Localization of human follicle-stimulating hormone in the testis

Localization of the follicle-stimulating hormone (FSH) molecule and its receptor (FSHR), as well as the role of FSH in Sertoli cell mitosis and maturation, has been demonstrated by several investigators in human and murine testis by detecting the localization of anti-FSH antibodies or [(131)I]-labeled FSH and by detecting FSH receptor (FSHR) mRNA by in situ hybridization, or FSHR by anti-FSHR antibodies. The presence of FSH in germinal cells is controversial or, in humans, excluded. We have investigated the distribution of the human FSH molecule and its receptor in human and mouse testicular cells under different experimental conditions, at the submicroscopical level, by using a better antigenicity conservative procedure. Thus, the distribution of FSH and of the messenger RNA for its receptor in Sertoli cells has now been clarified. In germinal cells, our observations demonstrate the presence of FSH and the FSHR mRNA: the first on the plasma membrane and in endocytotic vesicles, and the second scattered in the cytoplasm. The cells presenting the higher amount of positivity ranged from spermatogonia to spermatocytes, including round spermatids. Penetration was by the endocytosis via membrane vesicles in which the FSHR is present, whereas its messenger is largely present in the cytoplasm and is responsible for the binding and subsequent internalization of the FSH molecule. As a control, human FSH was administered in vitro to the Y1 mouse cell line, which was stably transfected with cDNA for FSHR and devoid of endogenous FSH. The FSH molecule has been localized by monoclonal antibodies on plasma membranes and vesicles, and the FSHR mRNA was found scattered in the cytoplasm after in situ hybridization. We can now conclude that FSH is present in Sertoli cells and in round germinal cells, both expressing the FSHR. FSH penetrates in a similar way in both kinds of cells via endocytosis, and is therefore subsequently localized in the same membranous organelles.     

The unique exon 10 of the human luteinizing hormone receptor is necessary for expression of the receptor protein at the plasma membrane in the human luteinizing hormone receptor, but deleterious when inserted into the human follicle-stimulating hormone receptor

The LH receptor (LHR) is a member of the family of G protein-coupled seven-times plasma membrane transversing receptors. Its gene consists of 11 exons, the last one encoding the transmembrane and intracellular domains of the receptor. The FSHR, and its gene, resemble structurally those of the LHR, with the exception that the sequences corresponding to exon 10 in LHR are missing in FSHR, which is thus encoded by a total of ten exons. Our recent studies on the marmoset monkey testis LHR cDNA indicated that an 81 bp nucleotide sequence, encoding the complete exon 10 of the LHR gene in other mammalian species, is absent in this species without affecting the LHR function. To study further the role of the exon 10 encoded sequences of the LHR in the gonadotropin receptor function, a deletion of exon 10 from the human LHR (hLHdeltaexon10R), and a chimeric hFSHR with exon 10 from hLHR inserted (hFSHLHexon10R), were constructed in expression vectors. The results presented here demonstrate that 293 cells transfected with the hLHdeltaexon10R display a decrease in the proportion of the receptor binding at the cell surface, compared with cells transfected with wild-type hLHR. However, the cells expressing hLHdeltaexon10R showed similar high affinity binding of [125I]iodo-hCG as those transfected with wild-type hLHR, in either intact cells or their detergent extracts. In addition, cells expressing the hLHdeltaexon10R and wild-type hLHR displayed similar dose-response of cAMP production to hCG stimulation. Cells transfected with chimeric hFSHLHexon10R showed barely detectable [125I]iodo-FSH binding in intact cells compared with those transfected with wild-type hFSHR. The FSH binding detected in cellular detergent extracts displayed 10-fold lower binding activity than wild-type receptors, in spite of similar level of immunoreactive FSHR protein expression in the transfected cells. The hFSHLHexon10R had a modest 5-fold lower binding affinity for FSH as compared with wild-type hFSHR. In conclusion, the present study indicates that the sequences encoding exon 10 of the hLHR are essential for the LHR expression at the plasma membrane, but deleterious for function if inserted into the hFSHR.     

Low levels of follicle-stimulating hormone receptor-activation inhibitors in serum and follicular fluid from normal controls and anovulatory patients with or without polycystic ovary syndrome

In patients with normogonadotropic anovulation, either with or without polycystic ovary syndrome (PCOS), factors interfering with FSH action may be involved in arrested follicle development. The aim of this study is to assess whether factors inhibiting FSH receptor activation are elevated in serum or follicular fluid from anovulatory patients, as compared with regularly cycling women. For this purpose, a Chinese hamster ovary cell line, stably transfected with the human FSH receptor, has been applied. FSH-stimulated cAMP secretion in culture medium was measured in the presence of serum or follicular fluid. Chinese hamster ovary cells were stimulated with a fixed concentration of FSH (3 or 6 mIU/mL) to mimic FSH levels in serum or follicular fluid. Samples were added in concentrations ranging from 3-90% vol/vol to approach protein concentrations occurring in serum or follicular fluid. In the presence of 10% vol/vol serum from regularly cycling women (n = 8), FSH-stimulated cAMP production was inhibited to 42 +/- 2% (mean +/- SEM of 2 experiments, each performed in duplicate) of cAMP production in the absence of serum, whereas a similar cAMP level (up to 38 +/- 4% of the serum-free level) was observed at higher concentrations of serum (30-90% vol/vol). The inhibition of FSH-stimulated cAMP production in the presence of serum samples from normogonadotropic anovulatory patients, without (n = 13) or with (n = 16) PCOS, was similar to controls. Follicular fluid samples (n = 57) obtained during the follicular phase in 25 regularly cycling women and follicular fluid samples (n = 25) from 5 PCOS patients were tested in a slightly modified assay system. In the presence of 10 or 30% (vol/vol) follicular fluid, FSH-stimulated cAMP levels were decreased to 68 +/- 2% and 55 +/- 2% (mean +/- SEM of a single experiment in triplicate) of the cAMP levels in the absence of follicular fluid, respectively. There was no correlation between the degree of cAMP inhibition and follicle size, steroid content (androstenedione or estradiol concentrations), or menstrual cycle phase. Furthermore, no differences in inhibition were found, comparing PCOS follicles with size- and steroid content-matched follicles obtained during the normal follicular phase. It is concluded that inhibition of FSH receptor activation by proteins present in serum or follicular fluid is constant (60 and 40%, respectively) and independent from the developmental stage of the follicle, either during the normal follicular phase or in patients with normogonadotropic anovulation. Inhibition of FSH receptor activation may be of limited significance for normal and arrested follicle development.     

Differential responses of granulosa cells from small and large follicles to follicle stimulating hormone (FSH) during the menstrual cycle and acyclicity: effects of tumour necrosis factor-alpha

This study determined effects of follicle stimulating hormone (FSH) alone and in combination with tumour necrosis factor (TNF), on granulosa cells from small (5-10 mm diameter) and large (>10-25 mm) follicles during follicular and luteal phases of the cycle and during periods of acyclicity. Granulosa cells were collected from ovaries of premenopausal women undergoing oophorectomy. The cells were cultured with human FSH (2 ng/ml) and testosterone (1 microM) in the presence or absence of human TNF-alpha (20 ng/ml). Media were removed at 48 and 96 h after culture and progesterone, oestradiol and cAMP in media were measured by radioimmunoassays. FSH stimulated the accumulation of oestradiol from granulosa cells of small follicles during the follicular and luteal phases but not during acyclicity; and TNF reduced oestradiol accumulation in the presence of FSH. Interestingly, in granulosa cells from small follicles, progesterone and cAMP secretion increased in response to FSH and neither was affected by TNF. Thus, TNF specifically inhibited the conversion of testosterone to oestradiol in granulosa cells from small follicles. FSH stimulated oestradiol production by granulosa cells of large follicles obtained only during the follicular phase of the cycle and TNF inhibited the FSH-induced oestradiol secretion. Granulosa cells obtained from large follicles during the luteal phase and during acyclicity did not accumulate oestradiol in response to FSH. However, FSH increased progesterone and cAMP secretion by granulosa cells obtained from large follicles during the follicular and luteal phases. During the luteal phase alone, TNF in combination with FSH increased progesterone accumulation above that of FSH alone. FSH did not increase progesterone, oestradiol or cAMP secretion by granulosa cells obtained from large follicles during acyclicity. Thus, FSH increases progesterone, oestradiol and cAMP secretion by granulosa cells of small follicles during the follicular and luteal phases and TNF appears to inhibit FSH-induced oestradiol secretion specifically in those cells. In large follicles, FSH-stimulated granulosa cell secretion of oestradiol is limited to the follicular phase and this effect can be inhibited by TNF. In addition, when granulosa cells of large follicles do not increase oestradiol secretion in response to FSH, TNF stimulates progesterone secretion.     

Accidental fatal drug overdoses in New York City: 1990-1992

Testicular tumorigenesis was observed in transgenic mice expressing the 6-kb mouse inhibin alpha-subunit promoter/Simian virus 40 T-antigen (SV40 Tag) fusion gene. The tumors were confined to Leydig cells using immunohistochemistry with anti-Tag antibody, specific binding of biotinylated hCG and histochemistry for 3 beta-hydroxysteroid dehydrogenase. Leydig cell hyperplasia and presence of Tag protein in the testicular interstitial tissue were already evident at 5 and 6.5 days of age, respectively. An immortalized cell line, BLT-1, was established from one testicular tumor. These cells expressed the LH receptor and P450scc mRNAs, and displayed LH-responsive cAMP and progesterone production, and low testosterone production. The cells also specifically bound 125I-labeled recombinant human LH with high affinity (36000 binding sites/cell), and the binding was regulated by 8Br-cAMP and hCG. This gonadal tumor model is valuable for further studies on endocrine functions of Leydig cells and their tumorigenesis in vivo and in vitro.     

Hypothalamus-pituitary-adrenal activity during human sleep: a coordinating role for the limbic hippocampal system

It is well known that deglycosylation of gonadotropins by enzymatic or chemical procedures or by deletion of sites for N-linked glycosylation produces antagonistic analogs which are able to interact strongly with the receptor and to inhibit binding of the wild-type hormone. In the present study, we analyzed the antagonistic properties of a naturally occurring basic follicle-stimulating hormone (FSH) charge isoform obtained after high-resolution chromatofocusing of human anterior pituitary glycoprotein extracts. Coincubation of increasing amounts of this isoform with a highly purified human pituitary FSH preparation or with recombinant human FSH at doses equivalent to their corresponding ED50 for estradiol and tissue-type plasminogen activator (tPA) production, inhibited FSH-induced estrogen production and tPA enzyme activity by cultured rat granulosa cells in a dose-dependent manner. These inhibitory effects were apparently exerted at steps following 3',5'-cyclic adenosine monophosphate (cAMP) formation and did not involve activation of the protein kinase C pathway since: (a) at low doses, this basic FSH isoform moderately increased FSH-induced cAMP production by cultured rat granulosa cells; (b) coincubation of the antagonist isoform with dibutyryl cAMP completely inhibited the effects of this cAMP analog on estrogen and tPA production; (c) the isoform was able to stimulate production of cAMP in a human fetal cell line expressing the recombinant human FSH receptor, and (d) the inhibitory effects of the isoform were not affected by staurosporine, a protein kinase C inhibitor. The effects of this isoform upon dibutyryl cAMP-induced estrogen and tPA production were blocked by the addition of a highly specific antibody directed against human FSH, further demonstrating that the antagonistic effects observed were due to FSH-like molecules. In contrast to the inhibitory effects exhibited by this basic FSH isoform, a more acidic FSH charge variant consistently acted as an agonist of pituitary and recombinant FSH on both estrogen production and induction of tPA enzyme activity. These results indicate that the anterior pituitary gland normally produces FSH isoforms which act as either agonists or antagonists of FSH at the target cell level.     

Bioassays of gonadotropins based on cloned receptors

Because of the microheterogeneities of gonadotropins, immunoreactive measurements of gonadotropins do not necessarily reflect their bioactivity. Follicle-stimulating hormone (FSH) bioassays have relied on measurement of aromatase activity in primary cultures of immature rat Sertoli cells or rat granulosa cells (GAB assay). Luteinizing hormone (LH) bioassays have relied on measurement of androgen production in primary cultures of rat interstitial testicular cells (RICT) or mouse Leydig cells. Those bioassays are cumbersome because they rely on primary culture and on indirect measurement of estradiol or testosterone by RIAs. The cloning of the cDNAs of FSH and LH receptors has allowed the establishment of cell lines expressing human receptors. The cotransfection of the recombinant gonadotropin receptor with a cAMP reporter gene allows a nonisotopic measurement of gonadotropin bioactivity. Furthermore, patient serum can be tested directly without prior extraction. We and other groups have developed a CHO cell line expressing the human FSH receptor and a luciferase reporter gene (CHO-FSHR). The CHO-FSHR assays is specific for FSH and free of serum interference up to a final concentration of 20%. The clinical sensitivity is 3 IU/l, the interCV 16%, the intraCV 8%. Studies were performed in normal women (n = 11) during the menstrual cycle using the CHO-FSHR cells. The ratio of bioactive to immunoactive FSH (B/I) equals 1.1 +/- 0.04 across the follicular and early luteal phase. During the mid to late luteal phase the mean B/I rises significantly to 1.65 +/- 0.07 (P < 0.001). Gonadotropin bioassays based on cloned receptors have been used to search for immunoglobulins, directed against the FSH or the LH receptors in premature ovarian failure patients. No blocking antibodies were found among the 38 women studied. A recent study of FSH bioactivity in patients with FSH secreting pituitary adenomas shows increased values of the B/I ratio. In summary, cell lines expressing the LH and the FSH human receptors are now available. Those homologous systems enable clinicians to study potential forms of mutated FSH or antibodies directed against gonadotropin receptors. Furthermore, bioassays based on cloned receptors are interesting tools to test anti-LH or anti-FSH molecules mainly in contraceptive research.     

A role for neurotransmitters in early follicular development: induction of functional follicle-stimulating hormone receptors in newly formed follicles of the rat ovary

The initiation of follicular growth in the mammalian ovary is a gonadotropin-independent phenomenon. Although some of the intraovarian signaling molecules that control the later phases of this process have been recently identified, the factors involved in the acquisition of gonadotropin receptors by early growing follicles have not been fully defined. In the rat, development of the ovarian innervation precedes the onset of folliculogenesis and occurs before follicles acquire responsiveness to gonadotropins. Because vasoactive intestinal polypeptide (VIP) and norepinephrine (NE), two of the neurotransmitters contained in ovarian nerves, are present in the ovary before the gland becomes responsive to gonadotropins, we sought to determine if VIP and/or NE are able to act on early follicles to facilitate the process of molecular differentiation that leads to gonadotropin dependency. In vitro exposure of 2-day-old rat ovaries to isoproterenol (ISO), a beta-adrenoreceptor agonist, or VIP, a neurotransmitter contained in both sympathetic and sensory nerves, increased the steady state levels of the messenger RNAs encoding cytochrome P-450 aromatase (P-450arom) and FSH receptors (FSHR) within 8 h of treatment. A similar effect was observed following forskolin-induced activation of cAMP formation. In situ hybridization experiments revealed that both the P-450arom and FSHR hybridization signals were localized to follicles. The increase in FSHR messenger RNA was accompanied by formation of functional receptor molecules, as demonstrated by the ability of FSH to stimulate cAMP formation in ovaries preexposed to either ISO or VIP, but not in untreated ovaries. The stimulatory effect of ISO and VIP on the formation of FSHR coupled to the cAMP generating system was not reproduced by phenylephrine, an alpha-adrenergic agonist, or secretin, a member of the VIP family not recognized by ovarian VIP receptors. Treatment of VIP-primed ovaries with FSH resulted in follicular growth, demonstrating that exposure of the gland to the neurotransmitter led to the formation of a functional complement of FSH receptors. These results suggest that ovarian nerves, acting via neurotransmitters coupled to the cAMP generating system, contribute to the differentiation process by which newly formed primary follicles acquire FSH receptors and responsiveness to FSH. Follicles that begin to grow in more densely innervated ovarian regions, may have a selective advantage over those not exposed to neurotransmitter-activated, cAMP-dependent signals and, thus, may become more rapidly subjected to gonadotropin control.     

Direct effects of estradiol and tamoxifen on gene expressions of inhibit alpha- and beta A-subunits in rat granulosa cells in vitro

To analyze direct effects of estrogen on gene expressions of inhibin subunits in vitro, the mRNA levels of inhibin alpha- and beta A-subunits were measured in rat granulosa cells cultured with FSH or estradiol. Through the culture process of the granulosa cells with serum-free medium, both the alpha- and beta A subunit mRNAs decreased, and were partially increased again by adding FSH to the culture medium. To examine whether these FSH effects are mediated via estrogen production, estradiol or tamoxifen was added to the cultured granulosa cells. After 36-h culture with estradiol, the inhibin alpha-subunit decreased but the inhibin beta A-subunit increased, in a dose-responsive manner. Tamoxifen showed completely opposite effects to estradiol, and a combination of estradiol and tamoxifen resulted in similar levels of inhibin-alpha and -beta A mRNAs to the control. These results indicate that estrogen would by a certain pathway affect gene expressions of the inhibin subunits in the rat granulosa cells, and may regulate the production of inhibin and activin through the paracrine system.     

A synthetic peptide corresponding to the third cytoplasmic loop (residues 533 to 555) of the testicular follicle-stimulating hormone receptor affects signal transduction in rat testis membranes and in intact cultured rat Sertoli cells

Involvement of the third cytoplasmic (3i) loop (residues 533 to 555) of the rat testicular FSH receptor in the mechanism of FSH signal transduction was examined using light membranes prepared from immature rat testes, monolayer cultures of rat Sertoli cells, and a synthetic peptide strategy. This region of the FSH receptor is structurally related to G protein-activator regions identified in other G protein-coupled receptors. FSHR-(533-555) peptide amide stimulated guanine nucleotide exchange in rat testis light membranes, presumably via its interaction with membrane-associated G protein. The peptide failed to inhibit FSH binding to testis membrane receptors, indicating that the nucleotide exchange effect was not a result of peptide interaction with receptor. When incubated with cultured Sertoli cells from immature rat testes, FSHR-(533-555) peptide amide consistently and significantly inhibited FSH stimulation of cAMP and estradiol biosynthesis, but failed to inhibit forskolin stimulation of each. The peptide effect, therefore, was not due to a direct interaction with adenylyl cyclase. Since FSHR-(533-555) peptide amide did not inhibit FSH binding to membrane receptor, these results imply entry of the peptide into the Sertoli cell, possibly by vesicular internalization or diffusion. Indeed, the inhibitory effects of FSHR-(533-555) peptide amide on FSH-stimulated estradiol biosynthesis were prevented by pretreating Sertoli cells with phenylarsine oxide, an inhibitor of FSH receptor internalization. FSHR-(533-555) was without effect on basal levels of cAMP and estradiol biosynthesis, indicating absence of toxicity at the concentrations tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential regulation of leucine-rich primary response gene 1 (LRPR1) mRNA expression in rat testis and ovary

In immature rat Sertoli cells, leucine-rich primary response gene 1 (LRPR1) represents a follicle stimulating hormone (FSH)-responsive gene; the function of the encoded protein is not yet known. LRPR1 mRNA expression is up-regulated very rapidly and specifically by FSH, both in cultured Sertoli cells and in vivo in regulation in more detail, in testis and ovary of fetal, immature, and adult rats. In addition, we have studied the expression of FSH receptor (FSHR) mRNA in relation to LRPR1 mRNA expression. In rat testis, LRPR1 mRNA and FSHR mRNA followed a similar expression pattern, during postnatal development and also at different stages of the spermatogenic cycle in the adult rat. Furthermore, after short-term challenge of the FSH signal transduction pathway in intact immature rats by injection with a relatively high dose of FSH, an inverse relationship between LRPR1 mRNA (up-regulation) and FSHR mRNA expression (down-regulation) was observed. Similar studies in the ovary provided completely different results. LRPR1 mRNA in the postnatal ovary is present well before expression of FSHR mRNA can be first detected. In addition, incubation of ovaries of immature rats with FSH or dibutyryl cyclic AMP (dbcAMP) did not result in up-regulation of LRPR1 mRNA expression. During fetal development, the LRPR1 mRNA expression pattern involved many more tissues, in contrast to the relatively tissue-specific expression of LRPR1 mRNA in gonads of 21 day old and adult rats. Moreover, LRPR1 mRNA expression could be detected as early as 12.5 days post-coitum, whereas FSHR mRNA is absent at this stage of fetal development. We concluded that the pronounced regulation of LRPR1 by FSH observed in the immature rat testis does not occur in the ovary. Furthermore, in the ovary LRPR1 mRNA expression does not appear to be dependent on FSH action. Finally, the LRPR1 gene product may play a general role during fetal development.     

In vitro and in vivo characterisation of glial cells immortalised with a temperature sensitive SV40 T antigen-containing retrovirus

An oncogene-carrying replication-defective retrovirus was used to establish immortalised lines of murine glial cells. Primary cultures of early postnatal cerebellar cells were infected with a retrovirus based on the Murine Moloney Leukemia Virus containing a temperature-sensitive mutant of the Simian Virus 40 large T antigen (SV40 T) oncogene and a gene coding for resistance to the antibiotic G418. Infected cells were selected in G418 and after several in vitro passages cells expressing the O4 antigen were established as a cell line. At a later time point O4-positive single-cell clones were established. Two different types of clones were obtained: 1) "plastic" clones consisting of cells which initially had a morphological and antigenic phenotype of young glial precursor cells but which gradually lost these features, and 2) "stable" cell clones including a clone with the immunological and electrophysiological characteristics of Schwann cells. Culture of the latter cells in the presence of 1 mM dibutyryl cyclic adenosine monophosphate for a period of at least 10 days induced a change in shape and a shift in antigen expression towards a more "differentiated" maturation stage. When the SV40 T O4-positive immortalised cell line isolated on the cell sorter was transplanted into demyelinated lesions in adult rats, cells were observed ensheathing axons and forming limited amounts of PNS-type myelin. Glial cells immortalised with a temperature-sensitive mutant of the SV40 T oncogene thus retain many physiological properties of their primary culture counterparts and can be induced to undergo limited differentiation in vitro and in vivo. These cell lines, which represent immature CNS glia or Schwann cells, are providing useful tools for investigating the role of cell surface antigens involved in neuron-glial interactions.     

A novel phenotype related to partial loss of function mutations of the follicle stimulating hormone receptor

A single natural loss of function mutation of the follicle stimulating hormone receptor (FSHR) has been described to date. Present in the Finnish population it markedly impairs receptor function, blocking follicle development at the primary stage and presenting as primary amenorrhea with atrophic ovaries. When Western European women with this phenotype were examined for FSHR mutations the result was negative, suggesting that other etiologies corresponding to this clinical pattern are markedly more frequent. We now describe a novel phenotype related to mutations provoking a partial loss of function of the FSHR. A woman with secondary amenorrhea had very high plasma gonadotropin concentrations (especially FSH), contrasting with normal sized ovaries and antral follicles up to 5 mm at ultrasonography. Histological and immunohistochemical examination of the ovaries showed normal follicular development up to the small antral stage and a disruption at further stages. The patient was found to carry compound heterozygotic mutations of the FSHR gene: Ile160Thr and Arg573Cys substitutions located, respectively, in the extracellular domain and in the third intracellular loop of the receptor. The mutated receptors, when expressed in COS-7 cells, showed partial functional impairment, consistent with the clinical and histological observations: the first mutation impaired cell surface expression and the second altered signal transduction of the receptor. This observation suggests that a limited FSH effect is sufficient to promote follicular growth up to the small antral stage. Further development necessitates strong FSH stimulation. The contrast between very high FSH levels and normal sized ovaries with antral follicles may thus be characteristic of such patients.     

New insights into the role of follicle-stimulating hormone in reproduction

Follicle-stimulating hormone (FSH) has been considered essential for normal ovarian follicular development and spermatogenesis. Although this concept remains valid, the recent reports of defects in genes encoding FSH and its receptor (FSHR) have altered the concepts, particularly with regard to the role of FSH in spermatogenesis. Complete FSH resistance caused by a mutation of the FSHR gene has demonstrated that normal FSH action is an absolute requirement for female fertility, but spermatogenesis and male fertility are possible without FSH action. Thus, while normal ovarian function is critically dependent on normal FSH action, male fertility is relatively independent of this hormone. The finding contradicts the potential of inhibition of FSH secretion or action as a means of male contraception.     

Thyroid hormone and follicle-stimulating hormone regulate Müllerian-inhibiting substance messenger ribonucleic acid expression in cultured neonatal rat Sertoli cells

Thyroid hormone is a major regulator of Sertoli cell development, and the present study sought to determine the role of T3 in Müllerian-inhibiting substance (MIS) messenger RNA (mRNA) expression. MIS, a Sertoli cell secretory protein that induces Müllerian duct regression and also may be critical for germ and Leydig cell development, is maximal perinatally, then decreases as Sertoli cells mature. The fall in MIS mRNA expression is delayed by hypothyroidism in vivo, indicating that T3 could regulate MIS mRNA. However, understanding of the hormonal regulation of MIS has been limited due partly to the lack of a primary Sertoli cell culture system in which sustained expression of MIS or its mRNA can be obtained. We have developed a Sertoli cell culture system for examining hormonal regulation of MIS mRNA. We then tested the effects of T3 and/or FSH treatment on MIS mRNA levels in this new system. Initial studies indicated that MIS mRNA production by 5-day-old rat Sertoli cells was minimal in vitro. Therefore, Sertoli cells from 2-day-old rats were cultured for 2 or 4 days. After 2 days in vitro, steady state MIS mRNA levels were decreased to 36% of the levels seen in freshly isolated Sertoli cells from 2-day-old rats. However, by day 4 of culture, steady state MIS mRNA production had recovered to 67% of that seen in freshly isolated 2-day-old Sertoli cells, which closely paralleled the decrease seen in MIS production in vivo from days 2-6. MIS mRNA levels were decreased 53%, 64%, and 86% in cultures treated with 0.01, 0.1, and 1.0 nM T3 (P < 0.05), respectively. This decrease in Sertoli cell MIS mRNA did not reflect a nonspecific effect on cell viability and/or activity, as shown by a dose-responsive increase in inhibin-alpha mRNA in these same cultures. FSH (2.5-100 ng/ml) also produced a dose-responsive decrease in MIS mRNA levels, and FSH and T3 together had an additive inhibitory effect on MIS mRNA levels, indicating that these hormones may act through distinct mechanisms. In summary, this is the first primary culture system in which sustained MIS mRNA production can be demonstrated, and it should prove useful for understanding the regulation of MIS in developing Sertoli cells. In addition, T3 and FSH are major regulators of the postnatal decrease in MIS production by the rat Sertoli cell, and these hormones may act through separate pathways.     

Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier

The objective of this study was to generate an immortal cell line representative of specialized human brain microvascular endothelia forming the blood-brain barrier (BBB) in vivo. Human capillary and microvascular endothelial cells (HCEC) were transfected with the plasmid pSV3-neo coding for the SV40 large T antigen and the neomycin gene. The neomycin-resistant transfected cells overcame proliferative senescence, and after a 6-8 wk period of crisis produced immortalization-competent cell colonies. Single-cell clones of near-diploid genotype were isolated from these colonies, propagated, and characterized. Immortalized HCEC (SV-HCEC) exhibited accelerated proliferation rates, but remained serum and anchorage dependent and retained the characteristic cobblestone morphology at confluence. SV-HCEC displayed a stable nuclear expression of SV40 large T antigen, lacked the invasiveness of transformed cells, and maintained major phenotypic properties of early passage control cells including expression of factor VIII-related antigen, uptake of acetylated low-density lipoprotein, binding of fluorescently labeled lectins, expression of transferrin receptor and transferrin receptor-mediated endocytosis, and high activities of the BBB-specific enzymes alkaline phosphatase and gamma-glutamyl transpeptidase. The diffusion of radiolabeled sucrose across SV-HCEC monolayers was fivefold lower than that observed with human lung microvascular endothelial cells. Furthermore, media conditioned by fetal human astrocytes increased the transendothelial electrical resistance of SV-HCEC monolayers by 2.5-fold. Therefore, this newly established human cell line expressing the specialized phenotype of BBB endothelium may serve as a readily available in vitro model for studying the properties of the human BBB.     

Efficient gene transfer into primary and immortalized human fetal glial cells using adeno-associated virus vectors: establishment of a glial cell line with a functional CD4 receptor

Adeno associated virus (AAV) is a non-pathogenic dependent parvovirus with a broad host range, capable of high levels of transduction and stable integration into the host cell genome. We have investigated the potential for using AAV as a vector for gene transfer into glial cells of the human fetal nervous system. Recombinant AAV vectors expression either the reporter gene beta-galactosidase or a human CD4 receptor were able to transduce both primary glial cells of the human fetal nervous system and an SV40 immortalized human fetal glial cell line (SVG). No difference in transduction efficiency was observed between the primary cells and the cell line which in both cases was as high as 95%. Stable transfectants of the glial cell line expressing the CD4 receptor were selected. An SVG/CD4 expressing line was then established. The presence of the CD4 receptor was confirmed by immunohistochemistry, Westerm immuno-blotting and flow cytometric analysis. The CD4 receptor was shown to be functional by infection of the SVG/CD4 cell line with the human immunodeficiency virus (HIV). Upon infection, the SVG/CD4 cells produced 20-fold higher levels of the HIV intracellular core antigen P24 than the CD4 negative parental cells and in addition formed syncytia. The use of AAV vectors should prove useful in biological investigations of human glial cells and offers promise as a means of ex vivo and in vivo gene delivery.