Biomedical subjects

M L Dufau

Publications and source records attributed to M L Dufau.

At least 37 records · Page 2Linked to original sources

PubMed1996

The genomic structure of the rat corticotropin releasing factor receptor. A member of the class II G protein-coupled receptors.

Isolation and structural characterization of the rat corticotropin releasing factor receptor (CRFR) gene was performed to determine the exon/intron organization of the coding region and the potential for splice variants. The CRFR gene contains 13 exons and 12 introns, and the positions of the exon/intron junctions are similar to those of other Class II G protein-coupled receptor genes including the parathyroid hormone and glucagon receptors. The promoter resides within 593 base pairs of the initiation codon and the major transcriptional start site at nucleotide -238. This domain does not possess a TATA box but contains multiple Sp1 and AP-2 sites upstream and downstream of the major transcriptional start site. Intron junctions were identified in the extracellular, transmembrane (TM), and cytoplasmic (C) domains of the CRFR, giving the potential for differential signal transduction by splice variants. CRFR cDNAs derived from rat Leydig cell mRNA included the pituitary Form A, which spans exons 1-13, and two splice variants with deletion of exon 3 or exons 7, 11, and 12. An evolutionary link between the intronless TM/C module of the glycoprotein hormone receptors and the intron-containing TM/C module of the CRFR is suggested by the common position of the luteinizing hormone receptor Form D alternate acceptor splice site and the CRFR intron 12.

Alternative Splicing↗

PubMed1996

Multiple and tissue-specific promoter control of gonadal and non-gonadal prolactin receptor gene expression.

Prolactin receptors (PRLRs) are widely expressed, and multiple mRNA transcripts encoding PRLRs are present in prolactin target tissues. The molecular basis for the control of the PRLR gene expression is currently unknown. Analyses of the 5'-untranslated regions of PRLR mRNAs expressed in gonadal and non-gonadal tissues and their genomic organization revealed three alternative first exons designated as E11, E12, and E13. Each of these exons is alternatively spliced to a common noncoding exon (exon 2, nucleotides -115 to -56) that precedes the third exon containing the translation initiation codon. Alternative utilization of exons E11, E12, and E13, as well as alternative splicing of exon 2, generates multiple 5'-untranslated regions in PRLR transcripts. These alternative first exons (E11, E12, and E13) were found to be utilized in a tissue-specific manner in vivo. E11 is predominantly expressed in the ovary, E12 is specifically expressed in the liver, and E13 is expressed as a predominant form in the Leydig cell and as a minor form in the ovary and liver. Genomic 5'-flanking regions containing the three putative PRLR gene promoters (PI, PII, and PIII) that initiate the transcription of E11, E12, and E13, respectively, were identified. E11 was found to initiate from a single site at -549, E12 from multiple sites at -405, -461, and -506, and E13 from two major sites at -340 and -351. These findings indicate that multiple promoters control transcription of the PRLR gene and provide a molecular basis for the differential regulation of PRLR expression in diverse tissues.

Alternative Splicing↗

PubMed1996

Requirement of cysteine residues in exons 1-6 of the extracellular domain of the luteinizing hormone receptor for gonadotropin binding.

The functional importance of cysteine residues in the extracellular domain and the extracellular loops (EL1 and EL2) to hormone binding of the rat luteinizing hormone receptor (LHR) was investigated. For this purpose, cysteines in the seven-transmembrane holoreceptor (Form A) and its hormone-binding splice variant (Form B) were replaced by serine residues, and mutant receptors were expressed in COS1 and/or insect cells. Within the extracellular domain, individual replacement of all four cysteines from Exon 1 abolished hormone binding activity, and replacement of Cys-109 and Cys-134 from exons 5 and 6 caused a 75% decrease in both cell surface and total cellular solubilized LHR hormone binding activity. Mutations of Cys-257 and -258 (Exon 9), Cys-321 and -331, and Cys-417 and -492 of EL1 and EL2, respectively (Exon 11), showed no surface hormone binding activity on intact cells, but exhibited wild type levels of total hormone binding activity when recovered from detergent-solubilized cellular extracts. This finding indicated that expression of high affinity LHR binding activity at the cell surface is independent of the acquisition of the high affinity binding conformation. Other cysteine residues, including Cys-282 (exon 10), and Cys-314 (exon 11) were not essential for hormone binding activity or plasma membrane insertion. This study demonstrates that the functional hormone binding domain utilizes all cysteines N-terminal to exon 7 and localizes the binding site to this N-terminal region of the extracellular domain.

Alternative Splicing↗

PubMed1995

Functional glycosylation sites of the rat luteinizing hormone receptor required for ligand binding.

The contribution of N-linked glycosylation to the ligand binding activity of the rat luteinizing hormone receptor (LHR) was studied in wild-type and mutant LHR expressed in mammalian (COS1) cells and overexpressed in insect (Sf9) cells. The binding affinities of the holoreceptor and its truncated splice variant (form B) lacking the transmembrane domain were equivalent in both cell lines. Tunicamycin-treated transfected Sf9 cells expressed a carbohydrate-free LH receptor that lacked hormone binding activity. Functional carbohydrate chains essential for binding activity were localized to glycosylation sites at Asn-173 and Asn-152. Glycosidase treatment of the double mutant N173Q/N152Q revealed the presence of at least one additional carbohydrate chain at Asn-269, Asn-277, or Asn-291 that does not contribute to hormone binding. Asn-77 was not glycosylated, but its mutation to Gln reduced hormone binding. LHR expressed in insect cells contained only high mannose carbohydrate chains, and those located at Asn-173 and Asn-152 were sufficient for high-affinity hormone binding. Enzymatic cleavage of glycosyl chains indicated that only the proximal N-acetylglucosamine residue, which is common to high mannose and complex carbohydrate forms, is necessary for acquisition of the high affinity conformation of the receptor. The carbohydrate chains of the LHR appear to be involved in intramolecular folding of the nascent receptor rather than in its interaction with the hormone.

Alternative Splicing↗

PubMed1995

Characterization of diverse functional elements in the upstream Sp1 domain of the rat luteinizing hormone receptor gene promoter.

Transcription of the luteinizing hormone receptor gene is dependent on Sp1-induced promoter activation from two Sp1 binding domains (Sp1(2), and Sp1(4)) within the 173-base pair promoter. Of the two Sp1 binding domains, the canonical GC box (GGGCGG) was determined by mutation to be the binding element for only the Sp1(2) domain. The Sp1 binding element within the Sp1(4) domain was identified by mutation and immunological/competition studies as the 5'-GGG GTG GGG that conforms to a Zif-268 like three zinc finger binding domain, rather than the canonical 3' Sp1(4) GC box (GGGCGG). The guanines in the third trinucleotide (GGG GTG GGG) were not required for Sp1 binding, although they increased binding affinity. Non-Sp1 protein(s) bind the 3' Sp1(4) GC box, and by themselves exhibit transcriptional activity. Tissue specific differences were localized to this non-Sp1 binding domain, which functionally substituted for the downstream activating M1 regulatory domain in non-expressing but not in expressing cells. Mutations of both non-Sp1 and M1 domains were required for inhibition of promoter activity in constructs that retained the Sp1 binding elements in non-expressing cells, indicating that together these domains may play a role in regulation of luteinizing hormone receptor gene expression.

Animals↗

PubMed1995

The rat 17 alpha-hydroxylase-17,20-desmolase (CYP17) active site: computerized homology modeling and site directed mutagenesis.

A homology model of the rat 17 alpha-hydroxylase-17,20 desmolase (CYP17) steroid binding domain was derived from the alpha/beta F supersecondary structural element of the 3 alpha/20 beta hydroxysteroid dehydrogenase (HSD) of Streptomyces hydrogenans that constitutes a major segment of the C19 steroid binding cavity. A CYP17 arginine-rich domain, including Arg346, Arg361 and Arg363, that has previously been shown to be important to CYP17 catalytic activity, is conserved in this HSD structural element between two HSD domains known to be important to C19 steroid binding. These two HSD motifs, in addition to a C-terminal domain at the apex of the steroid binding cavity, are also present in similar though not identical forms in the rat CYP17 sequence. The model was evaluated in terms of both hydroxylase/lyase activity and stability of CYP17 mutant proteins (Tyr334Phe, Phe343Ile, Arg357Ala, Arg361Ala, Asp345Ala), and further tested with mutagenesis of Glu353, Glu358, and Tyr431. Those amino acids located at folding junctions in the model steroid binding domain (Glu358, Arg361, and Tyr431) are each individually required to prevent degradation of the nascent protein, as well as for basic hydroxylase/lyase activity. Genomic analysis of the rat CYP17 gene reveals that this domain is contained in exon 6, and a correlation exists between the length of exon 6 and the boundaries of the HSD supersecondary element. These studies demonstrate that exon 6 of the rat CYP17 is essential for CYP17 activity, and may be structurally related to the NAD-linked prokaryote alpha/beta F supersecondary element.

Aldehyde-Lyases↗

PubMed1995

Structure and regulation of the luteinizing hormone receptor gene.

Studies of the mechanisms controlling the expression of the rat luteinizing hormone receptor gene were pursued by characterization of the gene structure and identification of regulatory protein binding domains in the 5'-non-coding region of the gene and of 3' non-coding functional domains responsible for generation of the major mRNA forms. The coding region of the rat LHR gene contains 10 introns and 11 exons, of which the first 10 exons comprise the hormone binding extracellular domain and exon 11, the seven transmembrane/G protein coupling module. Several alternative spliced variants of the LHR were identified that conform to deletions of complete and/or partial exons. Within the 6.2 kb of the 3'-non-coding region, two functional LHR pA domains (H1) and (H2) produce two sets of major mRNA transcripts, each coding for both holoreceptor and the form B splice variant. The H1 pA domain is unique to LHR and may represent a recombinant insertion domain. The functional efficiency of each pA domain is related to the specific pA signals, distal downstream elements, and tissue-specific factors. A TATA-less promoter region is present within the 173 bp 5'flanking region of the gene, with Initiator (Inr) elements at transcriptional start sites. Transcription is dependent on the binding of the Sp1 protein at two Sp1 domains that each contribute equally to transcript initiation. Promoter activity is regulated by at least three additional DNA domains, R (-1266 to -1307 bp), C-box (-42 to -73 bp) and M1 (-24 to -42 bp) that bind multiple trans-factors in a tissue-specific manner. Basal promoter activity is enhanced by a functional M1 domain in LHR-expressing mouse Leydig tumor cells (MLTC) but not in non-expressing CHO cells. C-box binding factors either inhibit promoter activity or block inhibition through overlapping but not identical DNA binding domains that carry AP-2 and NF-1 elements. Removal of the AP-2 element within the C-box results in MLTC-specific transcriptional activation that may involve an MTLC M1/C-box interaction. In addition, competition for C-box factors by an upstream regulatory element (R) that is only inhibitory in CHO cells, indicates that both C-box binding factors compete for this upstream (R) domain in a tissue-specific manner. Competition between the inhibitory and neutral DNA binding factors within both upstream (R) and promoter domains (C-box) could provide a mechanism for the control of LH receptor gene expression in gonadal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Growth hormone-releasing hormone in testicular interstitial and germ cells: potential paracrine modulation of follicle-stimulating hormone action on Sertoli cell function.

GH-releasing hormone (GHRH) is present in the interstitial and germ cells of the rat testis. In previous studies we found that GHRH is secreted from rat adult Leydig cells, in which it stimulates basal and LH-induced cAMP formation and steroidogenesis. In other studies cAMP production in Sertoli cells was found to be stimulated by GHRH. In the present report, we describe a potential paracrine action of GHRH in the Sertoli cell, with stimulation of cAMP formation in cultured adult and pubertal Sertoli cells. GHRH increased FSH-stimulated cAMP production in adult and pubertal cultures in a time-dependent manner. GHRH stimulation of basal and FSH-induced extracellular cAMP formation was more prominent in pubertal than in adult cultures. Immunocytochemical studies demonstrated the presence of GHRH-like immunoreactivity in rat interstitial cells from day 4 to adult life and in the acrosomal region of early and intermediate spermatids at stages III-VI of the seminiferous epithelium cycle. Immunoreactive GHRH was not observed in late spermatids and mature sperm or in Sertoli cells at any age. These results indicate that GHRH acts synergistically with FSH to promote cAMP production in Sertoli cells in culture. Testicular GHRH of Leydig and germ cell origin may be an important paracrine regulator of Sertoli cell function. Alternatively, GHRH present in germ cells may exert stage-specific intracrine functions.

Aging↗

PubMed1995

Calcium signaling in single rat Leydig cells.

The steroidogenic activity of the Leydig cell is regulated by glycoprotein and peptide hormones with the potential to activate both adenylate cyclase and phospholipase C. Although the control of androgen production by LH is clearly mediated by cAMP, the extent to which Ca(2+)-mobilizing stimuli control Leydig cell function is less well defined. The basal level of intracellular calcium ([Ca2+]i) in adult rat Leydig cells was 70-160 nM and was unaffected by high K+ or the dihydropyridine calcium channel agonist, Bay K 8644. These findings are consistent with the absence of voltage-sensitive calcium channels in the Leydig cell. In addition, no increase in [Ca2+]i was observed in cells treated with LH, CRF, and serotonin. However, both GnRH and endothelin-1 (ET-1) induced rapid and transient elevations of [Ca2+]i that were not associated with a sustained plateau phase and were unaffected by removal of Ca2+ from the incubation medium. The amplitude of the [Ca2+]i response was not altered by increasing concentrations of GnRH and ET-1, but the number of responsive cells increased progressively to a maximum of about 30% of the Leydig cell population. The calcium-mobilizing actions of GnRH and ET-1 were abolished by the GnRH and ETA receptor antagonists, [Dp-Glu1,D-Phe2,D- Trp3,6]GnRH and BQ-123, respectively. The majority of the cells expressed solely GnRH or ETA receptors, and about 10% expressed both receptors. GnRH-induced Ca2+ responses were observed almost exclusively in medium-sized Leydig cells, whereas ET-induced responses were most frequent in large Leydig cells. These data demonstrate that single Leydig cells expressing GnRH and ETA receptors exhibit monophasic [Ca2+]i responses that are activated in an all-or-none fashion. Such transient Ca2+ signaling may trigger short term cellular responses or could modulate the actions of gonadotropins acting through the cAMP signaling pathway.

Animals↗

PubMed1994

Transcriptional protein binding domains governing basal expression of the rat luteinizing hormone receptor gene.

The functional importance of specific protein binding domains on transcription within the GC-rich 173-base pair promoter of the luteinizing hormone receptor gene was studied by mutagenesis and gel retardation analysis. Transcription was dependent on the presence of two Sp1 elements in the promoter domain of transfected expressing mouse Leydig tumor cells (mLTC) and nonexpressing Chinese hamster ovary cells. Mutation of two protein binding domains located downstream of the Sp1 elements (M1 and C-box) revealed tissue-specific regulation of promoter activity by each domain. Also, gel retardation studies indicated the presence of multiple trans factors that bind to the C-box and M1 domains. Removal of the AP-2 element from the C-box resulted in mLTC-specific transcriptional activation that may involve an M1/C-box interaction. In addition, competition by overlapping NF-1 and AP-2 elements was demonstrable in both the C-box and upstream R domain for separate trans factors that exhibit neutral or inhibitory functions, respectively. Competition between the inhibitory and neutral DNA binding factors within both upstream and promoter domains may be responsible for a mechanism that controls the on/off state of luteinizing hormone receptor gene expression in gonadal cells. These studies reveal a complex pattern of transcriptional regulation that may reflect targeted mechanisms for the control of luteinizing hormone receptor gene expression.

Animals↗

PubMed1994

Sequence of the 3'-noncoding region of the luteinizing hormone receptor gene and identification of two polyadenylation domains that generate the major mRNA forms.

We present 6.2 kb of the 3'-noncoding region sequence of the rat luteinizing hormone receptor (LHR) gene and identification of two functional polyadenylation (pA) domains, H1 (nt 2368-2491) and H2 (nt 5579-5768) responsible for 3'-end processing of the 2.6/2.3 kb and the 5.8 kb LHR mRNA, respectively. Two identical copies of pA elements AAUAUA in H1 and of AAUAAA in H2 account for micro-heterogeneous poly(A) addition at each of the two pA regions. Both LH holoreceptor and major splice variant form B (lacking the first 266 bp of exon 11) are identified in H1-terminated (2.6 kb and 2.3 kb) and H2-terminated (5.8 kb) mRNA transcripts. A rodent repetitive DNA LINE R domain 3' of H1 within the major 5.8 kb species and a B2 element downstream of H2 were identified. Alignment of the 3'-noncoding region of LHR with TSH, FSH and beta 2-adrenergic receptors indicate that H1 pA signal is unique to the LHR and may represent an insertion domain.

Alternative Splicing↗

PubMed1994

Differential responses of biologically active luteinizing hormone secretion in older versus young men to interruption of androgen negative feedback.

To investigate the responsiveness of the healthy aging male hypothalamo-pituitary-gonadal axis to short term interruption of androgen negative feedback, we administered a selective nonsteroidal competitive antagonist of the androgen receptor, flutamide hydrochloride (250 mg, orally, three times daily for 3.5 days), to five older (aged 63-72 yr) and eight young (aged 21-30 yr) men. Pulsatile bioactive LH release was assessed by the rat interstitial cell testosterone bioassay in plasma sampled at 10-min intervals for 8 h overnight at baseline and after flutamide administration. Pituitary responsiveness was evaluated after two successive i.v. injections of 10 micrograms GnRH. Deconvolution analysis was used to estimate the number, amplitude, duration, and mass of bioactive LH secretory bursts and the half-life of biologically active hormone. At baseline, older men exhibited a significantly lower spontaneous bioactive LH secretory burst frequency than young men, with a median of 5 events/8 h (older) vs. 7.5 bursts/8 h (younger, P < 0.05). In older men, mean 8-h plasma bioactive LH concentrations increased significantly in response to flutamide (P = 0.006), and the 8-h calculated secretion rate of bioactive LH rose concomitantly. These increases were similar to responses in young men. However, during antiandrogen administration, the frequency of bioactive LH secretory bursts failed to rise in older men to the baseline value seen in young men. Moreover, older (but not young) men showed a significant prolongation of the LH secretory burst duration in response to flutamide treatment. On the other hand, the estimated half-life of endogenous bioactive LH increased significantly after flutamide ingestion in young compared to older individuals. After GnRH injections, older and young men secreted similar amounts of LH before flutamide administration, but during flutamide treatment, older men released more biologically active LH after the first GnRH stimulus [older men, 104 +/- 11 IU/L (median, 110); younger men, 44 +/- 8.4 IU/L (median, 40); P < 0.05]. Serum free testosterone concentrations rose significantly during flutamide exposure in both young and older men, but estradiol concentrations increased significantly only in young men. In summary, healthy older men exhibit a reduced (overnight) spontaneous bioactive LH secretory burst frequency. Pharmacological attenuation of androgen-mediated negative feedback increases mean serum free testosterone concentrations and plasma bioactive LH concentrations to a similar degree in older and young individuals, but different mechanisms operate in the two age groups. In older men, flutamide treatment amplifies the mass of bioactive LH secreted per burst by prolonging the LH secretory burst duration, whereas in young men, flutamide administration increases the apparent half-life of biologically active LHG significantly relative to values in older men. We conclude that competitive nonsteroidal blockade of the androgen receptor unmasks qualitatively altered mechanisms of increased bioactive LH release in healthy older men.

Adult↗

PubMed1993

Requirement of phenylalanine 343 for the preferential delta 4-lyase versus delta 5-lyase activity of rat CYP17.

Site-directed mutagenesis of a domain (amino acids 343-348) within the conserved region of rat CYP17 was performed to investigate species-specific differences between rat and human/bovine delta 4-versus delta 5-lyase activity. This domain displays substantial deviations between the rat and human/bovine/pig sequences and includes Arg346, which is known to be essential for delta 4-lyase (Kitamura, M., Buczko, E., and Dufau, M. L. (1991) Mol. Endocrinol. 5, 1373-1380) and delta 5-lyase activities. Analysis of the delta 4-lyase activity of mutant rat CYP17 cDNA expressed in nonsteroidogenic COS-1 cells revealed that substitution of Phe at position 343 in the rat with Ile of the human/bovine sequence resulted in a reduction in delta 4-lyase activity to levels in the range of the delta 5-supported reaction. This Phe343-->Ile mutant CYP17 did not exhibit changes either in delta 5-supported lyase activity or in delta 4- and delta 5-hydroxylase activities. Substitution of Asn344, Ser347, and His348 in rat CYP17 with the corresponding bovine amino acids Ser, Asn, and Arg did not enhance this effect. Thus, the reduced activity of the bovine CYP17 delta 4-lyase reaction can be mimicked in part in the rat polypeptide by the substitution of Phe343 with the bovine counterpart, Ile. Unlike the bovine CYP17-catalyzed reaction, the rat Phe343-->Ile mutant exhibited a low level lyase activity (kcat) that did not discriminate between delta 4- and delta 5-substrates. These results suggest that the presence of Phe343 enhances the delta 4-supported lyase activity possibly through stabilization of a delta 4-specific interaction.

Aldehyde-Lyases↗

PubMed1993

Promoter and regulatory regions of the rat luteinizing hormone receptor gene.

The region of transcriptional activity in the rat luteinizing hormone receptor gene was localized to the 5'-flanking 173-base pair (bp) sequence in transfected mouse Leydig tumor cells and non-expressing Chinese hamster ovarian (CHO) and HeLa cells. Repression of activity in all cell types was induced by at least two domains located between -174 and -990 bp. An additional inhibitory region between -1237 and -2056 bp observed only in CHO and HeLa cells may contribute to the absence of receptor expression in these cells. Analysis of the 173-bp region revealed a promoter domain between -1 and -137 bp containing no TATA or CAAT boxes, but with three SP1 sites and three initiator elements located at transcriptional start sites -14, -19, and -33. Gel retardation studies showed a protein-binding DNA domain about 30 bp upstream of the transcriptional start sites and an adjacent domain containing an SP1 element that are required for maximal activity in all cell types and may play a role in basal transcription. A second promoter domain (-120 to -173 bp) with a protein-binding SP1 element had minor activity in Leydig cells but was prominent in CHO and HeLa cells. Leydig cell-specific DNA-binding protein(s) for each of the promoter domains were detected and may be of importance in transcriptional regulation. The control of gene transcription by differential activation of these promoter domains could be involved in hormone-induced regulation of luteinizing hormone receptor expression in the gonads.

Animals↗

PubMed1993

Corticotropin-releasing factor: an antireproductive hormone of the testis.

Corticotropin-releasing factor (CRF), the key neuropeptide in the stress cascade, has major inhibitory actions on testicular function in addition to its known antireproductive effects at the central level (inhibition of sexual behavior and LH secretion). CRF is secreted by the Leydig cells of the testis and acts through high-affinity receptors at the Leydig cell membrane as a potent negative regulator of LH action, inhibiting gonadotropin-induced cAMP generation and androgen production. CRF is also a primary stimulus of beta-endorphin secretion by the Leydig cells, which in turn exerts paracrine inhibition of FSH action in the tubular compartment of the testis through high-affinity receptors in the Sertoli cells. CRF action in the Leydig cells involves a pertussis toxin-insensitive guanyl nucleotide regulatory unit. In contrast to CRF receptors in the brain, pituitary, and other peripheral tissues, those in the Leydig cell are not coupled to Gs. The inhibitory action of CRF in the Leydig cell is exerted through protein kinase C, at the level of the catalytic subunit of adenylate cyclase. The secretion of CRF by the Leydig cell is stimulated by LH, acting via release of serotonin (5HT) and autocrine activation of 5HT2 receptors. Serotonin acts on 5HT2 receptors in the Leydig cell to stimulate CRF secretion via a pertussis toxin insensitive G-protein and presumably through activation of phosphoinositide hydrolysis. The diversity of the biochemical responses to CRF and 5HT2 receptor activation (i.e., inhibition of adenylate cyclase at the cytoplasmic aspect of the cell membrane vs. stimulation of CRF release from secretion granules) may reflect the stimulation of different protein kinase C isoenzymes. The LH-->5HT-->CRF inhibitory loop serves to continuously buffer the stimulation of androgen production by gonadotropin. 5HT, the immediate stimulus of testicular CRF secretion, is released during stress and is locally increased in the testis in pathological conditions associated with impaired testicular function (i.e., orchitis, varicocele). Also, propranolol, the beta-adrenergic antagonist frequently used in the control of blood pressure in patients with hypertension and often associated with impotence, acts via a serotonergic mechanism to stimulate CRF secretion and causes marked inhibition of LH-induced cAMP production and steroidogenesis in cultured Leydig cells. These basic studies of 5HT and CRF are relevant to the pathogenesis of testicular dysfunction and for the development of antagonist therapies to block CRF production and its local antireproductive effects.

Animals↗

PubMed1993

Steroidal regulation of biologically active luteinizing hormone secretion in men and women.

The biological activity of circulating luteinizing hormone (LH) molecules can be assessed with high sensitivity, specificity, and precision by the in-vitro rat interstitial (Leydig)-cell testosterone (RICT) bioassay. Compared to immunoreactive estimates of the LH content of plasma, the living Leydig-cell bioassay integrates the functional in-vitro potency of all circulating LH isoforms, while simultaneously reflecting the effects of otherwise potentially confounding in-vivo antagonists of LH action on steroidogenesis. Here, we review the pathophysiological regulation by steroid hormones of bioactive LH secretion and clearance in men and women. Our investigations and those in the available literature indicate that: (i) exogenous (non-aromatizable) 5-alpha-dihydrotestosterone infusion suppresses LH bioactivity, whereas blockade of the endogenous androgen receptor with flutamide increases secretion of biopotent LH in men; (ii) endogenous androgen excess due to a masculinizing testosterone-secreting adrenal tumour in a post-menopausal woman reversibly suppressed plasma LH bioactivity markedly; (iii) exogenous oestradiol infusion will reduce whereas the antioestrogen, tamoxifen-HCl, can increase bioactive LH secretion in young men; (iv) the effect of anti-oestrogen is blunted in healthy older men; (v) endogenous hyperoestrogenism due to an adrenal oestrogen-secreting tumour in a middle-aged man with gynaecomastia profoundly but reversibly inhibited bioactive LH secretion; (vi) in post-menopausal women, exogenous oestrogen administration, whether oral (diethylstilbestrol), percutaneous (oestradiol), or intravaginal (oestradiol-impregnated silastic ring), exerts a temporally biphasic effect on basal bioactive LH secretion, i.e. acute suppression (within 24 h), subacute escape (at 5-10 days), and longer-term inhibition (30 days); (vii) during the normal menstrual cycle, bioactive LH secretion is pulsatile and both frequency and amplitude regulated; and (viii) oestrogen exposure can enhance gonadotrophin releasing hormone (GnRH)'s stimulatory action on biologically active LH secretion, resulting in so-called GnRH self-priming. Oestrogen's facilitative effects are achieved by a novel mechanism, in which oestrogen augments LH secretory burst mass and duration. Moreover, GnRH dose-LH secretory response studies show that oestrogen promotes an increase in GnRH efficacy (maximal effects) but not GnRH sensitivity (potency or half-maximally effective doses of GnRH). We conclude that steroid hormones are primary regulators of physiologically pulsatile bioactive LH secretion in healthy men and women. Moreover, steroids exert potent pathological effects on biologically active LH release in conditions of selective hyperandrogenism or hyperoestrogenism due to steroid-secreting adrenal or gonadal tumours.

Androgens↗

PubMed1993

Serotonergic inhibition of rat Leydig cell function by propranolol.

The beta-adrenergic antagonist propranolol binds to serotonin (5HT) receptors (5HT1B > 5HT1A > 5HT2) in brain membranes. We have recently demonstrated that 5HT acts through 5HT2 receptors in rat Leydig cells to release CRF, which, in turn, inhibits hCG-stimulated cAMP production and steroidogenesis. These observations prompted us to study the effects of propranolol on CRF secretion and cAMP and testosterone production in cultured rat Leydig cells. Treatment with (-)propranolol increased CRF release and inhibited basal and hCG-stimulated cAMP and steroidogenesis, with effects evident at 0.1 microM, an IC50 of 6 microM, and reduction of stimulated levels to near basal at 100 microM. These effects of the drug were prevented by pretreatment of cultures with the 5HT2 receptor antagonist ketanserin or a CRF antagonist or antiserum. Furthermore, propranolol increased the level of 5HT in the incubation medium of cultured Leydig cells. The (+)isomer of propranolol had minor effects on these parameters. Increasing concentrations of (-)propranolol displaced the binding of [125I] +/- 1-[2,5-dimethoxy-4-iodophyryl]-2-amino propane hydrochloride (DOI), a selective 5HT2 ligand, to Leydig cell membranes (IC50, 0.2 microM), and (+)propanolol showed weaker potency. The inhibitory actions of propranolol were exerted through its blockade of the Leydig cell 5HT2 low affinity receptor, which has functional properties of an autoreceptor, with consequent increases in 5HT and stimulation of CRF release through 5HT action at the high affinity site. The serotonergic actions of propranolol were prevented by DOI, an inhibitor of 5HT actions at the high affinity site. In addition, the propranolol-induced blockade of the low affinity site further increased the cAMP and steroidogenic responses to gonadotropin over those observed with DOI treatment alone. These studies demonstrate that propranolol acts as an antagonist at the Leydig cell low affinity 5HT2 receptor and stimulates CRF release via a serotonergic mechanism, with consequent inhibition of cAMP generation and steroidogenesis. This serotonergic action of the drug could contribute to the impairment of sexual function reported during propranolol treatment in man.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1993

Serotonin secretion from rat Leydig cells.

In rat Leydig cells, serotonin (5HT) binds to 5HT2 receptors and stimulates the secretion of CRF which in turn acts as an inhibitor of gonadotropin-induced cAMP formation and androgen production. In the present study we defined the regulation of 5HT secretion in cultured Leydig cells. Adult Leydig cells secreted considerable quantities of 5HT (100-150 pg/10(6) cells per 10 min). The release of 5HT was acutely stimulated by hCG (ED50, 1.1 pM) with maximal stimulation at 10 pM hCG (160%). Forskolin also increased (+220%) 5HT release from cultures (ED50, 50 nM) while TPA was much less effective (+20%), indicating a major role for cAMP in gonadotropin-induced 5HT release. This was confirmed by the finding that 8-Br cAMP (1 mM) was an effective stimulus of 5HT release (+360%). Similar increases of 5HT release by hCG were observed in the absence of extracellular Ca2+. However, ionomycin was a potent stimulus of 5HT release, indicating that elevation of cytoplasmic [Ca2+] could also induce amine secretion. The 5HT content of Leydig cells ranged from 300 to 350 pg/10(6) cells, and decreased during stimulation of 5HT release. Also, immunohistochemical studies revealed specific staining of 5 HT in interstitial cells of the adult rat testis. These studies demonstrated that rat Leydig cells contain and secrete 5HT, and that 5HT release is stimulated by gonadotropin acting primarily through a cAMP-mediated mechanism.

8-Bromo Cyclic Adenosine Monophosphate↗