Genes In Sertoli Cells Research Articles

Regulation of Sertoli Cell Steroidogenic Acute Regulatory (StAR)-Related Lipid Transfer (START-Domain-Containing) StARD1 and StARD4 Expression by Follicle Stimulating Hormone (FSH) Requires Multiple Protein Kinases.

Multiple mechanisms mediate gonadotropin (follicle stimulating hormone: FSH) regulation of Sertoli cell (SC) gene expression, although the temporal coordination of signalling components other than cAMP remain largely unknown. Cytokines within the seminiferous tubule participate in such physiological regulation, subsequently influencing germ cell development. Recently, we demonstrated differential regulation of Sertoli START-domain-containing proteins steroidogenic acute regulatory (StAR)-related lipid transfer (START-domain-containing) StARD1 and D5, but not D4, in response to cJun-N-terminal kinase (JNK) activation by interleukin (IL)-1beta. Herein differential START gene regulatory mechanisms in this specialized epithelial cell were further examined. In a time- and dose-dependent manner, both FSH and forskolin significantly increase expression of StARD1 and D4 mRNAs, but not that of D5. Whereas StARD1 protein increased in response to FSH and forskolin, no changes in steady state levels of D4 or D5 were observed. Unlike IL-1beta, FSH or forskolin increases in cAMP rapidly results in phosphorylation of cAMP response element binding protein, protein kinase C (PKC) and phosphatidylinositol 3 kinase (PI3K), but not JNK or p38 mitogen-activated protein kinase (MAPK). Also, in contrast to IL-1beta, neither FSH nor forskolin induces cyclooxygenase activity or prostaglandin (PG)E2 or PGF2alpha production. Interestingly, FSH and forskolin reduce basal phosphorylated extracellular signal regulated kinase levels. When PKA, PKC, or PI3K inhibitors are used with FSH or forskolin, induction of StARD1 and D4 expression is inhibited, consistent with transcriptional mechanisms dependent, in part, on activation of several kinases. The data demonstrate that Sertoli StARD1, D4 and D5 expression are differentially regulated. Testicular START-domain proteins may provide novel contraceptive targets or therapeutic interventions in male infertility secondary to lipid handling pathologies.

Cisplatin induces interleukin (IL)-1β and IL-6 expression through a cyclooxygenase 2 regulation of prostaglandin (PG)E 2, PGF 2α, PGD 2 and PGI 2 production pathway in rat sertoli cell

Cis-diaminedichloroplatinium (cisplatin, CDDP) is used mainly in the treatment of various cancers, such as testicular cancer or leukemia. It is well known that exposure to cisplatin results in impair of spermatogenesis. However, little is known about the signal mechanisms involved in CDDP regulation of Sertoli cell (SC) gene expression, which subsequently influences developing germ cells and spermatogenesis. The aim of present study was to determine how CDDP regulates SC paracrine/autocrine activities and subsequently influences developing germ cells and spermatogenesis.

GODZ-Mediated Palmitoylation of GABAAReceptors Is Required for Normal Assembly and Function of GABAergic Inhibitory Synapses

Golgi-specific DHHC (Asp-His-His-Cys) zinc finger protein (GODZ) is a DHHC family palmitoyl acyltransferase that is implicated in palmitoylation and regulated trafficking of diverse substrates that function either at inhibitory or excitatory synapses. Of particular interest is the gamma2 subunit of GABA(A) receptors, which is required for targeting these receptors to inhibitory synapses. Here, we report that GODZ and, to a lesser extent, its close paralog sertoli cell gene with a zinc finger domain-beta (SERZ-beta) are the main members of the DHHC family of enzymes that are able to palmitoylate the gamma2 subunit in heterologous cells. Yeast two-hybrid and colocalization assays in human embryonic kidney 293T (HEK293T) cells indicate that GODZ and SERZ-beta show indistinguishable palmitoylation-dependent interaction with the gamma2 subunit. After coexpression in HEK293T cells, they form homomultimers and heteromultimers, as shown by coimmunoprecipitation and in vivo cross-linking experiments. Analyses in neurons transfected with dominant-negative GODZ (GODZ(C157S)) or plasmid-based GODZ-specific RNAi indicate that GODZ is required for normal accumulation of GABA(A) receptors at synapses, for normal whole-cell and synaptic GABAergic inhibitory function and, indirectly, for GABAergic innervation. Unexpectedly, GODZ was found to be dispensable for normal postsynaptic AMPA receptor-mediated glutamatergic transmission. We conclude that GODZ-mediated palmitoylation of GABA(A) receptors and possibly other substrates contributes selectively to the formation and normal function of GABAergic inhibitory synapses.

Pattern of Orexin Expression and Direct Biological Actions of Orexin-A in Rat Testis

Orexins, hypothalamic neuropeptides initially involved in the control of food intake and sleep-wake cycle, have recently emerged as pleiotropic regulators of different biological systems, including the reproductive axis. Besides central actions, peripheral expression and functions of orexins have been reported, and prepro-orexin and orexin type-1 receptor mRNAs have been detected in the testis. However, the pattern of expression and biological actions of orexin in the male gonad remain mostly unexplored. In this study, we report analyses on testicular prepro-orexin mRNA expression and orexin-A immunoreactivity in different experimental settings, and on direct effects of orexin-A on seminiferous tubule functions. Expression of prepro-orexin mRNA was demonstrated in the rat testis at different stages of postnatal development, with negligible levels at early juvenile period and maximum values in adulthood. Likewise, orexin-A immunoreactivity was demonstrated along postnatal maturation, with strong peptide signal in Leydig cells and spermatocytes at specific stages of meiosis. Testicular expression of prepro-orexin mRNA appeared hormonally regulated; its levels decreased after hypophysectomy and increased after gonadotropin replacement and ghrelin stimulation. Finally, orexin-A suppressed the expression of key Sertoli cell genes, such as Müllerian-inhibiting substance and stem cell factor, and inhibited DNA synthesis in specific stages of the seminiferous epithelium. In conclusion, we provide evidence for the regulated expression of orexin in the rat testis and its potential involvement in the control of seminiferous tubule functions. Together with our recent results on the expression of orexin type-1 receptor in the rat testis, our data further document a novel testicular site of action of orexins in the control of male reproductive axis.

Nuclear receptors Sf1 and Dax1 function cooperatively to mediate somatic cell differentiation during testis development

Mutations of orphan nuclear receptors SF1 and DAX1 each cause adrenal insufficiency and gonadal dysgenesis in humans, although the pathological features are distinct. Because Dax1 antagonizes Sf1-mediated transcription in vitro, we hypothesized that Dax1 deficiency would compensate for allelic loss of Sf1. In studies of the developing testis, expression of the fetal Leydig cell markers Cyp17 and Cyp11a1 was reduced in heterozygous Sf1-deficient mice at E13.5, consistent with dose-dependent effects of Sf1. In Sf1/Dax1 (Sf1 heterozygous and Dax1-deleted) double mutant gonads, the expression of these genes was unexpectedly reduced further, indicating that loss of Dax1 did not compensate for reduced Sf1 activity. The Sertoli cell product Dhh was reduced in Sf1 heterozygotes at E11.5, and it was undetectable in Sf1/Dax1 double mutants, indicating that Sf1 and Dax1 function cooperatively to induce Dhh expression. Similarly, Amh expression was reduced in both Sf1 and Dax1 single mutants at E11.5, and it was not rescued by the Sf1/Dax1 double mutant. By contrast, Sox9 was expressed in single and in double mutants, suggesting that various Sertoli cell genes are differentially sensitive to Sf1 and Dax1 function. Reduced expression of Dhh and Amh was transient, and was largely restored by E12.5. Similarly, there was recovery of fetal Leydig cell markers by E14.5, indicating that loss of Sf1/Dax1 delays but does not preclude fetal Leydig cell development. Thus, although Sf1 and Dax1 function as transcriptional antagonists for many target genes in vitro, they act independently or cooperatively in vivo during male gonadal development.

The Helix-Loop-Helix Inhibitor of Differentiation (ID) Proteins Induce Post-Mitotic Terminally Differentiated Sertoli Cells to Re-Enter the Cell Cycle and Proliferate

Prior to puberty the Sertoli cells undergo active cell proliferation, and at the onset of puberty they become a terminally differentiated postmitotic cell population that support spermatogenesis. The molecular mechanisms involved in the postmitotic block of pubertal and adult Sertoli cells are unknown. The four known helix-loop-helix ID proteins (i.e., Id1, Id2, Id3, and Id4) are considered dominant negative regulators of cellular differentiation pathways and act as positive regulators of cellular proliferation. ID proteins are expressed at low levels by postpubertal Sertoli cells and are transiently induced by serum. The hypothesis tested was that ID proteins can induce a terminally differentiated postmitotic Sertoli cell to reenter the cell cycle if they are constitutively expressed. To test this hypothesis, ID1 and ID2 were stably integrated and individually overexpressed in postmitotic rat Sertoli cells. Overexpression of ID1 or ID2 allowed postmitotic Sertoli cells to reenter the cell cycle and undergo mitosis. The cells continued to proliferate even after 300 cell doublings. The functional markers of Sertoli cell differentiation such as transferrin, inhibin alpha, Sert1, and androgen binding protein (ABP) continued to be expressed by the proliferating Sertoli cells, but at lower levels. FSH receptor expression was lost in the proliferating Sertoli cell-Id lines. Some Sertoli cell genes, such as cyclic protein 2 (cathepsin L) and Sry-related HMG box protein-11 (Sox11) increase in expression. At no stage of proliferation did the cells exhibit senescence. The expression profile as determined with a microarray protocol of the Sertoli cell-Id lines suggested an overall increase in cell cycle genes and a decrease in growth inhibitory genes. These results demonstrate that overexpression of ID1 and ID2 genes in a postmitotic, terminally differentiated cell type have the capacity to induce reentry into the cell cycle. The observations are discussed in regards to potential future applications in model systems of terminally differentiated cell types such as neurons or myocytes.

Retinol-induced mdr1 and mdr3 modulation in cultured rat Sertoli cells is attenuated by free radical scavengers

The effects of retinol on modulation of mdr genes in Sertoli cells were investigated. The hypothesis that free radical scavengers may attenuate the effect of retinol was also tested. Sertoli cells isolated from 15-day-old Wistar rats were cultured for 48 h and then treated with retinol for 24 h with or without free radical scavengers (1 mM mannitol, 0.1 mM Trolox or superoxide dismutase [200 U/ml]). Expression of mdr1, mdr2 and mdr3 genes was monitored by RT-PCR. Mitochondrial superoxide production was used as an index of ROS production. Expression of mdr1 and mdr3 was inhibited by retinol treatment (7 μM, 24 h), while mdr2 was not detected in response to any of the treatments. We also observed that retinol supplementation (7 μM, 24 h) increased superoxide production. The observed inhibition of mdr genes was attenuated by all co-treatments, suggesting that retinol-induced ROS are required for inhibition of mdr1 and mdr3 expression. The results suggest that retinol may play an important role in the modulation of the mdr gene family in cultured rat Sertoli cells and that these effects appear to be mediated by ROS.

The Id2 Transcriptional Repressor Is Induced by Follicle-stimulating Hormone and cAMP

Id (inhibitor of DNA binding/differentiation) proteins repress differentiation and promote cell division by dimerizing with and inhibiting the action of basic helix-loop-helix transcription factors including those that bind to E-box motifs. Of the four characterized Id proteins, only Id2 is found in the nucleus of Sertoli cells that support the development of spermatozoa in the testis. Differential display analysis of rat primary Sertoli cell mRNA identified Id2 as being inducible by forskolin, a stimulator of cAMP production. Northern blot analysis confirmed that Id2 mRNA expression peaked in Sertoli cells 6-12 h after stimulation with forskolin or follicle-stimulating hormone (FSH), the major physiological stimulator of cAMP in Sertoli cells. Similarly, Id2 promoter activity in Sertoli cells was induced after forskolin or FSH stimulation as well as by overexpression of protein kinase A. Forskolin induction of the Id2 promoter required sequences located between positions -122 and -82. Protein(s) of 40-45 kDa were found to bind two activated transcription factor/cAMP-response element-like sites and a GATA motif within the regulatory region. The induction of the Id2 gene by FSH corresponded with a decrease in protein binding to an E-box consensus motif and decreased E-box-mediated transcription. Together, these findings raise the possibility that FSH-mediated induction of Id2 and resultant inhibition of basic helix-loop-helix transcription factor-regulated genes in Sertoli cells may contribute to the regulation of spermatogenesis.

Identification of a novel Sertoli cell gene product SERT that influences follicle stimulating hormone actions

Sertoli cells provide the cytoarchitectural support and microenvironment necessary for the process of spermatogenesis and have a central role in male sex determination. Characterization of Sertoli cell specific gene products provides insight into the unique functions of this testicular cell type. The current study reports the identification of a novel Sertoli cell gene product that is termed Sertoli cell specific gene (SERT). The SERT cDNA sequence shows no homology to any of the known gene sequences in the GenBank database. Some homology was found with short sequences in the expressed sequence tag (EST) database. A motif analysis demonstrates a signature to an RNA binding protein sex lethal (sx1) involved in Drosophila sex determination. The expression pattern of SERT was examined with reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis. SERT expression was found to be specific to the testis and absent in other tissues examined. In the testis, the approximate 1.5-kb SERT transcript was localized to the Sertoli cells. A 1.1-kb spliced form of SERT was also identified by reverse transcription polymerase chain reaction analysis. The genomic structure analysis demonstrates the presence of at least three exons with 85% sequence homology between mouse and rat sequences. Treatment of cultured Sertoli cells with follicle stimulating hormone (FSH) significantly increased the expression of SERT mRNA levels. The potential role of SERT in Sertoli cell function was investigated with the use of a transferrin promoter reporter construct. Transferrin expression is a marker of Sertoli cell differentiated function. An antisense oligonucleotide to SERT significantly inhibited FSH and cAMP induced transferrin promoter activation, while control oligonucleotides had no effect. In summary, a novel gene product expressed primarily by Sertoli cells, SERT, was identified with a potential RNA binding protein motif similar to sex lethal that appears to have a role in maintaining hormone (e.g. FSH) actions in Sertoli cells.

Effects of flutamide in the rat testis on the expression of occludin, an integral member of the tight junctions

In an effort to uncover the gonadal impairment by the antiandrogen flutamide (FM) in males, the effect of subacute administration of FM on the expression of tight junction (TJ) genes that build the blood–testis barrier (BTB) was investigated in adult rat testis. At 13 weeks old of age male rats were given vehicle (corn oil) or FM (25 mg/kg per day, in corn oil) orally for 6 days. At 8 days (D8) after the first dose, testicular expression of the occludin, claudin-1, and -11 was analyzed by semiquantitative RT-PCR. The testicular weight of the FM-treated rats on D8 was a little but significantly higher than in the control group. On D8 the expression of occludin in the FM-treated animals was significantly decreased but claudin-1 and -11 were not altered significantly. Because FM administration inhibits germ cell differentiation, it is likely that the down-regulated occludin expression in FM-rat testes may be attributed to the alteration in the paracrine interaction between Sertoli cells and germ cells in testis. It also emphasized that FM might have differentially affected the transcription of TJ genes in Sertoli cells building the BTB. These findings provide a rationale for a number of observations on the gonadal impairment by FM in males and suggest that FM is potentially harmful to spermatogenesis by alteration of the BTB.

A 3-kilobase region derived from the rat cathepsin L gene directs in vivo expression of a reporter gene in sertoli cells in a manner comparable to that of the endogenous gene.

During mammalian spermatogenesis, the transcription of several genes in Sertoli cells is turned on and off as the adjacent male germ cells progress through the stages of the cycle of the seminiferous epithelium. A requirement for defining how germ cells regulate this process is the identification of a promoter that confers, in vivo, accurate, stage-specific gene expression in Sertoli cells. To date, such a promoter has not been identified. Using transgenic mice, we show that the 3-kilobase genomic fragment immediately upstream of the rat cathepsin L translation start site directs expression of the reporter gene, beta-galactosidase, only in Sertoli cells. The expression pattern of the reporter gene recapitulated that of the endogenous gene in Sertoli cells as 75% of the seminiferous tubules that contained X-gal positive Sertoli cells were at stages VI-VIII and beta-galactosidase enzymatic activity was 4-fold higher in mature testes compared with immature testes. This is, to our knowledge, the first identification of a promoter region that contains all of the regulatory elements required for accurate, stage-specific gene expression in Sertoli cells.

Sertoli and granulosa cell-specific Cre recombinase activity in transgenic mice.

We have established transgenic mice expressing the Cre recombinase under the control of the anti-Müllerian hormone (AMH) gene promoter. Cre activity and specificity were evaluated by different means. In AMH-Cre mice, expression of the Cre recombinase mRNA was confined to the testis and ovary. AMH-Cre mice were crossed with reporter transgenic lines and the offspring exhibited Cre-mediated recombination only in the testis and the ovary. In male, histochemical analysis indicated that recombination occurred in every Sertoli cells. In female, Cre-mediated recombination was restricted to granulosa cells, but the protein was not evenly active in every cells. From these results, we conclude that potentially, this transgenic line possessing AMH promoter-driven expression of the Cre recombinase is a powerful tool to delete genes in Sertoli cells only, in order to study Sertoli cell gene function during mammalian spermatogenesis.

Identification of a Novel Gene Product, Sertoli Cell Gene with a Zinc Finger Domain, That Is Important for FSH Activation of Testicular Sertoli Cells

Identification of a Novel Gene Product, Sertoli Cell Gene with a Zinc Finger Domain, That Is Important for FSH Activation of Testicular Sertoli Cells

Identification of a novel gene product, Sertoli cell gene with a zinc finger domain, that is important for FSH activation of testicular Sertoli cells.

Sertoli cells provide the cytoarchitectural support and microenvironment necessary for the process of spermatogenesis. A novel, ubiquitously expressed cDNA clone was isolated from Sertoli cells and termed Sertoli cell gene with a zinc finger domain (SERZ). A significant homology of SERZ was found with a mouse genomic sequence that suggested the presence of at least 10 exons. An open reading frame at the 5'-end of the cDNA, termed SERZ-alpha, had a cryptic basic helix-loop-helix (bHLH) domain, but no start codon. When a start codon was engineered into the 5'-end of the cDNA, an in vitro translation product of SERZ-alpha was obtained. The longest second open reading frame with an ATG start site at 304 bp from the 5'-end coded for a 308-amino acid SERZ-beta polypeptide. Motif analysis and BLAST search of SERZ-beta showed significant homology to the DHHC domain of conserved zinc finger proteins. A number of potential phosphorylation sites were observed in the SERZ-beta polypeptide sequence. The long 5'-untranslated region of SERZ-beta prompted an investigation of both potential alternate polypeptide products, SERZ-alpha and SERZ-beta. Both SERZ-alpha and SERZ-beta proteins were detected with specific antibodies to SERZ-beta and the 5'-end open reading frame SERZ-alpha in a Western blot analysis of total Sertoli cell proteins. The presence of the SERZ-beta polypeptide was also confirmed by in vitro translation of the cDNA, but SERZ-alpha was not translated in vitro in the absence of an engineered start codon. The expression pattern of SERZ mRNA was observed in all tissues examined. The transcript size of SERZ as determined by Northern blot analysis is approximately 2.7 kb. An antisense oligonucleotide to SERZ was found not to influence basal levels of transferrin promoter activation, but significantly blocked FSH-induced transferrin promoter activation. SERZ mRNA expression was not regulated by FSH treatment of Sertoli cell cultures. In summary, a novel gene product, SERZ, was identified that appears to have a role in maintaining Sertoli cell differentiated functions and mediating FSH actions. Translation of SERZ may give rise to two gene products; however, the SERZ-beta containing the zinc finger domain is probably the principal product of the SERZ gene.

Adenovirus-mediated gene delivery and in vitro microinsemination produce offspring from infertile male mice.

Sertoli cells play a pivotal role in spermatogenesis through their interactions with germ cells. To set up a strategy for treating male infertility caused by Sertoli cell dysfunction, we developed a Sertoli cell gene transfer system by using an adenovirus vector, which maintained long-term transgene expression in the testes of infertile mice. Introduction of an adenovirus carrying the mouse Steel (Sl) gene into Sertoli cells restored partial spermatogenesis in infertile Steel/Steel(dickie) (Sl/Sl(d)) mutant mouse testes. Although these males remained infertile, round spermatids and spermatozoa from the testes produced normal fertile offspring after intracytoplasmic injection into oocytes. None of the offspring showed evidence of germ line transmission of adenoviral DNA. Thus, we demonstrate a successful treatment for infertility by using a gene therapy vector. Therefore, adenovirus-mediated gene delivery into Sertoli cells not only provides an efficient and convenient means for studying germ cell-Sertoli cell interactions through manipulation of the germ cell microenvironment in vivo, but also is a useful method to treat male infertility resulting from a Sertoli cell defect.

Expression of the basic helix-loop-helix protein REBalpha in rat testicular Sertoli cells.

Sertoli cell differentiation is initiated in the embryo to promote testicular development and male sex determination. In the adult, Sertoli cells are critical for maintenance of the spermatogenic process. Previously, Sertoli cell differentiation has been shown to be regulated in part by basic helix-loop-helix (bHLH) transcription factors. This was based on the observation that promoters of a number of Sertoli cell genes contained bHLH-responsive E-box response elements and that overexpression of Id, a negatively acting HLH protein, down-regulates Sertoli cell differentiated functions. Analysis of Sertoli cell bHLH proteins demonstrated the expression of REBalpha in Sertoli cells. REBalpha and REBbeta are spliced variants of the REB gene that is implicated in cell-specific gene expression as part of dimeric bHLH complexes acting on E-box response elements. Although both the transcripts of the REB gene are widely expressed, differential expression of the REB gene transcripts REBalpha and REBbeta has been shown. In the current study, a polymerase chain reaction (PCR)-based approach demonstrated that REB gene transcripts are expressed in the testis. Characterization of the REB transcripts suggested that REBalpha is the major splice variant in Sertoli cells. PCR primers specifically designed to amplify either REBalpha or REBbeta demonstrated that Sertoli cells express only REBalpha, not REBbeta. REBbeta was present in the RNA samples obtained from whole testis, suggesting expression in other testicular cell types. A Northern blot analysis of RNA from Sertoli cells treated with or without FSH or cAMP demonstrated that REBalpha is not hormone responsive. REBalpha was also found to be expressed in germ cells and peritubular cells. An immunocytochemical analysis demonstrated that REBalpha is predominantly expressed by Sertoli cells within the seminiferous tubules. The activity of REBalpha in Sertoli cells was demonstrated with an E-box gel shift with Sertoli cell nuclear extracts. The E-box gel shift was found to contain REBalpha and E47/E12 bHLH proteins. In summary, the Sertoli cell is one of the first cells shown to specifically express the REBalpha isoform of the REB gene. The results are discussed in relation to the possibility that Sertoli cells may express a cell-specific bHLH protein that can preferentially dimerize with REBalpha.

Isoform-specific regulation of the CCAAT/enhancer-binding protein family of transcription factors by 3',5'-cyclic adenosine monophosphate in Sertoli cells.

The C/EBP (CCAAT/enhancer-binding protein) family of transcription factors is important for differentiation, lipid biosynthesis, and metabolism. Here, we demonstrate for the first time the presence of C/EBP alpha, beta, delta, and zeta messenger RNA (mRNA) and protein in Sertoli cell primary cultures. Treatment with FSH or 8-CPTcAMP strongly induced C/EBP beta mRNA above basal levels with rapid and transient kinetics in Sertoli cell primary cultures as well as in whole testes from hypophysectomized rats. Whereas C/EBP beta mRNA was induced approximately 50-fold, C/EBP delta mRNA was induced 5- to 8-fold by cAMP in Sertoli cells. Messenger RNA for C/EBP beta and delta were induced by inhibition of protein synthesis with cycloheximide and cycloheximide acted synergistically with cAMP. Immunoblots with C/EBP antibodies demonstrated a strong induction of C/EBP beta, delta, and zeta by cAMP. Electrophoretic mobility shift analysis of nuclear proteins from cAMP-treated Sertoli cells using a C/EBP consensus oligonucleotide and antibodies revealed specific binding of C/EBP/DNA complexes, the majority of which were supershifted by C/EBP beta antibody. Transfections of Sertoli cells with a C/EBP reporter construct showed approximately 3-fold induction of reporter gene activity by cAMP. In contrast, the reporter gene vector with a mutated form of the C/EBP binding site, was almost unresponsive to cAMP in transfections of Sertoli cells. Furthermore, C/EBP beta expression increased the activities of two promoters known to be cAMP-responsive in Sertoli cells. Thus, the early induction of C/EBP isoforms by cAMP may play a role in FSH-dependent regulation of late response genes in Sertoli cells.

Rat pachytene spermatocytes down-regulate a polo-like kinase and up-regulate a thiol-specific antioxidant protein, whereas sertoli cells down-regulate a phosphodiesterase and up-regulate an oxidative stress protein after exposure to methoxyethanol and methoxyacetic acid.

2-Methoxyethanol (ME) and its metabolite, methoxyacetic acid (MAA), produce testicular lesions characterized by pachytene spermatocyte degeneration. To understand the molecular basis of this action on meiotic prophase cells, mRNA differential display was used to identify gene expression changes in control and treated cells. When pachytene spermatocytes were cultured with 5 mM ME or 5 mM MAA for 24 h, two complementary DNAs (cDNAs), of 557 nucleotides (clone 5) and 388 nucleotides (clone 6), were up-regulated; and a cDNA of 648 nucleotides (clone 1) was down-regulated. The altered expression pattern shown by differential display was confirmed by Northern blotting. Sequence analyses indicate that clones 1 and 6 have 83% and 79% homology at the nucleotide level to a polo-like kinase and a thiol-specific antioxidant, respectively. Clone 5 shows no homology to any known gene in the database. Messenger RNAs (mRNAs) encoding the thiol-specific antioxidant and clone 5 are up-regulated within 30 min of the addition of MAA, whereas the polo-like kinase mRNA decreased to undetectable levels after 6 h. Changes in Sertoli cell gene expression were also detected when Sertoli cells were cultured with 5 mM ME or MAA for 24 h. Two cDNAs, of 367 nucleotides (clone 2) and 676 nucleotides (clone 3), were up-regulated; and a cDNA of 538 nucleotides (clone 4) was down-regulated. Homology searches revealed that clones 3 and 4 have 90 and 91% homology at the nucleotide level to an oxidative stress protein and a phosphodiesterase (PDE), respectively. Northern blotting confirmed the differential display expression pattern for the PDE and oxidative stress protein. mRNAs for the latter were induced within 30 min, and PDE mRNAs were down-regulated within one h, after the addition of MAA. To determine whether the changes in gene expression seen with cells in culture also occur in vivo, rats were given a single oral dose of 250 mg/kg ME or MAA. After 24 h, total testis RNAs from control and treated rats were purified and hybridized. The expression patterns seen in vivo for the differentially expressed cDNAs were identical to those seen in vitro. We conclude that, although pachytene spermatocytes seem to be selectively affected by ME and MAA, changes in gene expression are also detected in Sertoli cells, suggesting that the action(s) of ME or MAA on pachytene spermatocytes could be mediated through Sertoli cells.

Identification and regulation of testicular interferon-gamma (IFNgamma) receptor subunits: IFNgamma enhances interferon regulatory factor-1 and interleukin-1beta converting enzyme expression.

Interferon-gamma (IFNgamma) transmits its signal through a specific cell surface receptor (IFNgammaR), which consists of a primary ligand binding alpha-chain (IFNgammaR alpha) and a signaling beta-chain (IFNgammaR beta). Recent studies identified the cytokines IFNgamma, interleukin-6 (IL-6), IL-1alpha, and tumor necrosis factor-alpha in testicular cells. Therefore, we: 1) examined the expression of IFNgammaR alpha and IFNgammaR beta subunits in freshly isolated and purified rat testicular cells; 2) examined the differential regulation of receptor components by cytokines using primary cultures of Sertoli cells; 3) identified the cell signaling pathway components of testicular IFNgammaR; and 4) characterized the functional role of testicular IFNgamma using primary Sertoli cells. We demonstrated the messenger RNAs for both chains of IFNgammaR in rat testicular cells using Northern hybridization analysis. Western blot analysis and immunocytochemistry showed that both specific IFNgammaR protein subunits were present in cultured primary Leydig and Sertoli cells prepared from the testes of immature rats. The expression of both IFNgammaR component messenger RNAs in cultured Sertoli cells was increased by its specific ligand (IFNgamma), as well as IL-1alpha and tumor necrosis factor-alpha, in both a time- and dose-dependent manner. IFNgamma-activation of the Janus (JAK) tyrosine kinases, JAK1 and JAK2 proteins, indicate that IFNgammaR, expressed in the Sertoli cell, is functional. Moreover, IFNgamma modulates the expression of interferon regulatory factor (IRF)-1 and IL-1beta converting enzyme genes in Sertoli cells. Thus, our data are suggestive of a role(s) for IFN-gamma in the regulation of distinct gene expression and cell-specific sensitivity to apoptosis in the testis.

Coexpression of multiple Sertoli cell and Leydig cell marker genes in the spontaneous testicular tumor of F344 rat: evidence for phenotypical bifurcation of the interstitial cell tumor.

The development of testicular tumor has been frequently observed in some laboratory rat strains. In the present study, we have further characterized the testicular tumor that spontaneously develops in the F344 rat (F344/Jcl). Tumor cells first appeared in the interstitium and developed into multifocal nodular lesions. In the later stage, the whole testes were occupied by tumor cells that consisted of three different types of cells in morphological appearance: large clear type, small eosinophilic type and intermediate type. To determine the character of these cells, we examined the expression of marker genes for Sertoli cells (e.g., transferrin) and Leydig cells (e.g., 3β–hydroxysteroid dehydrogenase 1 (3β–HSD 1)). Transferrin and 3β–HSD 1 mRNAs were found in all 8 tumor samples analyzed hy northern blotting. By in situ hybridization, we observed a substantial amount of 3β–HSD 1 mRNA and little or no transferrin mRNA in the large clear cells. In contrast, the small eosinophilic cells showed little or no 3β–HSD 1 mRNA and a large amount of transferrin mRNA, suggesting that the tumor was a mixture of at least two types of cells. Other Sertoli cell marker genes, such as cyclic protein 2 and sulfated glycoprotein 2, were expressed in all 8 tumors analyzed, and testin and steel factor (SLF), the c–kit receptor ligand, were also expressed in some of the tumors (testin, 75%; SLF, 25%), while other Leydig cell markers, LH receptor and c–kit, were expressed in 87% and 80% of the tumors, respectively. These results indicate that the spontaneous testicular tumor of F344 rat is of interstitium origin, showing phenotypical bifurcation possibly via transdifferentiation.