli cells are allowed to stretch, spermatogonial cell cohorts attached to Sertoli cell surfaces remain viable and exhibit typical cell oscillatory movements with a maximal oscillation radial length of 0.8 microm throughout the duration of the experiments. However, spermatogonial cell viability decreased when Sertoli cells were compelled to contract by preventing cell spreading onto a non-adhesive substrate. A video-microscopy analysis of spermatogonial cells progenies cocultured with contracted Sertoli cells revealed that conjoined members of the cohorts displayed a typical apoptotic sequence preceded by vigorous oscillatory cell movements (maximal oscillation radial length: 1.5 microm) followed by the release of apoptotic bodies and cessation of cell movements. This sequence of events occurred in a single cell. Upon completion of this sequence, another member of the cohort initiated the same cell death course until all members completed the cell death sequence. A similar apoptotic sequence was observed following addition of Fas (CD95/APO-1) antibody (ligand agonist) to the cocultures. Fragmentation of the actin-containing cytoskeleton was observed by indirect immunofluorescence in apoptotic spermatogonial cell cohorts, independent from the activating mechanism. We conclude that by forcing Sertoli cells to contract or by adding an apoptosis inducer to the cocultures, individual members of a spermatogonial cell cohort switch on a death (apoptosis) program in a coordinated fashion.

Actins↗

PubMed1999

Basic helix-loop-helix proteins can act at the E-box within the serum response element of the c-fos promoter to influence hormone-induced promoter activation in Sertoli cells.

The Sertoli cell is a terminally differentiated testicular cell in the adult required to maintain the process of spermatogenesis. Previously basic helix-loop-helix (bHLH) factors and c-fos have been shown to influence Sertoli cell-differentiated functions. The induction of Sertoli cell differentiation appears to involve the serum response element (SRE) of the c-fos promoter to activate c-fos and intermediate bHLH factor(s) that regulate down-stream Sertoli cell-differentiated genes (e.g. transferrin expression). The SRE of the c-fos promoter is influenced through the serum response factor (SRF). Interestingly, an E-box nucleotide sequence is present within the SRE. bHLH proteins act through E-box elements, and the current study investigates the possibility that bHLH proteins may directly influence the SRE of the c-fos promoter. The activation of the c-fos promoter in Sertoli cells was found to be inhibited with the overexpression of the inhibitory HLH protein Id. Analysis of major response elements within the c-fos promoter demonstrated that the expression of Id specifically inhibited the activation of SRE in Sertoli cells and no other elements tested. Mutations in the E-box of the SRE also inhibited the activation of SRE, suggesting the direct role of bHLH proteins in regulating SRE activity in Sertoli cells. In contrast, the activation of SRE containing a mutated E-box was comparable to wild-type SRE in control stromal cells. Analysis of SRE oligonucleotide gel mobility shift assays with nuclear extracts from Sertoli cells demonstrated the presence of both the SRF and the ubiquitously expressed bHLH protein E12/E47. In contrast, no E12/E47 was detected in the SRE oligonucleotide gel shift using control stromal cell nuclear extracts. Observations suggest the binding of E12/E47 to SRE may be a cell-specific event. The SRF and bHLH proteins appear to bind to the SRE and activate the c-fos promoter in Sertoli cells. Observations provide evidence that a bHLH protein can interact with the SRE of the c-fos promoter to influence hormone-induced promoter activation. Cross-talk between these nuclear transcription factors appears to be instrumental in the control of Sertoli cell-differentiated functions.

Animals↗

PubMedDate not supplied

Establishment and characterization of neonatal mouse sertoli cell lines.

Sertoli cells isolated from 6-day postpartum mouse testes were conditionally immortalized with the simian virus 40 large tumor antigen gene (SV40-LTAg) under the control of a promoter inducible with ponasterone A, an analog of ecdysone. This strategy produced 2 cell lines, which exhibited mixed phenotypes. We first tested the conditional expression of the LTAg gene in the presence or absence of ponasterone A. The results showed that both cell lines expressed LTAg when the inducer was present in the culture media. When ponasterone A was removed, the majority of the cells died. After 60 generations, however, the continued expression of LTAg in the absence of the hormone indicated that unknown changes may have occurred in the genome of the cells. One of the cell lines was further subcloned, resulting in 7 new lines exhibiting a morphology resembling that of Sertoli cells in tissue culture. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on RNA collected from each cell line in order to determine which cells were phenotypically similar to Sertoli cells in vivo. All cell lines expressed the products of the Sertoli cell-specific genes stem cell factor (SCF) and sulfated glycoprotein-2 (SGP-2), in addition to alpha-inhibin, GATA-1, and steroidogenic factor-1. Further, the lines express growth and differentiation factors known to act upon germ cells in vivo and in vitro such as leukemia inhibitory factor (LIF), transforming growth factor beta (TGF-beta), and basic fibroblast growth factor (bFGF). Moreover, when used as feeder layers in cocultures, at least 2 of these lines are able to maintain the viability of type A spermatogonia for at least 7 days and to support the first steps of spermatogonial differentiation.

Alkaline Phosphatase↗

PubMed2003

Isolation of sertoli cells from adult rat testes: an approach to ex vivo studies of Sertoli cell function.

Much of what is known about the molecular regulation and function of adult Sertoli cells has been inferred from in vitro studies of immature Sertoli cells. However, adult and immature cells differ in significant ways and, moreover, many Sertoli cell functions are regulated by conditions that are difficult to replicate in vitro. Our objective was to develop a procedure to isolate Sertoli cells rapidly and in sufficient number and purity to make it possible to assess Sertoli cell function immediately after the isolation of the cells. The isolation procedure described herein takes less than 4 h and does not require culturing the cells. From a single 4-mo-old adult rat, we routinely obtain 7.0 +/- 0.4 x 10(6) Sertoli cells per testis, and from a 21-mo-old rat, 7.2 +/- 0.4 x 10(6) Sertoli cells per testis. The purity, determined by morphologic analyses of plastic-embedded cells or after staining for tyrosine-tubulin or vimentin, averaged 80%. The contaminants typically included germ cells (10%) and myoid cells (10%). The germ cell-expressed genes protamine-2 and hemiferrin were not detected in the Sertoli cell preparations by Northern blot analyses, but the Sertoli cell-expressed genes clusterin, cathepsin L, and transferrin were highly expressed. Transferrin mRNA levels were greater in Sertoli cells isolated from aged than from young adult rats, consistent with previous analyses of whole testes; and cathepsin L mRNA levels were far more highly expressed in Sertoli cells isolated from stages VI-VII than from other stages of the cycle of the seminiferous epithelium, also consistent with previous analyses of whole testes and isolated tubules. These studies indicate that the freshly isolated cells retain differentiated function, and thus it should be possible to assess the in vivo function of adult Sertoli cells by isolating the Sertoli cells and immediately assessing their function.

Animals↗

PubMedDate not supplied

Enzymatic digestion of bradykinin by rat Sertoli cell cultures.

Sertoli cells play a key role in spermatogenesis. To study the involvement of the kallikrein-kinin system in the testis, the pattern of bradykinin-inactivating kininases in rat Sertoli cells was investigated. Exogenous bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) is cleaved at Pro7-Phe8, Phe5-Ser6, and Gly4-Phe5, as demonstrated by high performance liquid chromatography analysis. Degradation of bradykinin was strongly inhibited by phosphoramidon and thiorphan, which are specific inhibitors of neutral metalloendopeptidase-24.11. The kininase type II-specific inhibitors, captopril and enalapril, were only partially effective in preventing peptidolysis. This indicates that the main kininases responsible for rapid bradykinin inactivation are neutral metalloendopeptidase and, to a lesser extent, kininase type II. Neutral metalloendopeptidase and kininase type II were shown to be located on Sertoli cell membranes. A low degree of bradykinin degradation was detected by simultaneous inhibition of neutral metalloendopeptidase-24.11 and kininase II, pointing out the involvement of further peptidases with minor activities. This remaining activity is probably not due to the action of kininase type I or cysteine proteases, as shown by specific inhibitors. The data presented indicate the occurrence of membrane-bound kininases, which are an important part of the kallikrein-kinin system, in rat Sertoli cell cultures.

Amino Acid Sequence↗

PubMed1993

Activin is produced by rat Sertoli cells in vitro and can act as an autocrine regulator of Sertoli cell function.

The production of activin by Sertoli cells isolated from 21-day-old rats was studied using the mesoderm-inducing activity of activin on Xenopus laevis animal cap explants, immunoprecipitation and Western blotting. Furthermore, the effects of recombinant bovine activin-A on rat Sertoli cell aromatase activity and FSH and androgen receptor gene expression were examined. Animal cap explants from Xenopus laevis blastulas elongated after culture in conditioned medium of Sertoli cells cultured with or without ovine FSH or conditioned medium of the mouse Sertoli cell-derived TM4 cell line. Animal cap explants cultured in control medium remained spherical. This elongation was also found in the more than 10-kilodalton fraction of the conditioned medium and after heating for 10 min at 95 C, indicating that heat-stable activin-like bioactivity is present in the culture medium. Immunoprecipitation of [35S]methionine-labeled proteins and Western blotting of Sertoli cell-conditioned medium with polyclonal antisera against the inhibition beta-subunits indicated the presence of 24- to 25-kilodalton activin-like immunoreactive material. Sertoli cell aromatase activity was dose-dependently stimulated by ovine FSH after 72 h of culture. Recombinant bovine activin-A partly inhibited this stimulation in a dose-dependent way. This inhibition was also found after 24 h of culture. Furthermore, basal and FSH-stimulated androgen receptor mRNA expression in Sertoli cells and binding of the synthetic androgen R1881 to Sertoli cells were decreased after 24 h of culture in the presence of recombinant bovine activin-A. In the same experiments, FSH receptor mRNA expression was not significantly affected. These results indicate that activin can act as an autocrine regulator of Sertoli cell function.

Activins↗