Amal K. Mukhopadhyay

Male germ cells and photoreceptors, both dependent on close cell–cell interactions, degenerate upon ClC‐2 Cl− channel disruption

The functions of some CLC Cl− channels are evident from human diseases that result from their mutations, but the role of the broadly expressed ClC‐2 Cl− channel is less clear. Several important functions have been attributed to ClC‐2, but contrary to these expectations ClC‐2‐deficient mice lacked overt abnormalities except for a severe degeneration of the retina and the testes, which led to selective male infertility. Seminiferous tubules did not develop lumina and germ cells failed to complete meiosis. Beginning around puberty there was a massive death of primary spermatocytes and later also of spermatogonia. Tubules were filled with abnormal Sertoli cells, which normally express ClC‐2 in patches adjacent to germ cells. In the retina, photoreceptors lacked normal outer segments and degenerated between days P10 and P30. The current across the retinal pigment epithelium was severely reduced at P36. Thus, ClC‐2 disruption entails the death of two cell types which depend on supporting cells that form the blood–testes and blood–retina barriers. We propose that ClC‐2 is crucial for controlling the ionic environment of these cells.

VASCULAR ENDOTHELIAL GROWTH FACTOR AND ITS RECEPTORS IN NORMAL HUMAN TESTICULAR TISSUE

Expression of vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), and its receptors Flt-1 and KDR (Flk-1 in mouse) and their localization in the human testis were analyzed by means of reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. VEGF mRNA was detected in the human testicular tissue and in fragments of seminiferous tubules by means of RT-PCR, while fragments of blood vessels isolated from testes were negative. Western blotting procedure using a specific VEGF antibody, revealed two protein bands corresponding to 24 and 49 kDa in the extracts prepared from the whole testis and in the seminiferous tubules while no such bands were found in isolated fragments of human testicular blood vessels. Also immunohistochemically, human testicular blood vessels show no VEGF immunoreactivity, while Leydig cells and Sertoli cells were positive. The mRNA of the VEGF receptor Flt-1 was found to be expressed in human testicular tissue, in isolated fragments of testicular blood vessels and in seminiferous tubules as determined by RT-PCR procedure. In accordance with these results, the Flt-1 protein was immunohistochemically localized in Leydig, Sertoli and perivascular cells. Endothelial cells of certain segments of human testicular microvasculature also stained positive for Flt-1. Expression of VEGF receptor, KDR, could be demonstrated in human testicular tissue, in isolated seminiferous tubules and in isolated fragments of human testicular blood vessels by means of RT-PCR. Immunohistochemically, the KDR protein was localized in endothelial cells and perivascular cells of capillaries within the lamina propria of seminiferous tubules. Leydig cells and Sertoli cells show KDR immunoreactivity, too. Thus we demonstrate the presence of both types of VEGF receptors Flt-1 and KDR on Leydig as well as on Sertoli cells which are normal non-endothelial cells, suggesting hitherto unrecognized and novel functions for such receptors. The results obtained permit us to suggest VEGF as a paracrine mitogenic and angiogenic factor, responsible for modulating the capillarization of the human testicular tissue and maintaining the functions of testicular microvasculature. VEGF may also influence the permeability of capillaries passing through the groups of Leydig cells and those localized within the lamina propria of human seminiferous tubules. The differences in the expression pattern of the VEGF receptors in the human testicular tissue probably reflect different VEGF effects in different compartments of human testis.

Testosterone production by mouse Leydig cells is stimulated in vitro by atrial natriuretic factor

The synthetic atrial peptides, rat atrial natriuretic peptide, atriopeptin I and atriopeptin II, stimulated testosterone production by mouse Leydig cells in a time‐ and concentration‐dependent manner. The maximum stimulation of the steroidogenesis in response to the peptides was 6–10‐fold over the basal level, as compared with 20–24‐fold stimulation obtained with saturating concentrations of hCG. The stimulation of steroidogenesis by the most potent peptide, atriopeptin II, was markedly enhanced in the presence of the phosphodiesterase inhibitor, 3‐isobutyl‐1‐methylxanthine, suggesting an involvement of cyclic nucleotides. However, neither basal nor hCG‐stimulated levels of cAMP were altered by the peptide, though testosterone production in response to submaximal concentrations of hCG was increased in the presence of atriopeptin II. The nature of the second messenger involved and the mechanism of action of the atrial peptides may by elucidated by further research in progress.

Stimulatory Effect of Progesterone on the Expression of Steroidogenic Acute Regulatory Protein in MA-10 Leydig Cells

Abstract The steroidogenic acute regulatory protein (StAR), by virtue of its ability to facilitate the intramitochondrial transport of cholesterol, plays an important role in regulating steroid hormone biosynthesis in steroidogenic cells. In agreement with published data, both StAR expression and progesterone production in MA-10 mouse Leydig tumor cells could be stimulated with hCG and 8Br-cAMP. Addition of aminoglutethimide, an inhibitor of cholesterol side chain cleavage (P450scc) enzyme, not only resulted in a drastic inhibition of progesterone production but also in an attenuation of StAR expression in response to either hCG or 8-Br-cAMP. Therefore, we addressed the question of whether progesterone, the end product of the steroidogenic cascade in these cells, could be in a position to regulate the StAR gene expression. In MA-10 cells, we report here that progesterone in microgram amounts can induce StAR gene expression in a time- and dose-dependent manner. StAR expression in response to a maximally effective concentration of progesterone of 10 μg/ml was highest at 6 h and started decreasing thereafter. The effect of progesterone on StAR protein and StAR mRNA induction was mimicked by its synthetic analog, progestin R5020, but not by other steroids, including dexamethasone, estradiol, testosterone, and dihydrotestosterone. Dexamethasone, in contrast, was able to inhibit StAR expression in MA-10 cells. Surprisingly, RU486, a potent antagonist of progesterone and glucocorticoid action, had a stimulatory effect on StAR mRNA levels. Reverse transcription-polymerase chain reaction analysis demonstrated the absence of the classical form of progesterone receptor in MA-10 cells. Thus, for the first time, a direct stimulatory effect of a steroid on StAR gene expression has been demonstrated. Furthermore, these results provide a new insight, indicating that progesterone mediates the activation of StAR expression exerted presumably through a novel, nonclassical progesterone receptor in mouse Leydig cells.

Inhibitory effects of TNFα on mouse tumor Leydig cells: possible role of ceramide in the mechanism of action

TNF alpha is reported to inhibit steroidogenesis in mouse Leydig cells. In primary cells this inhibition resulted mainly from a reduced expression of Cyp-17 gene. Mouse tumor Leydig cells, MA-10, being free of macrophages and lacking Cyp-17, appear to be an excellent model to investigate those effects of TNF alpha which are independent of either macrophages or Cyp-17. We report here that TNF alpha receptors are expressed in this cell line. Treatment of the cells with TNF alpha had no effect on basal progesterone production. In contrast, LH-, 8Br-cAMP and forskolin-stimulated progesterone production was inhibited by TNF alpha. Neither enzymes involved in the conversion of cholesterol to pregnenolone nor hormone-induced hydrolysis of [14C] cholesterol-ester were affected by TNF alpha. The hormone-induced expression of StAR protein was diminished in mitochondrial fractions from TNF alpha-treated cells. Also cell permeable ceramides markedly inhibited StAR protein levels. We show further that TNF alpha was able to induce [14C]-ceramide accumulation in MA-10 cells and suggest that this sphingolipid may be considered as a transmitter of TNF alpha signals to the StAR protein.

Lysophosphatidic acid and its role in reproduction

Abstract Lysophosphatidic acid (LPA) belongs to a new family of lipid mediators that are endogenous growth factors and that elicit diverse biological effects, usually via the activation of G protein-coupled receptors. LPA can be generated after cell activation through the hydrolysis of preexisting phospholipids in the membranes of stimulated cells. A dramatic elevation of LPA levels was found in serum of patients suffering from ovarian carcinoma. Because these high LPA amounts can be detected as early as stage I of the disease, LPA has been introduced as a new marker for ovarian cancer. Progression of the malignancy is correlated with a differential expression of various LPA receptor subtypes. The presence of LPA in the follicular fluid of healthy individuals implicates that this biological mediator may be relevant to normal ovarian physiology. LPA induces proliferation and mitogenic signaling of prostate cancer cells, and a novel LPA receptor isoform has been recognized in healthy prostate tissues. This evidence indicates multiple roles for LPA in both male and female reproductive physiology and pathology. In this review, we summarize the literature on LPA generation, the way it is degraded, and the mechanisms by which signals are transduced by various LPA receptors in reproductive tissues, and we discuss possible future research directions in these areas.

Selective translocation of non-conventional protein kinase C isoenzymes by gonadotropin-releasing hormone (GnRH) in the gonadotrope-derived αT3-1 cell line

Gonadotropin-releasing hormone acts via G-protein coupled receptors to stimulate polyphosphoinositide-specific phospholipase C (PIC) with consequent elevation of cytosolic Ca2+ and activation of protein kinase C (PKC). Whereas Ca2+ is known to mediate stimulation of exocytotic gonadotropin release by GnRH, the identity of the PKC isoenzymes activated by GnRH and their physiological role in gonadotropes are poorly understood. In many systems translocation of PKC (from cytosolic to particulate fractions of cellular homogenates) has been taken as evidence of hormonal activation of PKC and down regulation of PKC (by prolonged treatment with PKC-activating phorbol esters) has been used extensively to investigate the role of PKC in hormone action. Here we have assessed the influence of GnRH and phorbol esters on translocation and down regulation of PKC isoenzymes identified by Western blotting with isoenzyme-specific antibodies in alpha T3-1 cells (a gonadotrope-derived cell line). These cells were found to posses PKCs alpha, epsilon and zeta but not beta, delta (present in rat pituitaries) or gamma (present in rat brains). In short-term stimulations (10 min), the PKC-activating phorbol esters, PMA and PDBu, caused concentration-dependent increases in the proportion of PKC alpha and PKC epsilon recovered from the particulate fraction of alpha T3-1 cells, but did not induce measurable translocation of PKC zeta. The inactive phorbol ester 4 alpha PDBu did not cause translocation of any of these isoenzymes. GnRH treatment induced a concentration-dependent increase in the proportion of particulate PKC epsilon and PKC zeta but had no measurable effect on PKC alpha translocation. In longer incubations (6-48 h) GnRH failed to cause measurable down-regulation of these isoenzymes whereas PMA treatment led to a clear down regulation of PKCs alpha and epsilon (albeit with different kinetics). The data demonstrate the differential activation and down regulation of PKC isoenzymes by GnRH versus PMA, which are clearly pertinent to the design of experiments intended to address the role of such isoenzymes in GnRH action. Moreover, they provide the first demonstration of hormonal regulation of an atypical PKC isoenzyme (PKC zeta) in pituitary cells.

The expression of the RLF/INSL3 gene is reduced in Leydig cells of the aging rat testis

The relaxin-like factor (RLF), which is the product of the INSL3 gene, is highly expressed in the fetal and adult-type Leydig cells of all species so far examined. In adult testes it is upregulated at puberty but appears subsequently to be expressed in a constitutive manner, independently of acute changes in the hypothalamic-pituitary-gonadal (HPG) axis. Functional hypogonadism with decreased testosterone is prevalent in the aging male. In order to test whether this is a property of the HPG axis, or of the Leydig cells themselves, RLF/INSL3 was used as an independent marker to assess rat Leydig cell differentiation status. Hybridization analysis showed that in the testes of old (2 years) rats, RLF/INSL3 mRNA expression was dramatically reduced, compared to young (3 months) animals. This was also evident at the protein level using immunohistochemistry. The results suggest that increasing functional hypogonadism in older male mammals is likely caused by a dedifferentiation of the Leydig cells themselves.

Regulation of Vascular Endothelial Growth Factor Production by Leydig Cells In Vitro: The Role of Protein Kinase A and Mitogen-Activated Protein Kinase Cascade

Abstract We previously reported the presence of vascular endothelial growth factor (VEGF) in testicular cells, and high concentrations of VEGF have been measured in semen, although its role in male reproduction remains obscure. In the present study we focus on understanding the mechanism of VEGF production by mouse Leydig cells cultured in vitro. Production of VEGF protein in medium by testicular cells was markedly increased by the addition of hCG in a time- and dose-dependent manner. Gonadotropin-stimulated VEGF production was mediated by cAMP-dependent protein kinase A (PKA), as evidenced by the effect of hCG being mimicked by 8Br-cAMP and being abolished in the presence of a PKA-specific inhibitor, H-89. Protein kinase C was not involved, as evidenced by phorbol 12-myristate 13-acetate having no influence on VEGF production by Leydig cells. In addition to hCG, atrial natriuretic peptide was also able to stimulate VEGF production, suggesting that cGMP is able to cross-activate PKA. A specific Src kinase inhibitor, PP2, could completely block the stimulatory effects of both gonadotropin and 8Br-cAMP on VEGF production by Leydig cells, implying an involvement of the Src kinase pathway. Furthermore, addition of U0126, an inhibitor of MEK 1/2, abolished the increase in VEGF production stimulated by both hCG and 8Br-cAMP. A similar inhibitory effect was observed by the addition of SB203580, a p38 mitogen-activated protein kinase inhibitor. Thus, in conclusion, Leydig cells are able to produce VEGF by a process under gonadotropic control, and PKA plays a key role in this process. Downstream of PKA, it appears that both MEK 1/2 and Src kinase-dependent pathways are involved, although further research will be necessary to determine the precise link between PKA and other kinases involved.

Central Nervous System-Specific Glycosylation of the Type A Natriuretic Peptide Receptor

Physiological effects of atrial natriuretic peptide (ANP) are thought to be mediated by binding to and activation of a widely expressed membrane receptor, termed guanylyl cyclase (GC)-A. During comparative analyses of ANP receptor expression in various rat and bovine tissues, by UV light-induced affinity cross-linking to 125I-ANP, we uncovered a size heterogeneity of GC-A, detectable as a 130-kDa protein in peripheral and as a 122-kDa protein in central nervous system tissues. This heterogeneity could be explained by differences in N-linked glycosylation, because treatments with N-glycosidase F reduced the apparent molecular weights of both receptor variants to the same value of 116,000. As judged by displacement experiments, the two receptor glycoforms did not differ in their binding affinities for natriuretic peptides. Assessments of GC activities did not reveal any difference in ANP-induced generation of the second messenger, cyclic GMP. The examination of GC-A expression in brain during ontogeny revea...