Biomedical subjects

I T Huhtaniemi

Publications and source records attributed to I T Huhtaniemi.

At least 19 recordsLinked to original sources

PubMed1999

Molecular mechanisms of thyroid hormone-stimulated steroidogenesis in mouse leydig tumor cells. Involvement of the steroidogenic acute regulatory (StAR) protein.

Using a mouse Leydig tumor cell line, we explored the mechanisms involved in thyroid hormone-induced steroidogenic acute regulatory (StAR) protein gene expression, and steroidogenesis. Triiodothyronine (T3) induced a approximately 3.6-fold increase in the steady-state level of StAR mRNA which paralleled with those of the acute steroid response ( approximately 4.0-fold), as monitored by quantitative reverse transcriptase-polymerase chain reaction assay and progesterone production, respectively. The T3-stimulated progesterone production was effectively inhibited by actinomycin-D or cycloheximide, indicating the requirement of on-going mRNA and protein synthesis. T3 displayed the highest affinity of [125I]iodo-T3 binding and was most potent in stimulating StAR mRNA expression. In accordance, T3 significantly increased testosterone production in primary cultures of adult mouse Leydig cells. The T3 and human chorionic gonadotropin (hCG) effects on StAR expression were similar in magnitude and additive. Cells expressing steroidogenic factor 1 (SF-1) showed marginal elevation of StAR expression, but coordinately increased T3-induced StAR mRNA expression and progesterone levels. In contrast, overexpression of DAX-1 markedly diminished the SF-1 mRNA expression, and concomitantly abolished T3-mediated responses. Noteworthy, T3 augmented the SF-1 mRNA expression while inhibition of the latter by DAX-1 strongly impaired T3 action. Northern hybridization analysis revealed four StAR transcripts which increased 3-6-fold following T3 stimulation. These observations clearly identified a regulatory cascade of thyroid hormone-stimulated StAR expression and steroidogenesis that provides novel insight into the importance of a thyroid-gonadal connection in the hormonal control of Leydig cell steroidogenesis.

Animals↗

PubMed1999

Many LH peaks are needed to physiologically stimulate testosterone secretion: modulation by fasting and NPY.

The pulsatile luteinizing hormone (LH) and testosterone secretions were studied during serial blood collections performed at 7-min time intervals in the male rat. In fed rats, a discontinuous pattern of LH secretion was observed. Periods without secretion alternated with active secretory episodes consisting in trains of three to four LH peaks that triggered testosterone secretion usually 1-2 h later. The magnitude of the testosterone response was not correlated with the amplitude of the LH peaks. Isolated, single peaks of LH did not evoke clear testosterone responses. Forty-eight hours after initiation of fasting, testosterone secretion was markedly decreased, but integrated LH secretion was only partly reduced. Chronic infusion of neuropeptide Y (NPY; 18 microgram/day, icv) reduced testosterone secretion to very low levels and abolished pulsatile LH secretion or testosterone response to isolated LH peaks. In conclusion, the stimulation of testosterone secretion by LH necessitates several LH peaks organized in a proper sequence, and the testosterone response is not immediate. Low testosterone secretion in fasting rats appears to result from disappearance of coordinated, multiple LH peaks of sufficient size. Inhibition of the gonadotropic axis achieved by central NPY administration is due to either absence of LH peak "clusters" or occurrence of nonfunctional single LH peaks.

Activity Cycles↗

PubMed1999

Expression and regulation of transcription factors GATA-4 and GATA-6 in developing mouse testis.

Previous studies have shown that transcription factors GATA-4 and GATA-6 are expressed in granulosa and thecal cells of the mouse ovary and that GATA-4 expression in ovarian tissue is regulated by gonadotropins. Given the emerging role of GATA-4 and GATA-6 in gonadal cells, we have now studied the expression and regulation of these factors in the mouse testis and testicular cell lines. In situ hybridization demonstrated GATA-4 messenger RNA (mRNA) in the fetal testis at 13.5 days postcoitum. Both GATA-4 and GATA-6 transcripts were observed in late fetal, neonatal, juvenile, and adult Sertoli cells. In addition, GATA-4 mRNA was detected in interstitial cells throughout development. Immunohistochemistry demonstrated GATA-4 protein in both Sertoli and Leydig cells in postnatal animals. The regulation of GATA-4 and GATA-6 expression was explored using established testicular cell lines. Treatment of Leydig tumor cell lines with hCG resulted in a modest, but statistically significant, increase in the steady state level of GATA-4 mRNA, comparable to the previously described effect of FSH on GATA-4 expression in Sertoli cell lines. Gonadotropin or androgen action was not, however, a prerequisite for the basal expression of GATA-4 or GATA-6 in the testis, as their presence in Sertoli and Leydig cells was demonstrated in genetically hypogonadal hpg mice, in rats treated with GnRH receptor antagonist, and in Sertoli cells after chemical abolition of Leydig cells. Cotransfection studies using a GATA-4 expression plasmid and an inhibin alpha promoter/reporter gene construct in Leydig and granulosa tumor cell lines revealed that the inhibin alpha promoter harboring essential GATA-binding sites can be trans-activated by GATA-4. In light of these results, we propose that transcription factors GATA-4 and GATA-6 play differing roles in the maturation and function of testicular somatic cells.

Androgens↗

PubMed1999

Functional assessment of the calcium messenger system in cultured mouse Leydig tumor cells: regulation of human chorionic gonadotropin-induced expression of the steroidogenic acute regulatory protein.

The steroidogenic acute regulatory (StAR) protein, a 30-kDa mitochondrial factor, is a key regulator of steroid hormone biosynthesis, facilitating the transfer of cholesterol from the outer to the inner mitochondrial membrane. StAR protein expression is restricted to steroidogenic tissues, and it responds to hormonal stimulation through different second messenger pathways. The present study was designed to explore the mechanisms of extracellular calcium (Ca2+) involved in the hCG-stimulated expression of StAR protein and steroidogenesis in a mouse Leydig tumor cell line (mLTC-1). Extracellular Ca2+ (1.5 mmol/liter) enhanced the hCG (50 microg/liter)-induced increases in StAR messenger RNA (mRNA) and protein levels (1.7 +/- 0.3-fold; 4 h), as monitored by quantitative RT-PCR and immunoblotting. The potentiating effect of Ca2+ on the hCG-stimulated StAR response correlated with the acute progesterone (P) response. In accordance, omission of Ca2+ from the extracellular medium by specific Ca2+ chelators, EDTA or EGTA (4 mmol/liter each), markedly diminished the hCG-stimulated P production. The Ca2+ effect on hCG-induced StAR mRNA expression was dramatically suppressed by 10 micromol/liter verapamil, a Ca2+ channel blocker. The Ca2+-mobilizing agonist, potassium (K+; 4 mmol/liter), greatly increased the hCG responses of StAR expression and P production, which conversely were attenuated by Ca2+ antagonists, further supporting the involvement of intracellular free Ca2+ ([Ca2+]i) in these responses. The interaction of Ca2+ or K+ with hCG accounted for a clear increase in the StAR protein level (1.4-1.8-fold; 4 h) compared with that after hCG stimulation. Inhibition of protein synthesis by cycloheximide (CHX) drastically diminished the hCG-induced StAR protein content, indicating the requirement for on-going protein synthesis for hCG action. The transmembrane uptake of 45Ca2+ was increased by 26% with hCG and was strongly inhibited by verapamil. [Ca2+]i moderately augmented the response to hCG in fura-2/AM-loaded mLTC-1 cells within 30-40 sec, reaching a plateau within 1-3 min. Interestingly, the calcium ionophore (A23187) clearly increased (P < 0.01) StAR mRNA expression, in additive fashion with hCG. Northern hybridization analysis revealed four StAR transcripts at 3.4, 2.7, 1.6, and 1.4 kb, with the 1.6-kb band corresponding to the functional StAR protein; all of them were up-regulated 3- to 5-fold upon hCG stimulation, with a further increase in the presence of Ca2+. The mechanism of the Ca2+ effect on hCG-stimulated StAR expression and P production was evaluated by assessing the involvement of the nuclear orphan receptor, steroidogenic factor 1 (SF-1). Stimulation of hCG significantly elevated (2.1 +/- 0.3-fold) the SF-1 mRNA level, which was further augmented in the presence of Ca2+, whereas EGTA and verapamil completely abolished the increase caused by Ca2+. Cells expressing SF-1 marginally increased StAR expression, but coordinately elevated StAR mRNA levels in response to hCG and hCG plus Ca2+ compared with those in mock-transfected cells. On the other hand, overexpression of the nuclear receptor DAX-1 remarkably diminished (P < 0.0001) the endogenous SF-1 mRNA level as well as hCG-induced StAR mRNA expression. In summary, our results provide evidence that extracellular Ca2+ rapidly increases [Ca2+]i after hCG stimulation, presumably through opening of the transmembrane Ca2+ channel. Neither extracellular Ca2+ nor K+ alone has a noticeable effect on StAR expression and steroidogenesis, whereas they clearly potentiate hCG induction. The Ca2+-mediated increase in hCG involved in StAR expression and P production is well correlated to the levels of SF-1 expression. The stimulatory effect of hCG that rapidly increases [Ca2+]i is responsible at least in part for the regulation of SF-1-mediated StAR expression that consequently regulates steroidogenesis in mouse Leydig tumor cells.

Animals↗

PubMed1999

Luteinizing hormone and different genetic variants, as indicators of frailty in healthy elderly men.

We investigated the possible clinical correlates between the serum LH concentration and characteristics of frailty and determined the presence and concentration of a genetic LH variant in an independently living population of elderly men. After exclusion of subjects with severe mobility problems and signs of dementia, 403 healthy men (aged 73-94 yr) were randomly selected from a population-based sample. Total testosterone (T), sex hormone-binding globulin (SHBG), and leptin were determined by RIA. Non-SHBG-bound T was calculated. LH and the presence of the genetic LH variant were measured using immunofluorometric assays. The characteristics of frailty were leg extensor strength using dynamometry, bone mineral density of total body and proximal femur, and body composition, including lean mass and fat mass, measured by dual energy x-ray absorptiometry. Disability was further assessed by the Modified Health Assessment Questionnaire and by a measure of physical performance. LH significantly increased with age and inversely correlated with T and non-SHBG-bound T. LH was inversely related to muscle strength and lean mass, and both relations were independent of T. LH was positively related to self-reported disability (Modified Health Assessment Questionnaire); 12.5% of the study population was heterozygous for the LH variant allele. T levels and the degree of frailty were not different in the wild-type LH group compared with the heterozygote LH variant group. A significant positive relation between LH and fat mass as well as leptin was only present in the heterozygote LH variant group. In conclusion, serum LH levels increases with age in independently living elderly men and correlates inversely with a variety of indicators of frailty. The observed relation between LH and frailty, independent of T, suggests that LH reflects serum androgen activity in a different way than T, possibly reflecting more closely the combined feedback effect of estrogen and androgen. A difference in biological response between the two LH forms is suggested, as a difference exists in the relation between LH and fat mass, respectively, and leptin in the heterozygote LH variant subjects vs. the wild-type LH subjects.

Aged↗

PubMed1999

A new contributing factor to polycystic ovary syndrome: the genetic variant of luteinizing hormone.

Although the etiology of polycystic ovary syndrome (PCOS) is still unclear, LH is considered to play a central role in its pathogenesis. An immunologically anomalous form of LH, with two point mutations in the LHbeta gene, has been recently described. This genetic variant of LH (v-LH), of wide geographic distribution, is functionally different from wild-type (wt) LH. To assess the role of the v-LH in PCOS, we analyzed its frequency in groups of PCOS patients from Finland, The Netherlands, the United Kingdom, and the United States. The LH status was determined by two immunofluorometric assays from a total of 1466 subjects. The carrier frequency of the v-LH allele in the whole study population was 18.5%, being highest (28.9%) in Finland and lowest (11.2%) in The Netherlands. In the individual countries, the frequency of v-LH was similar in obese and nonobese controls, but in The Netherlands and Finland, it was 5- to 7-fold lower in obese PCOS subjects compared with that in the other groups (2-4.5% vs. 10.3-33.3%; P < 0.05). A similar tendency was found in the United States (5.7% vs. 11.1-25.0%), but not in the United Kingdom. The overall high prevalence of v-LH in healthy women and women with PCOS suggests that it is compatible with fertility. The similar frequency of v-LH in healthy nonobese and obese women indicates that obesity per se is not related to the variant. In contrast, the lower frequency of v-LH in obese PCOS patients suggests that v-LH somehow protects obese women from developing symptomatic PCOS. However, the regional differences in this finding between patients with apparently similar diagnostic criteria emphasizes the multifactorial nature of this syndrome, and that its pathogenesis may vary according to the genetic background. Although the definitive role of v-LH in PCOS remains to be proven, its determination may improve the prediction of risk of PCOS, especially in obese women.

Adolescent↗

PubMed1999

Reciprocal changes in the expression of transcription factors GATA-4 and GATA-6 accompany adrenocortical tumorigenesis in mice and humans.

While certain genetic changes are frequently found in adrenocortical carcinoma cells, the molecular basis of adrenocortical tumorigenesis remains poorly understood. Given that the transcription factors GATA-4 and GATA-6 have been implicated in gene expression and cellular differentiation in a variety of tissues, including endocrine organs such as testis, we have now examined their expression in the developing adrenal gland, as well as in adrenocortical cell lines and tumors from mice and humans. Northern blot analysis and in situ hybridization revealed abundant GATA-6 mRNA in the fetal and postnatal adrenal cortex of the mouse. In contrast, little or no GATA-4 expression was detected in adrenal tissue during normal development. In vivo stimulation with ACTH or suppression with dexamethasone did not affect the expression of GATA-4 or GATA-6 in the murine adrenal gland. To assess whether changes in the expression of GATA-4 or GATA-6 accompany adrenocortical tumorigenesis, we employed an established mouse model. When gonadectomized, inhibin alpha/SV40 T-antigen transgenic mice develop adrenocortical tumors in a gonadotropin-dependent fashion. In striking contrast to the normal adrenal glands, GATA-6 mRNA was absent from adrenocortical tumors or tumor-derived cell lines, while GATA-4 mRNA and protein were abundantly expressed in the tumors and tumor cell lines. Analogous results were obtained with human tissue samples; GATA-4 expression was detected in human adrenocortical carcinomas but not in normal tissue, adenomas, or pheochromocytomas. Taken together these results suggest different roles for GATA-4 and GATA-6 in the adrenal gland, and implicate GATA-4 in adrenal tumorigenesis. Immunohistochemical detection of GATA-4 may serve as a useful marker in the differential diagnosis of human adrenal tumors.

Adrenal Cortex↗

PubMed1999

Genetic variant of luteinizing hormone in Czech Republic.

OBJECTIVE: To evaluate the frequency of luteinizing hormone (LH) variant in males and females in West region of the Czech Republic. This species of mutated LH concerns borderline alterations in pituitary-gonadal function, including higher risk for the development of more aggressive forms of prostate carcinoma in males. METHODS: The examined normal population consisted of randomly selected 82 males and 175 females (age range of 14 to 72 years). Variant LH was determined by immunofluorimetric method using two pairs of monoclonal antibodies, one of which detecting both wild-type (w+) and variant LH, while the other detecting only w+-LH. RESULTS: The carrier frequency of the variant LH allele in the population sample was 17.5 % (12.2 % in males and 20.6 % in females) which was within the range of the European prevalence. CONCLUSION: The prevalence of the common variant of LH in the investigated region of West Bohemia was close to that of other Middle-European and North-European populations.

Adolescent↗

PubMed1998

Mechanisms of desensitization of follicle-stimulating hormone (FSH) action in a murine granulosa cell line stably transfected with the human FSH receptor complementary deoxyribonucleic acid.

The desensitization of follicle-stimulating hormone (FSH)-evoked cAMP synthesis occurs upon continuous or repeated hormonal stimulation, and it involves the hormone-receptor interaction and post-receptor events. These mechanisms were studied in a murine granulosa cell line (KK-1) stably transfected with the human FSH receptor (hFSHR) complementary deoxyribonucleic acid (cDNA) under a powerful viral promoter. Hence, the FSHR transcriptional regulation was eliminated from the experimental model. Stimulation of the cells with recombinant human FSH (rhFSH) or a phorbol ester, 12-O-tetradecanoylphorbol-13 acetate (TPA), resulted in clear desensitization, i.e. subsequent rhFSH-stimulated cAMP formation was 73.4 +/-2.2%, (P < 0.001) and 66.3 +/-3.4%, (P < 0.0001), respectively, of that of cells preincubated in medium. TPA prestimulation evoked also clear inhibition (65-74% of control) of rhFSH or forskolin (a non-specific activator of adenylate cyclase) induced progesterone production. The suppression by TPA preincubation of the rhFSH-induced cAMP synthesis was completely abolished by the protein kinase C (PKC) inhibitor staurosporine (STR). Preincubation with STR exhibited a significant (P < 0.0001) increasing effect on the rhFSH-stimulated cAMP accumulation. The specific involvement of PKC was further evidenced by other inhibitors, all of them exerted significant elevation of cAMP synthesis following rhFSH restimulation. Furthermore, only the PKC beta isoform appeared to be constitutively expressed in these cells during desensitization. Prestimulation of the G-protein activity by sodium fluoride (NaF) or cholera toxin (CT), followed by rhFSH challenge, accounted for a decrease in the cAMP-mediated responsiveness, down to 69.4 +/- 2.8 or 74.2 +/- 1.9%, of control (P < 0.001), respectively, indicating that the post-receptor events are critical for desensitization. [125I]iodo-rhFSH binding to the cells did not change significantly during desensitization and the different stimulations. In contrast, approximately 50% increase (P < 0.001) occurred in the steady-state levels of FSHR mRNA in the cells stimulated with FSH. This was apparently due to prolonged half-time of mRNA, and not to altered transcription, since the FSHR cDNA was driven by a powerful viral promoter. In accordance, the cells transfected with Simian Virus (SV40) promoter-driven luciferase gene did not display alterations in luciferase activity following stimulatory treatments. The effects of the post-receptor stimulations (NaF or CT) on [125I]iodo-rhFSH binding were minor (8-12% reduction). Taken together, these data provide evidence that the agonist-responsive hFSHR desensitization appears through a PKC-beta isoform-mediated modulation of cAMP production. The desensitization of FSH action involves modifications of functional properties of the existing components of the FSH signal transduction complex, and does not require concomitant suppression of transcription or translation of the FSHR gene.

Animals↗

PubMed1998

Inactivating FSH receptor mutations and gonadal dysfunction.

A variety of mutations and polymorphisms of genes regulating female and male reproductive functions have been discovered during the last few years. These include several inactivating and activating mutations in LH receptor genes. The first mutation of FSH receptor (FSHR) gene was discovered in six Finnish families. This inactivating Ala189Val transition in the extracellular receptor domain causes primary amenorrhea, arrest of follicular development and infertility in homozygous women. In contrast to females, this mutation did not cause absolute infertility in males but only suppressed spermatogenesis. Another inactivating mutation of the FSHR gene has been found at position 191 (Asn191Ile) in a healthy fertile woman. The studies on inactivating FSHR mutations demonstrate that normal ovarian function is critically dependent on FSH while, in contrast to earlier views, male fertility is less strictly dependent on normal FSH action.

Alleles↗

PubMed1998

Transgenic mouse models for gonadal tumorigenesis.

The versatile transgenic (TG) techniques allow the production of in vivo animal models for a variety of diseases, including malignant tumors, through tissue-specific expression of oncogenes. We have created a TG mouse model for gonadal somatic cell tumors by expressing the powerful viral oncogene, Simian virus 40 T-antigen (Tag) under regulation of the murine inhibin alpha-subunit promoter (inh alpha). Ovarian granulosa and theca cell tumors were formed in the female, and those of testicular Leydig cells, in the male TG mice at the age of 5-6 months, with 100% penetrance. The tumors produced high levels of inhibin peptides, especially the alpha-subunit, and were steroidogenically active, mainly producing progesterone. The gonadal tumorigenesis was gonadotropin-dependent, since TG mice rendered gonadotropin-deficient by crossbreeding them into the hypogonadotropic hpg genetic background, or by treating them with a gonadotropin-releasing hormone (GnRH) antagonist, did not develop tumors. In order to study the possibility of using the tumor mouse model for testing gene therapy, we created another TG mouse model expressing under the same inhibin-alpha promoter the Herpes Simplex virus (HSV) thymidine kinase (TK) transgene. The inh alpha/HSV-TK mice were crossbred with the inh alpha/Tag mice and the double mutant mice also developed gonadal tumors. When they were treated with antiherpes drugs (acyclovir or gancyclovir), further growth of the tumors was blocked. These preliminary findings prove the principle that tumor ablation in our TG mouse model can be achieved by transduction of the HSV-TK gene into the tumor cells. Besides studies of formation, regulation and therapy of the tumors in vivo, immortalized cell lines derived from them provide models for studies of gonadal somatic cell functions in vitro.

Animals↗

PubMed1998

Persistence of biological activity of biotinylated human chorionic gonadotropin and its use for visualization of rat luteinizing hormone receptors in tissue sections.

Biotinylation of antibodies is an established method for producing systems for detection of antigens. We currently aim to develop liposomal targeting vectors for gene transfer into transgenic gonadal tumor cells expressing the luteinizing hormone (LH) receptor (R). We have biotinylated (B) human chorionic gonadotrophin (hCG) to obtain a selective targeting molecule to be attached to biotinylated liposomes via an avidin-streptavidin bridge. The biotinylation was performed by combining biotin isothiocyanate (BITC) and hCG in alkaline reaction buffer in a 100:1 (BITC:hCG) molar ratio. B-hCG maintained its ability to bind specifically to rat testicular membranes and was also bound to streptavidin-coated polypropylene wells. cAMP production was induced in BLT-1 Leydig tumor cells in vitro after stimulation with B-hCG, as a sign of persistent bioactivity. Frozen sections of rat testicular and ovarian tissues and skeletal muscle were labeled by incubating for 2 hr at 37 degrees C with 10 ng/microliter B-hCG. The binding was subsequently visualized by the avidin-biotin-peroxidase system, followed by silver enhancement of Ni-DAB staining. In rat testicular and ovarian sections, labeling was observed in structures known to strongly express the LH-R, i.e., Leydig cells, corpora lutea, and blood vessels. The labeling was blocked by preincubation with a 100-fold excess of the native hormone, and by injecting the rats sc with a high dose of hCG (1000 IU/kg) 48 hr before sacrifice. Skeletal muscle, used as negative control, was not labeled. These data demonstrate that the bioactivity of hCG is relatively well preserved after biotinylation. The biotinylated gonadotropin offers a new nonradioactive alternative for visualization of bioactive LH receptors in tissue sections.

Animals↗

PubMed1998

Mutations of follicle-stimulating hormone and its receptor: effects on gonadal function.

The physiological functions of the two gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are quite well known in both males and females. Nevertheless, the recent unraveling of structures of the gonadotropin and gonadotropin receptor genes has provided us with tools to obtain completely new information on the functions of these hormones at the molecular level. Several gain- and loss-of-function mutations of these genes have been discovered in humans, and the first knock-out experiments with these genes in the mouse have been recently published. The purpose of the present review is to describe the current state of knowledge on human mutations of FSH and its receptor, as well as on relevant animal models.

Animals↗

PubMed1997

Worldwide frequency of a common genetic variant of luteinizing hormone: an international collaborative research. International Collaborative Research Group.

OBJECTIVE: To determine the worldwide frequency of a common immunological LH variant because of two point mutations in the LH beta-subunit gene (Trp8Arg and Ile15Thr). DESIGN: Cross-sectional study on LH status (variant and wild-type) in serum (or DNA) samples from Finland (Finns and Lapps), Estonia, Poland, Sweden, The Netherlands, United Kingdom, Italy, South Africa (blacks), Thailand, China, Japan, and the United States (Hispanics and blacks). SETTING: Academic research environment. PATIENT(S): Ambulatory adult men and women (n = 2,936) with minor illnesses and no known endocrinological disorders. INTERVENTION: A single blood sample was collected from each subject. MAIN OUTCOME MEASURE(S): The LH status was determined by two immunofluorometric assays using monoclonal antibodies. One (assay 1) only recognizes the wild-type LH, the other (assay 2) recognizes equally variant and wild-type LH. The ratio of assay 1 to assay 2 indicates the LH status: wild-type, > 0.9; heterozygote, 0.2 to 0.9; and homozygote, < 0.15. One population (Lapps) was studied by DNA analysis using polymerase chain reaction and allele-specific oligonucleotide hybridization. RESULT(S): The carrier frequency of the variant LH beta allele varied from 7.1% in U.S. Hispanics to 41.9% in Lapps of northern Finland. The variant LH beta allele tended to be more common in populations from Northern Europe as compared with those from Asia. CONCLUSION(S): The high frequency of the LH beta variant worldwide makes it an important confounding factor when obtaining disproportionately low LH levels with some immunometric assays. The LH variant may contribute to some pathologies of the pituitary-gonadal function.

Adolescent↗

PubMed1997

Men homozygous for an inactivating mutation of the follicle-stimulating hormone (FSH) receptor gene present variable suppression of spermatogenesis and fertility.

Gonadal function is controlled by the two pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). While LH mainly regulates gonadal steroidogenesis, FSH is considered essential for folliculogenesis in the female and spermatogenesis in the male. We recently discovered that an inactivating point mutation in the FSH receptor (R) gene causes a recessively inherited form of hypergonadotropic ovarian failure in homozygous females. This 566C-->T mutation, predicting an alanine to valine substitution, is located in exon 7 of the FSHR gene, in the region encoding the extracellular domain of the receptor molecule. Functional testing showed a clear-cut reduction in ligand binding and signal transduction by the mutated receptor. Hence, lack of FSH function is incompatible with ovarian follicular maturation and female fertility. In the male, FSH is generally considered essential for the pubertal initiation of spermatogenesis and maintenance of quantitatively normal sperm production in adults. We report here the first characterization of males homozygous for an inactivating FSHR mutation. They have variable degrees of spermatogenic failure, but, surprisingly, do not show azoospermia or absolute infertility. These results question the essential role of FSH for the initiation of spermatogenesis, and demonstrate that FSH is more important for female than for male fertility.

Adult↗

PubMed1997

Cloning and functional expression of the luteinizing hormone receptor complementary deoxyribonucleic acid from the marmoset monkey testis: absence of sequences encoding exon 10 in other species.

Based on sequence homologies among the human, porcine, rat, and mouse genes for the LH receptor (LHR), overlapping partial fragments of LHR complementary DNAs (cDNAs) were multiplied from marmoset monkey testicular RNA using reverse transcription-PCR. Ligations of the individual cDNA fragments generated a full-length monkey LHR cDNA (2031 bp) containing the complete amino acid-coding sequence (676 amino acids). Northern hybridization analysis of monkey testicular RNA, using a complementary RNA probe corresponding to the full-length cDNA, demonstrated major transcripts of 5.5 and 1.4 kilobases and minor ones of 4.0, 2.7, and 1.9 kilobases. Sequence analysis of the monkey LHR cDNA revealed a striking feature, i.e. the absence of an 81-bp nucleotide sequence corresponding to exon 10, present in the LHR cDNAs of all other species studied to date. The monkey LHR cDNA displayed 83-94% overall sequence homology with the other mammalian LHR cDNAs. Reverse transcription-PCR with human exon 10-specific primers demonstrated the total absence of this sequence from the monkey LHR messenger RNA. Southern hybridization of monkey genomic DNA using a human exon 10 probe demonstrated its presence in the monkey gene and that it is totally spliced out from the primary transcript. COS cells transfected with the monkey LHR cDNA showed similar high affinity (Kd = 0.25 nmol/liter) of [125I]iodo-hCG binding as those transfected with human LHR cDNA (Kd = 0.20 nmol/liter). The cells expressing the recombinant monkey and human LHR displayed similar responses of extracellular cAMP and inositol trisphosphate to hCG. In conclusion, marmoset monkey LHR seems to lack the sequence corresponding to exon 10 of the LHR gene in other mammalian species. The truncation does not alter LHR function, as the monkey receptor protein bound hCG and evoked cAMP and inositol trisphosphate responses comparable to those of the human LHR containing the exon 10-encoded structure. As the sequence homologous to exon 10 is missing in the other two glycoprotein receptors, i.e. those of FSH and TSH, this extra exon is apparently inserted into the LHR messenger RNA of some species during evolution from intronic sequences by a change in alternative splicing.

Alternative Splicing↗

PubMed1997

Molecular mechanisms of reappearance of luteinizing hormone receptor expression and function in rat testis after selective Leydig cell destruction by ethylene dimethane sulfonate.

Considering the major role of LH in the control of Leydig cell (LC) development and function, we aimed to characterize further the pattern of LH receptor (LHR) expression in two experimental paradigms: the rat treated with ethylene dimethane sulfonate (EDS), in which the selective destruction of preexisting mature LCs induces the proliferation and differentiation of newly formed LCs, a process that takes place in the presence of high levels of gonadotropins; and the EDS-rat treated with a high dose of testosterone (EDS + T), in which the LH secretion is suppressed, and consequently LC development after EDS arrested. In EDS rats, serum T was suppressed and testicular LHR binding became undetectable on days 5 and 15 after treatment. The pattern of LHR messenger RNA (mRNA) expression was profoundly modified: only one of the splice variants [1.8-kilobase (kb)] persisted, whereas the others disappeared. On days 20 and 45 after EDS, along with LC repopulation, serum T and LHR binding recovered, and the pattern of LHR mRNA expression gradually returned to that resembling controls. In EDS + T rats, a similar drop in testicular LHR binding and change in the pattern of LHR mRNA expression was detected on days 5 and 15 after treatment. However, on days 20 and 45, no recovery either in LHR binding or in expression of the longer LHR mRNA splice variants was observed, showing that LH is needed to induce LHR expression in repopulating LCs, at least to a quantitatively significant level. To gain further insight into the mechanism(s) by which LH acts on LC precursors, the translational status of the 1.8-kb LHR transcript, persistently expressed after EDS, was analyzed and compared with that of the 6.8-kb message. In polysome distribution analysis of total testicular RNA, the 6.8-kb LHR message was highly associated with polysomes, whereas the 1.8-kb variant was mainly localized to prepolysomal fractions, both in control and EDS testes, thus predicting lower translational efficiency. In addition, considering that only LCs express LHRs in the testis, the time course of the reappearance of functional receptors was mapped by evaluating testicular responsiveness to human recombinant LH in vitro. No response to LH stimulation was detected 5 days after EDS. However, cAMP response to LH was observed on days 15 and 20, regardless of the presence of high (EDS) or suppressed (EDS + T) LH in the donor animal. Hence, the appearance of functional LHRs, qualitatively, can take place in the absence of measurable LH levels. In EDS-treated rats, the appearance of the cAMP response coincided with those of pregnenolone, progesterone, and T. In contrast, no LH-induced steroid release was observed in EDS + T rats, indicating that steroidogenic response in developing LC requires LH priming. In conclusion, the appearance of functional LHRs, at a low level of expression, in LC precursors is an LH-independent developmental event, essential for the subsequent LH-dependent maturational steps, including the onset of steroidogenesis and increased LHR expression. In addition, our results cast doubt on a major functional role of the truncated (1.8-kb) form of LHR mRNA, which persists after EDS at a high level of expression, in the early Leydig cell precursors.

Analysis of Variance↗

PubMed1997

Expression and hormonal regulation of transcription factors GATA-4 and GATA-6 in the mouse ovary.

Two members of the GATA-binding family of transcription factors, GATA-4 and GATA-6, are expressed in the vertebrate ovary. To gain insight into the role of these factors in ovarian cell differentiation and function, we used in situ hybridization to determine the patterns of expression of GATA-4 and GATA-6 in mouse ovary during development and in response to hormonal stimulation. GATA-4 messenger RNA (mRNA) was first evident in the ovary around the time of birth. In the adult ovary, abundant GATA-4 mRNA was detected in granulosa cells of primary and antral follicles, with lesser amounts of GATA-4 message detected in theca cells, germinal epithelium, and interstitial cells. Little or no GATA-4 mRNA was found in corpus luteum. GATA-6 message exhibited a different distribution in the ovary, with abundant expression evident in both granulosa cells and corpora lutea. Stimulation of 3-week-old females with PMSG or estrogen enhanced follicular expression of GATA-4 and GATA-6 transcripts. Subsequent induction of ovulation with human CG resulted in a decrease in GATA-4 mRNA expression in granulosa cells, whereas GATA-6 mRNA expression persisted in granulosa cells after ovulation and in corpora lutea. Moreover, follicular apoptosis was associated with a decrease in the expression of GATA-4 but not GATA-6 message. Stimulation of cultured gonadal cell lines with FSH resulted in increased expression of GATA-4 message, whereas GATA-6 mRNA expression was not affected. In light of these findings, the established role of other GATA-binding proteins in hematopoetic cell differentiation and apoptosis, and the presence of conserved GATA motifs in the promoters of genes expressed selectively in ovary, we propose that GATA-4 and GATA-6 play distinct roles in follicular development and luteinization.

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