Primary Cultures Of Sertoli Cells Research Articles

Hormonal Regulation of Rat Testicular γ-Glutamyl-Transpeptidase “In Vivo” and “In Vitro”/Die hormonelle Regulation von testikulärer γ-Glutamyl-Transpeptidase bei Ratten “in vivo” und “in vitro”

The hormonal regulation of gamma-glutamyl transpeptidase (gamma-GTP), an enzyme marker of Sertoli cells, was studied in immature rats that received 50 micrograms/day of testosterone propionate (TP) during 6 days to suppress pituitary LH and FSH. Suppression of LH was monitored indirectly by the determination of intratesticular levels of testosterone and suppression of FSH by radioimmunoassay of serum FSH. Enzyme activity in the testis decreased in parallel to intratesticular testosterone suppression, and it did recover up to control values when animals received 500 micrograms/day of TP, a dose that was able to maintain intratesticular testosterone at normal levels. beta-glucuronidase, another enzyme marker of Sertoli cells, was not affected by these treatments. A significant decrease in gamma-GTP was detected 24h after significant suppression of intratesticular testosterone and it returned to control levels 2 days after increasing the dose of TP to 500 micrograms/day. Administration of FSH to rats with depletion of intratesticular testosterone was able to maintain testicular gamma-GTP at control levels. An stimulatory action of FSH could also be demonstrated in primary Sertoli cell cultures. It is concluded that testicular gamma-GTP is under the regulation of both androgens and FSH while beta-glucuronidase is not. Eventhough the function of gamma-GTP in the testis is not known, the key role that it plays in other tissues suggests that it might be important in the regulation of Sertoli cell-germ cell interactions.

Intracellular pH regulation in human Sertoli cells: role of membrane transporters

Sertoli cells are responsible for regulating a wide range of processes that lead to the differentiation of male germ cells into spermatozoa. Intracellular pH (pHi) is an important parameter in cell physiology regulating namely cell metabolism and differentiation. However, pHi regulation mechanisms in Sertoli cells have not yet been systematically elucidated. In this work, pHi was determined in primary cultures of human Sertoli cells. Sertoli cells were exposed to weak acids, which caused a rapid acidification of the intracellular milieu. pHi then recovered by a mechanism that was shown to be particularly sensitive to the presence of the inhibitor DIDS (4,4'-diisothiocyanostilbene disulfonic acid). In the presence of amiloride and PSA (picrylsulfonic acid), pHi recovery was also significantly affected. These results indicate that, in the experimental conditions used, pHi is regulated by the action of an Na(+)-driven HCO(3)(-)/Cl(-) exchanger and an Na(+)/HCO(3)(-) co-transporter and also by the action of the Na(+)/H(+) exchanger. On the other hand, pHi recovery was only slightly affected by concanamycin A, suggesting that V-Type ATPases do not have a relevant action on pHi regulation in human Sertoli cells, and was independent of the presence of bumetanide, suggesting that the inhibition of the Na(+)/K(+)/Cl(-) co-transporter does not affect pHi recovery, not even indirectly via the shift of ionic gradients. Finally, pHi was shown to be sensitive to the removal of external Cl(-), but not of Na(+) or K(+), evidencing the presence of a membrane Cl(-)-dependent base extruder, namely the Na(+)-independent HCO(3)(-)/Cl(-) exchanger, and its role on pHi maintenance on these cells.

Par3/Par6 polarity complex coordinates apical ectoplasmic specialization and blood–testis barrier restructuring during spermatogenesis

The Par3/Par6/aPKC and the CRB3/Pals1/PATJ polarity complexes are involved in regulating apical ectoplasmic specialization (ES) and blood-testis barrier (BTB) restructuring in the testis. Par6 was a component of the apical ES and the BTB. However, its level was considerably diminished at both sites at stage VIII of the cycle. Par6 also formed a stable complex with Pals1 and JAM-C (a component of the apical ES) in normal testes. When rats were treated with adjudin to induce apical ES restructuring without compromising the BTB, Par6 staining virtually disappeared at the apical ES in misaligned spermatids before their depletion. Additionally, the Par6/Pals1 complex became tightly associated with Src kinase, rendering a loss of association of the Par6/Pals1 complex with JAM-C, thereby destabilizing apical ES to facilitate spermatid loss. Primary Sertoli cell cultures with established functional BTB, but without apical ES, were next used to assess the Par6-based complex on BTB dynamics. When either Par6 or Par3 was knocked down by RNAi in Sertoli cell epithelium, a significant loss of the corresponding protein by approximately 60% in cells vs. controls was detected, alongside with a decline in aPKC after Par6, but not Par3, knockdown. This Par3 or Par6 knockdown also led to a transient loss of selected BTB proteins at the cell-cell interface, thereby compromising the BTB integrity. These findings illustrate that the Par6/Par3-based polarity complex likely coordinates the events of apical ES and BTB restructuring that take place concurrently at the opposing ends of adjacent Sertoli cells in the seminiferous epithelium during spermatogenesis.

Adrenomedullin in the Rat Testis. II: Its Production, Actions on Inhibin Secretion, Regulation by Follicle-Stimulating Hormone, and Its Interaction with Endothelin 1 in the Sertoli Cell1

The present study demonstrates the expression of adrenomedullin (ADM) in the rat Sertoli cells and its effect on inhibin production. The regulation of ADM by FSH and its interaction with endothelin 1 (EDN1) in the rat Sertoli cells have also been established. Primary culture of Sertoli cells secreted 414+/-27 pg immunoreactive ADM per 10(6) cells in 24 h and expressed Adm mRNA. In addition, the Sertoli cell was shown to co-express mRNAs encoding for the calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying proteins (RAMPs) 1-3. These may account for the specific binding of ADM to the Sertoli cells. Administration of ADM to Sertoli cells resulted in an enhancement of basal and FSH-stimulated inhibin B production. On the other hand, the production of ADM and the mRNA levels of Calcrl and Ramp2 in the Sertoli cells were suppressed by FSH. The results suggest that ADM, via its control in the secretion of inhibin B, may play a role in regulating spermatogenesis as well as the hypothalamus-pituitary-gonad feedback system. In addition, like in the Leydig cell, ADM and EDN1 were found to regulate the production of each other in opposite directions in the Sertoli cells, suggesting the presence of yet another local regulatory mechanism in the rat testis that may be important in modulating testicular functions regulated by gonadotropins.

Effects of dexamethasone and dibutyryl cAMP on stanniocalcin-1 mRNA expression in rat primary Sertoli and Leydig cells

In this study, STC1 expression levels were determined at various postnatal developmental stages in rat ovaries and testes. The expression pattern of STC1 was found to be sexually dimorphic. In addition we examined the effect of exogenous glucocorticoids, dexamethasone (DEX), testosterone, follicle stimulating hormone (FSH) and dibutyryl cAMP (dbcAMP) on STC1 expression in rat primary Sertoli cell cultures. Our results showed that DEX (via glucocorticoids receptor) stimulated while dbcAMP inhibited STC1 expression in the Sertoli cells. Moreover testosterone and FSH treatment had no significant effect on STC1 expression in the cells. The inhibitory effect of dbcAMP to STC1 expression was consistently demonstrated in rat primary Leydig cell cultures. In human chorinoic gonadotropin (hCG) or dbcAMP treated Leydig cells, a significant reduction of STC1 expression was accompanied with an induction of testosterone secretion. The actions of hCG/dbcAMP were mediated by PKA, as the effect can be rescued by a co-treatment with H89. Using the Sertoli cell model, the modulation of STC1 expression by DEX or dbcAMP was found to be regulated at the transcriptional level as actinomycin D treatment significantly reduced the respective effect. In addition, both DEX and dbcAMP mediated effects were found to be abolished by cycloheximide co-treatment. These indicated that the regulation in the both treatments were indirect and may require prior protein synthesis. We suggested that the de novo synthesis of other protein(s) and mRNA may be involved in the regulation of the steady-state levels of STC1 mRNA in the treatments. Taken together, this study provides the first evidence of the regulation of STC1 expression in the male reproductive system.

FSH-Sensitive Murine Sertoli Cell Lines Immortalized by Human Telomerase Gene hTERT Express the Androgen Receptor in Response to TNF-α Stimulation

Sertoli cells are regulated by follicular stimulating hormone (FSH) and testosterone secreted by the pituitary gland and Leydig cells, respectively. However, the expression of the FSH receptor and androgen receptor were undetectable in both primary cultured Sertoli cells and Sertoli cell lines immortalized by SV40 large T antigen. Two Sertoli cell lines, B6Sc-2 and B6Sc-3, were established from the testis of 19-day-old C57BL/6 mice testis by immortalization with human telomere reverse transcriptase. These Sertoli cell lines expressed FSH receptors and the total phosphoprotein patterns were converted after FSH treatment. Additionally, immunological methods demonstrated that these cell lines expressed characteristic Sertoli cell proteins, such as tyrosine-tubulin, vimentin and stem cell factor (SCF). Reverse transcription-polymerase chain reaction (RT-PCR) also indicates that they express Sertoli specific mRNAs, such as Amh, claudin11 and ZO-1. The expression of the androgen receptor in both B6Sc-2 and B6Sc-3 cells could be induced by TNF-alpha treatment. The present results indicate that these Sertoli cell lines are more native than others and may thus provide useful tools for in vitro studies.

Isolation, culture, and characterization of canine Sertoli cells

Primary Sertoli cell cultures have been established from several animals including the sheep and rhesus monkey; however, not for the domestic dog, Canis familiaris. Sertoli cells are the only readily accessible cell type in the body which expresses all six type IV collagens. These collagens play key roles in tissue structure, basement membrane formation, and filtration. The study of these genes is necessary to determine their exact roles and regulation in the aforementioned functions and to investigate diseases associated with mutations in these genes. For such studies, a cell culture system is a requisite tool. Therefore, Sertoli cells were targeted, and a culture was established from cells isolated from canine testes. Cultures maintained consistent morphology and steady growth for up to seven passages. Cultured cells were identified as Sertoli cells through positive Western blot results for SOX9 and Clusterin B proteins and transcript sequence verification of SOX9 as well as the presence of type IV collagen transcripts. Primary cultures of canine Sertoli cells will provide a useful tool for study of the function and regulation of collagen genes and will permit new research pertaining to canine health while also serving as a model for the study of human diseases.

Coxsackievirus and Adenovirus Receptor Is Up-Regulated in Migratory Germ Cells during Passage of the Blood-Testis Barrier

The coxsackievirus and adenovirus receptor (CAR) is a cell adhesion molecule expressed in epithelial tight junctions and other cell-cell contacts. Using indirect immunofluorescence, quantitative RT-PCR, and Western blots, the expression and distribution of CAR in developing and adult testis are examined. CAR is highly expressed in both Sertoli and germ cells during perinatal and postnatal development, followed by a rapid down-regulation of both mRNA and protein levels. Interestingly, we find that CAR is a previously unknown downstream target for FSH because CAR mRNA levels were induced in primary cultures of FSH-stimulated Sertoli cells. In contrast to other epithelia, CAR is not a general component of tight junctions in the seminiferous epithelium, and Sertoli cells in the adult testis do not express CAR. Instead, CAR expression is stage dependent and specifically found in migratory germ cells. RT-PCR also demonstrated the presence of junctional adhesion molecule-like (JAML) in the testis. JAML was previously reported by others to form a functional complex with CAR regulating transepithelial migration of leukocytes. The expression of JAML in the testis suggests that a similar functional complex might be present during germ cell migration across the blood-testis barrier. Finally, an intermediate compartment occupied by CAR-positive, migrating germ cells and flanked by two occludin-containing junctions is identified. Together, these results implicate a function for CAR in testis morphogenesis and in migration of germ cells across the blood-testis barrier during spermatogenesis.

Relaxin family peptide receptors Rxfp1 and Rxfp2: mapping of the mRNA and protein distribution in the reproductive tract of the male rat

BackgroundRelaxin is the endogenous ligand of the G-protein coupled receptor RXFP1, previously known as LGR7. In humans relaxin can also activate, but with lower affinity, the closely related receptor for the insulin-like peptide from Leydig cells, RXFP2, previously known as LGR8. The lack of relaxin impairs male fertility but the precise distribution and the function of relaxin receptors in the male reproductive tract is not known. We investigated the distribution of Rxfp1 and Rxfp2 in the reproductive tract of the male rat and the function of relaxin in the vas deferens, a tissue with high expression of both receptors.MethodsThe presence of mRNA for Rxfp1 and Rxfp2 was investigated in testes, cultured Sertoli cells, epididymis, vas deferens, seminal vesicle, prostate, and spermatozoa by RT-PCR and Southern blot. Protein expression in the testis, vas deferens, primary culture of Sertoli cells, and spermatozoa was assessed by immunohistochemistry and immunofluorescence. The role of relaxin in the vas deferens was evaluated by contractility studies and radioimmunoassay of cAMP production. The effect of relaxin on mRNA levels for metalloproteinase-7 was measured by Northern blot.ResultsTranscripts for Rxfp1 and Rxfp2 were present in almost all parts of the male reproductive tract, with high levels in testis and vas deferens. Both receptors were immunolocalized in late stage germ cells but not in mature spermatozoa, although mRNAs for both receptors were also present in mature spermatozoa. Rxfp1 but not Rxfp2 was detected in cultured Sertoli cells. Strong immunostaining for Rxfp1 and Rxfp2 was seen in muscular and epithelial layers of the vas deferens and in arteriolar walls. Relaxin did not affect contractility and cyclic AMP production of the vas deferens, but increased the levels of mRNA for metalloproteinase-7.ConclusionRxfp1 and Rxfp2 are widely and similarly distributed throughout the male reproductive tract. Our results suggest that Rxfp1 on spermatids and Sertoli cells may be important in spermatogenesis. Relaxin in the vas deferens does not affect contractility, but may affect vascular compliance and collagen and matrix remodeling.

Common and distinct factors regulate expression of mRNA for ETV5 and GDNF, Sertoli cell proteins essential for spermatogonial stem cell maintenance

Ets variant gene 5 (ETV5) and glial cell-derived neurotrophic factor (GDNF) are produced in Sertoli cells and required for maintenance and self-renewal of spermatogonial stem cells (SSCs) in mice. Fibroblast growth factors (FGFs) have been reported to stimulate Etv5 mRNA expression, and FSH was shown to stimulate Gdnf mRNA in Sertoli cell cultures, but there is no other information on factors that regulate these key Sertoli cell proteins necessary for stem cell maintenance. In this study, we investigated regulation of ETV5 and GDNF using the TM4 murine Sertoli cell line. FGF2 stimulated a time- and dose-dependent increase in Etv5 mRNA expression, with a maximal 8.3-fold increase at 6 h following 25 ng/ml FGF2 treatment. This FGF2 dose also stimulated Gdnf mRNA at 48 h. FGF2 effects on Etv5 and Gdnf mRNA were partially mediated through mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K)-signaling cascades. Specific inhibitors of MAPK (PD98059) and PI3K (wortmannin) pathways reduced Etv5 and Gdnf mRNA expression in FGF2-treated cells. Epidermal growth factor (EGF) stimulated Etv5 mRNA but not Gdnf mRNA. TNFα and IL-1β stimulated Gdnf mRNA, but had no effect on Etv5 mRNA. Other hormonal regulators of Sertoli cells such as testosterone, triiodothyronine and activin A did not affect Etv5 or Gdnf mRNA expression. Results with primary Sertoli cell cultures confirmed findings obtained with the TM4 cell line, validating the use of the TM4 model to examine regulation of Etv5 and Gdnf mRNA expression. In conclusion, we have identified common and unique pathways that regulate Etv5 and Gdnf mRNA in Sertoli cells, and FGFs are emerging as key regulators of the Sertoli cell proteins that control SSCs.

Phosphoprotein enriched in astrocytes-15 is expressed in mouse testis and protects spermatocytes from apoptosis

Phosphoprotein enriched in astrocytes (PEA-15) is a 15 kDa acidic serine-phosphorylated protein expressed in different cell types, especially in the CN. We initially detected the expression of PEA-15 in primary cultures of Sertoli cells. To assess the presence and localization of PEA-15 in the mouse testis, we studied the expression pattern of the PEA-15 protein by immunohistochemistry and mRNA by in situ hybridization. Both the protein and the mRNA of PEA-15 were localized in the cytoplasm of Sertoli cells, all types of spermatogonia, and spermatocytes up till zygotene phase of the meiotic prophase. Subsequently, with ongoing development of the spermatocytes, the expression decreased and was very low in the cytoplasm of diplotene spermatocytes. To analyze the possible role of PEA-15 in the developing testis, null mutants for PEA-15 were examined. As the PEA-15 C terminus contains residues for ERK binding, we studied possible differences between the localization of the ERK2 protein in wild type (WT) and PEA-15(-/-)mice. In the WT testis, ERK2 was localized in the cytoplasm of Sertoli cells, B spermatogonia, preleptotene, leptotene, and zygotene spermatocytes, whereas in the KO testis, ERK2 was primarily localized in the nuclei of these cells and only little staining remained in the cytoplasm. Moreover, in PEA-15-deficient mice, significantly increased numbers of apoptotic spermatocytes were found, indicating an anti-apoptotic role of PEA-15 during the meiotic prophase. The increased numbers of apoptotic spermatocytes were not found at a specific step in the meiotic prophase.

Coxsackie and adenovirus receptor (CAR) is a product of Sertoli and germ cells in rat testes which is localized at the Sertoli–Sertoli and Sertoli–germ cell interface

The coxsackie and adenovirus receptor (CAR), a putative cell–cell adhesion molecule, has attracted wide interest due to its importance in viral pathogenesis and in mediating adenoviral gene delivery. However, the distribution pattern and physiological function of CAR in the testis is still not clear. Here, we identified CAR in Sertoli cells and germ cells of rats. In vivo studies have shown that CAR resides at the blood–testis barrier as well as at the ectoplasmic specialization. The persistent expression of CAR in rat testes from neonatal period throughout adulthood implicates its role in spermatogenesis. Using primary Sertoli cell cultures, we observed a significant induction of CAR during the formation of Sertoli cell epithelium. Furthermore, CAR was seen to be concentrated at inter-Sertoli cell junctions, co-localizing with tight junction protein marker ZO-1 and adherens junction protein N-cadherin. CAR was also found to be associated with proteins of Src kinase family and its protein level declined after TNFα treatment in Sertoli cell cultures. Immunofluorescent staining of isolated germ cells has revealed the presence of CAR on spermatogonia, spermatocytes, round spermatids and elongate spermatids. Taken together, we propose that CAR functions as an adhesion molecule in maintaining the inter-Sertoli cell junctions at the basal compartment of the seminiferous epithelium. In addition, CAR may confer adhesion between Sertoli and germ cells at the Sertoli–germ cell interface. It is possible that the receptor utilized by viral pathogens to breakthrough the epithelial barrier was also employed by developing germ cells to migrate through the inter-Sertoli cell junctions.

The Cathepsin L First Intron Stimulates Gene Expression in Rat Sertoli Cells1

Large amounts of cathepsin L (CTSL), a cysteine protease required for quantitatively normal spermatogenesis, are synthesized by mouse and rat Sertoli cells during stages VI to VII of the cycle of the seminiferous epithelium. We previously demonstrated that all of the regulatory elements required in vivo for both Sertoli cell- and stage-specific expression of the Ctsl gene are present within a ~3-kb genomic fragment that contains 2065 nucleotides upstream of the transcription start site and 977 nucleotides of downstream sequence. Most of the downstream region encodes the first intron. In this study, transient transfection assays using primary Sertoli cell cultures and the TM4 Sertoli cell line established that the Ctsl first intron increased reporter gene activity by ~5-fold. While the intron-mediated enhancement in reporter gene activity was not restricted to the Ctsl promoter, positioning the first intron upstream of the Ctsl promoter in either orientation abolished its stimulatory activity, suggesting that it does not contain a typical enhancer. Mutating the 5'-splice site of the Ctsl first intron or replacing the first intron by the Ctsl fourth intron abolished the stimulatory effect. Finally, the intron-dependent increase in reporter gene activity could be explained in part by an increase in the amounts of total RNA and transcript polyadenylation. Results from this study suggest that the stimulatory effect mediated by the Ctsl first intron may explain in part why Sertoli cells in seminiferous tubules at stages VI to VII produce high levels of CTSL.

Expression of stress inducible protein 1 (Stip1) in the mouse testis

Phthalate esters are considered endocrine disruptors that interfere with the endocrine balance and development of the mammalian testis. Mono-2-ethylhexyl phthalate (MEHP), the active metabolite of the ubiquitously used plasticizer di-2-ethylhexyl phthalate (DEHP), acts upon Sertoli cells as initial target. By subtractive cDNA libraries we identified genes deregulated as response to MEHP in primary cultures of mouse Sertoli cells. The expression of mouse stress inducible protein 1 (Stip1) was detected as upregulated as a result of MEHP exposure. Stip1 is a cochaperone protein that is homologous to the human heat shock cognate protein 70 (hsc70)/heat shock protein 90 (hsp90)-organizing protein (Hop). To assess the presence and localization of Stip1 in mouse testis and its potential role in stress defense, we studied the expression pattern of the Stip1 protein by immunohistochemistry and of the mRNA by in situ hybridization. Both the protein and the mRNA of Stip1 were mainly found in the cytoplasm of all types of spermatogonia and spermatocytes up till zygotene, the expression decreased during late pachytene and was very weak in diplotene spermatocytes and round spermatids. Interestingly, this expression pattern resembled the pattern of stress sensitivity of spermatogenic cells in that the most sensitive cell types show the weakest expression of Stip1. This suggests an important role for Stip1 in the ability of germ cells to survive in stress conditions including high temperatures.

The Effect of Species-Specific FSH Administration During In vitro Maturation of Bovine Oocytes on Embryonic Developmental Capability

Follicle-stimulating hormone (FSH) is a heterodimeric glycoprotein synthesized and secreted by the anterior pituitary gland under hypothalamic control. This hormone is involved in the regulation of essential mammalian reproductive processes such as gametogenesis, follicular growth, and ovulation. An important aspect of the structure of FSH is its double species-specificity, at both the amino acid and sugar level. In fact, both the amino acid sequence and the oligosaccharide structure significantly differ among species (Green and Baenziger, 1988; Wallis, 2001). For example, bovine (bFSH) and human FSH (hFSH) differ by 45 amino acids (sequence identity: α-subunit 75%; β-subunit 90%), and in addition they show strikingly different profiles with respect to their oligosaccharide side-chains (the relative distribution of sialylated, sulfated and neutral Asn-linked oligosaccharides in human FSH is 88%, 7% and 5%, while in the bovine it is 56%, 13% and 31%). Both protein and sugar structures affect the biological activity of FSH (e.g. hormonereceptor interactions)(Ulloa-Aguirre et al., 2003), however, the molecular basis of FSH functions in domestic animals are usually studied by heterologous experimental models. Examples are the in vitro bioassays based on rat and mouse primary cultures of Sertoli and granulosa cells, and the bioassays based on recombinant cell lines expressing the cloned FSH rat and human receptors (Christin-Maitre and Bouchard, 1996). Another non speciesspecific application involves the use of supraphysiological doses of recombinant hFSH or pituitary pig FSH during in vitro bovine embryo production (IVP) protocols (Ali and Sirard, 2002). The objective of the present study was to develop a species-specific in vitro bioassay to study the molecular mechanisms underlying the biological activity of bovine FSH (bFSH). We used cumulus-oocyte complexes (COCs) isolated from bovine ovaries in order to evaluate the ability of pituitary bFSH to stimulate cumulus expansion, and blastocyst development

Differential Interactions between Transforming Growth Factor-β3/TβR1, TAB1, and CD2AP Disrupt Blood-Testis Barrier and Sertoli-Germ Cell Adhesion

The biochemical basis that regulates the timely and selective opening of the blood-testis barrier (BTB) to migrating preleptotene/leptotene spermatocytes at stage VIII of the epithelial cycle in adult rat testes is virtually unknown. Recent studies have shown that cytokines (e.g. transforming growth factor (TGF)-beta3) may play a crucial role in this event. However, much of this information relies on the use of toxicants (e.g. CdCl(2)), making it difficult to relay these findings to normal testicular physiology. Here we report that overexpression of TGF-beta3 in primary Sertoli cells cultured in vitro indeed perturbed the tight junction (TJ) barrier with a concomitant decline in the production of BTB constituent proteins as follows: occludin, N-cadherin, and ZO-1. Additionally, local administration of TGF-beta3 to testes in vivo was shown to reversibly perturb the BTB integrity and Sertoli-germ cell adhesion via the p38 MAPK and ERK signaling pathways. Most importantly, the simultaneous activation of p38 and ERK signaling pathways is dependent on the association of the TGF-beta3-TbetaR1 complex with adaptors TAB1 and CD2AP because if TbetaR1 was associated preferentially with CD2AP, only Sertoli-germ cell adhesion was perturbed without compromising the BTB. Collectively, these data illustrate that local production of TGF-beta3, and perhaps other TGF-betas and cytokines, by Sertoli and germ cells into the microenvironment at the BTB during spermatogenesis transiently perturbs the BTB and Sertoli-germ cell adhesion to facilitate germ cell migration when the activated TbetaRI interacts with adaptors TAB1 and CD2AP. However, TGF-beta3 selectively disrupts Sertoli-germ cell adhesion in the seminiferous epithelium to facilitate germ cell migration without compromising BTB when TbetaRI interacts only with adaptor CD2AP.

Transfection of Sertoli cells ex vivo; Opening the door to the in vitro study of junction turnover in the seminiferous epithelium

Sertoli cells interact with adjacent Sertoli cells and with spermatids through elaborate intercellular adhesion junctions termed ectoplasmic specializations (ESs). Among adhesion molecules present at the sites is Nectin-2. Tubulobulbar complexes have been proposed to play a role in junction internalization. Here we establish a culture model for exploring the role of tubulobulbar complexes during junction turnover. Cell clumps enriched for Sertoli cells were isolated from ten day-old rat pups and cultured on Matrigel coated Transwells. These cells established a columnar morphology and formed identifiable basal ESs. Significantly, structures resembling tubulobulbar complexes developed at the junction sites. We generated eGFP-tagged nectin-2 constructs and transfected them first into Hela cells (cells that do not normally form adherens junctions) and then into our primary cultures of morphologically differentiated Sertoli cells. In Hela cells, eGFP-Nectin-2 localized to sites of intercellular contact between adjacent cells indicating that the protein was expressed and targeted to the plasma membrane where it appeared to establish adherens junctions. In Sertoli cells, the protein was localized to areas consistent with the position of ESs and to tubular structures associated with them. Primary Sertoli cell cultures may be a model for studying the role of tubulobulbar complexes in junction turnover. CIHR MOP 62728

Follicle-Stimulating Hormone-Induced Aromatase in Immature Rat Sertoli Cells Requires an Active Phosphatidylinositol 3-Kinase Pathway and Is Inhibited via the Mitogen-Activated Protein Kinase Signaling Pathway

Postnatal development and function of testicular Sertoli cells are regulated primarily by FSH. During this early period of development, estrogens play a role in proliferation of somatic cells, which contributes significantly to testicular development. Growth factors like epidermal growth factor (EGF) are produced in the testis and play a role in regulation of estradiol production and male fertility. Although these divergent factors modulate gonadal function, little is known about their mechanism of action in Sertoli cells. The present study investigates the intracellular events that take place down-stream of FSH and EGF receptors in Sertoli cells isolated from immature (10-d-old) rats, and examines which intracellular signals may be involved in their effects on aromatase activity and estradiol production in immature rat Sertoli cells. Primary cultures of rat Sertoli cells were treated with FSH in combination with EGF and signaling pathway-specific inhibitors. Levels of estradiol production, aromatase mRNA (Cyp19a1), and aromatase protein (CYP19A1) were determined. Western blot analysis was performed to determine the effects of FSH and EGF on levels of activated (phosphorylated) AKT1 and p42 ERK2 and p44 ERK1, also named MAPK1 and MAPK3, respectively. The stimulatory actions of FSH on aromatase mRNA, aromatase protein, and estradiol production were blocked by inhibition of the phosphatidylinositol 3-kinase/AKT1 signaling pathway. In contrast, inhibition of ERK signaling augmented the stimulatory effects of FSH on estradiol production, aromatase mRNA, and protein levels. Furthermore, EGF inhibited the expression of aromatase mRNA and protein in response to FSH, and these inhibitory effects of EGF were critically dependent on the activation of the ERK signaling pathway. We conclude that an active phosphatidylinositol 3-kinase /AKT signaling pathway is required for the stimulatory actions of FSH, whereas an active ERK/MAPK pathway inhibits estradiol production and aromatase expression in immature Sertoli cells.

Transcriptional Regulation of the Estrogen Receptor α Gene by Testosterone in Cultures of Primary Rat Sertoli Cells

Background: We wanted to identify the presence of the estrogen receptor (ER) α in Sertoli cells and gain insight on the regulation of the ERα gene expression by testosterone in Sertoli cells. The transcriptional regulation of the ERα gene was investigated in primary Sertoli cell cultures by in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR). Methods: Primary Sertoli cell culture was performed. The expression levels of ERα and ERβ mRNA in Sertoli cells were detected by Northern blot, RT-PCR, immunocytochemistry and in situ hybridization. Results: The ovary, testis and epididymis showed a moderate to high expression of ERα while the prostate, ovary and LNCap cells showed the ERβ expression. ERα mRNA and protein were detected in the germ cells and Sertoli cells by in situ hybridization and immunocytochemistry. The level of ERα mRNA was gradually decreased in a time-dependent manner after testosterone treatment, and the changes of ERα mRNA were dependent on the concentration of testosterone. Androgen binding protein and testosterone-repressive prostate message-2 (TRPM-2) mRNA were reduced at 24 hour by estradiol, while the transferrin mRNA was not affected. ERα mRNA was strongly detectable in the testes of 7 days-old-rats, but it was gradually decreased from 14 to 21 days of age. The primary Sertoli cells also showed the same pattern. The ERα gene expression was also regulated by testosterone in the Sertoli cells prepared from the 14- and 21-day old rats. Conclusions: These results suggest that ER α is transcriptionally regulated by testosterone and it may play some role in the Sertoli cells. (J Kor Soc Endocrinol 21:106~115, 2006)

Dedifferentiation of Adult Monkey Sertoli Cells through Activation of Extracellularly Regulated Kinase 1/2 Induced by Heat Treatment

Sertoli cells play a key role in triggering and regulating the process of spermatogenesis. Failure of a Sertoli cell to mature functionally will presumably render it incapable of supporting germ cell survival and development that appeared after puberty. Expression of cytokeratin 18 (ck-18) intermediate filaments indicates a state of undifferentiation usually observed in Sertoli cells of prepubertal testis. In this study we demonstrated that local testicular heat treatment of adult monkey with water at 43 C for 30 min once daily for 2 consecutive days was capable of activating reexpression of ck-18 in Sertoli cells, which was coincident with activation of ERK1/2 and Akt kinases. Using primary Sertoli cell culture isolated from adult monkey testis, we further confirmed that the heat treatment of the cells at 43 C could also induce ck-18 reexpression, which was similar to the in vivo treatment. ERK MAPK was also induced by the heat treatment in a time- and protein kinase A (PKA)-dependent manner. After blocking the ERK MAPK signaling pathway, an inhibition of ck-18 expression in the cultured Sertoli cells was observed, and this inhibitory effect was also detected by blocking the PKA activation. However, ck-18 activation in Sertoli cells remained unaltered when the phosphatidylinositol 3-kinase/Akt pathway was blocked. In conclusion, the heat treatment of adult monkey Sertoli cells are capable of inducing a reversible change in the Sertoli cells from an adult differentiated state to an immature-like dedifferentiated state through PKA-ERK MAPK-dependent pathways but not via the phosphatidylinositol 3-kinase/Akt pathway.