Luteinizing Hormone Receptor Gene Research Articles

Effectiveness of Anastrozole and Cyproterone Acetate in Two Brothers with Familial Male Precocious Puberty

Testotoxicosis is a rare form of precocious puberty caused by a constitutively activating mutation in the luteinizing hormone receptor (LHR) gene. Symptoms include rapid virilization, accelerated growth and reduced adult height. We describe a rare association of testotoxicosis with a metaphyseal chondrodysplasia called cartilage-hair hypoplasia (CHH) and report two brothers with testotoxicosis after 4 years of treatment. The brothers had a T577I mutation in the LHR gene. One brother also presented CHH. The older brother was treated with ketoconazole, then with the aromatase inhibitor anastrozole and the anti-androgen cyproterone acetate. The younger brother received this combination as first-line therapy. Clinical improvements included reductions in growth velocity and bone maturation rate, which should result in taller adult stature. Tolerance was good. Combined treatment with anastrozole and cyproterone acetate is effective in improving the prognosis of adult height in testotoxicosis.

Effects of polymorphisms of LHR and FSHR genes on sexual precocity in a Bos taurus x Bos indicusbeef composite population

The purpose of the present research was to investigate the effects of polymorphisms of luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) genes, evaluated by polymerase chain reaction-restriction fragment length polymorphism in European-Zebu composite beef heifers from six different breed compositions. The polymorphism site analysis from digestion with HhaI and AluI restriction endonucleases allowed the genotype identification for LHR (TT, CT and CC) and FSHR (GG, CG and CC) genes. A high frequency of heterozygous animals was recorded in all breed compositions for both genes, except in two compositions for LHR. The probability of pregnancy (PP) at first breeding was used to evaluate the polymorphism effect on sexual precocity. The PP was analyzed as a binary trait, with a value of 1 (success) assigned to heifers that were diagnosed pregnant by rectal palpation and a value of 0 (failure) assigned to those that were not pregnant at that time. Heterozygous heifers showed a higher pregnancy rate (67 and 66% for LHR and FSHR genes, respectively), but no significant effects were observed for the genes studied (P = 0.9188 and 0.8831 for LHR and FSHR, respectively) on the PP. These results do not justify the inclusion of LHR and FSHR restriction fragment length polymorphism markers in selection programs for sexual precocity in beef heifers. Nevertheless, these markers make possible the genotype characterization and may be used in additional studies to evaluate the genetic structure in other bovine populations.

Expression of gonadotropin and gonadotropin receptor genes during early sexual maturation in male Atlantic salmon parr

Atlantic salmon males may mature already as small parr in freshwater. Sexual maturation in teleosts as in vertebrates is characterized by the activation of the brain-pituitary-gonad axis. The endocrine regulation of early puberty is still not well understood. In the present study, one-summer-old male Atlantic salmon parr were sampled regularly from December several months prior to the beginning of spermatogenesis until spawning in October. Pituitary expression levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) beta subunit genes were analyzed in parallel with testis expression of FSH receptor (FSHR) and LH receptor (LHR) genes by RT-PCR and plasma 11-ketostestosterone (11-KT) was measured. Expression levels of FSHbeta, low during winter and spring started to increase prior to the onset of gonadal growth at the end of May while LHbeta mRNA levels were hardly detectable. Both gonadotropin receptor genes were expressed in immature testis with FSHR transcripts being more abundant (8-fold). FSHR transcript levels increased in parallel to FSHbeta levels from early spermatogenesis onwards, while LHR mRNA started to increase prior to any large changes in LHbeta expression. Both transcript levels of LHbeta and LHR were highest during spermiation. Plasma 11-KT increased at the beginning of spermatogenesis reaching highest levels at spermiogenesis suggesting a possible role of FSH in inducing 11-KT production during early spermatogenesis while LH stimulates via its specific receptor 11-KT production at spermiogenesis. The commitment into sexual maturation appears to be dependant on both the presence of FSHR in immature testis and the increase of FSH expression.

New molecular variants of hypothalamus–pituitary–gonad axis genes and their association with early puberty phenotype in Bos taurus indicus (Nellore)

Endocrine system plays a major role in the control of reproductive functions which are regulated by the hypothalamus–pituitary–gonad axis and its interactions. FSH and LH receptor genes are expressed at the gonads and GnRH receptor gene is expressed at the anterior pituitary gland. Misense mutations of the FSH, LH or GnRH receptors, activating or inactivating their functions in mammals, are potentially useful to allow the understanding of the role of this group of gonadotropins in reproductive phenotypes as early puberty and birth interval length. In the present study, polymorphisms in bovine exon 11 and 3′UTR of LHR, exon 10 and 3′UTR of FSHR and GnRHR genes were characterized with some of them resulting in changes in the aminoacidic chain. These polymorphic sites were found in a Bos taurus indicus (Nellore) female population by means of PCR–SSCP and DNA sequencing. Association between nucleotidic/aminoacidic changes and early puberty were determined by Chi-square analysis. It was found association between FSHR 3′UTR polymorphisms at position 2181, 2248 and 2249 bp and early puberty phenotype ( p < 0.05). The presence of these new molecular markers might be considered in further studies to validate its correlation with early puberty or other reproduction associated phenotypes in cattle breeds.

Genomic Checkpoints for Exon 10 Usage in the Luteinizing Hormone Receptor Type 1 and Type 2

Alternative splicing is a hallmark of glycoprotein hormone receptor gene regulation, but its molecular mechanism is unknown. The LH receptor (LHR) gene possesses 11 exons, but exon 10 is constitutively skipped in the New World monkey lineage (LHR type 2), whereas it is constitutively spliced in the human (LHR type 1). This study identifies the regulatory elements of exon 10 usage. Sequencing of genomic marmoset DNA revealed that the cryptic LHR exon 10 is highly homologous to exon 10 from other species and displays intact splice sites. Functional studies using a minigene approach excluded the contribution of intronic, marmoset-specific long interspersed nucleotide-1 elements to exon 10 skipping. Sequencing of the genomic regions surrounding exon 10 from several primate lineages, sequence comparisons including the human and mouse LHR gene, revealed the presence of unique nucleotides at 3'-intronic position -19 and -10 and at position +26 within exon 10 of the marmoset LHR. Exon trap experiments and in vitro mutagenesis of these nucleotides resulted in the identification of a composite regulatory element of splicing consisting of cis-acting elements represented by two polypyrimidine tracts and a trans-acting element within exon 10, which affect the secondary RNA structure. Changes within this complex resulted either in constitutive exon inclusion, constitutive skipping, or alternative splicing of exon 10. This work delineates the molecular pathway leading to intronization of exon 10 in the LHR type 2 and reveals, for the first time, the essential function of regulatory and structural elements involved in glycoprotein hormone receptor splicing.

Phosphatidylinositol 3-Kinase/Protein Kinase Cζ-Induced Phosphorylation of Sp1 and p107 Repressor Release Have a Critical Role in Histone Deacetylase Inhibitor-Mediated Derepression of Transcription of the Luteinizing Hormone Receptor Gene

We have demonstrated that silencing of luteinizing hormone receptor (LHR) gene transcription is mediated via a proximal Sp1 site at its promoter. Trichostatin A (TSA) induced histone acetylation and gene activation in JAR cells that prevailed in the absence of changes in Sp1/Sp3 expression, their binding activity, disassociation of the histone deacetylase/mSin3A complex from the Sp1 site, or demethylation of the promoter. This indicated a different mechanism involved in TSA-induced derepression. The present studies have revealed that phosphatidylinositol 3-kinase/protein kinase Czeta (PI3K/PKCzeta)-mediated Sp1 phosphorylation accounts for Sp1 site-dependent LHR gene activation. TSA caused marked phosphorylation of Sp1 at serine 641 in JAR and MCF-7 cells. Blockade of PI3K or PKCzeta activity by specific inhibitors, kinase-deficient mutants, or small interfering RNA abolished the effect of TSA on the LHR gene and Sp1 phosphorylation. PKCzeta was shown to associate with Sp1, and this association was enhanced by TSA. Sp1 phosphorylation at serine 641 was required for the release of the pRb homologue p107 from the LHR gene promoter, while p107 acted as a repressor of the LHR gene. Inhibition of PKCzeta activity blocked the dissociation of p107 from the LHR gene promoter and markedly reduced Sp1 phosphorylation and transcription. These results have demonstrated that phosphorylation of Sp1 by PI3K/PKCzeta is critical for TSA-activated LHR gene expression. These studies have revealed a novel mechanism of TSA action through derecruitment of a repressor from the LHR gene promoter in a PI3K/PKCzeta-induced Sp1 phosphorylation-dependent manner.

Exclusion of coding-region mutations in luteinizing hormone and follicle-stimulating hormone receptor genes as the cause of ovarian hyperstimulation syndrome

To sequence the coding regions of the luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) genes to find out if polymorphisms in them are responsible for the severe form of ovarian hyperstimulation syndrome (OHSS) in Swedish patients. A mutation analysis of gonadotropin receptor genes from women undergoing gonadotropin treatment. The Fertility Unit of Karolinska University Hospital Huddinge, Stockholm, Sweden. A set of 10 well-characterized patients with severe OHSS, and 10 control women who did not develop OHSS after FSH stimulation. An additional 11 patients and 41 control women were screened for a two-amino-acid insertion in the first exon of the LHR gene. None. Changes in the sequence of the receptor genes between patients and controls. No association was found between polymorphisms of the coding region of LHR or FSHR genes and the development of OHSS. Incidence of the two-amino-acid insertion in the first exon of the LHR gene was slightly higher in patients than in controls, but no statistically significant difference was seen. LHR and FSHR coding polymorphisms are not a major cause of severe OHSS in Swedish patients.

Mutational analysis of BMP15 and GDF9 as candidate genes for premature ovarian failure

Mutational screening of the bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) genes in a population with premature ovarian failure (POF) identified no new mutations. However, three single nucleotide polymorphisms in the BMP15 gene, two in the 5' untranslated region (31T>G and 71C>G) and another in exon 1 (387G>A), were found to be common in both POF and control groups.

Phosphatidylinositol 3-Kinase/Protein Kinase Cζ-Induced Phosphorylation of Sp1 and p107 Repressor Release Have a Critical Role in Histone Deacetylase Inhibitor-Mediated Depression of Transcription of the Luteinizing Hormone Receptor Gene

We have demonstrated that silencing of luteinizing hormone receptor (LHR) gene transcription is mediated via a proximal Sp1 site at its promoter. Trichostatin A (TSA) induced histone acetylation and gene activation in JAR cells that prevailed in the absence of changes in Sp1/Sp3 expression, their binding activity, disassociation of the histone deacetylase/mSin3A complex from the Sp1 site, or demethylation of the promoter. This indicated a different mechanism involved in TSA-induced derepression. The present studies have revealed that phosphatidylinositol 3-kinase/protein kinase Czeta (PI3K/PKCzeta)-mediated Sp1 phosphorylation accounts for Sp1 site-dependent LHR gene activation. TSA caused marked phosphorylation of Sp1 at serine 641 in JAR and MCF-7 cells. Blockade of PI3K or PKCzeta activity by specific inhibitors, kinase-deficient mutants, or small interfering RNA abolished the effect of TSA on the LHR gene and Sp1 phosphorylation. PKCzeta was shown to associate with Sp1, and this association was enhanced by TSA. Sp1 phosphorylation at serine 641 was required for the release of the pRb homologue p107 from the LHR gene promoter, while p107 acted as a repressor of the LHR gene. Inhibition of PKCzeta activity blocked the dissociation of p107 from the LHR gene promoter and markedly reduced Sp1 phosphorylation and transcription. These results have demonstrated that phosphorylation of Sp1 by PI3K/PKCzeta is critical for TSA-activated LHR gene expression. These studies have revealed a novel mechanism of TSA action through derecruitment of a repressor from the LHR gene promoter in a PI3K/PKCzeta-induced Sp1 phosphorylation-dependent manner.

Activating Mutations in the Luteinizing Hormone Receptor Gene: A Human Model of Non-Follicle-Stimulating Hormone-Dependent Inhibin Production and Germ Cell Maturation

Familial male-limited precocious puberty is a dominant autosomal genetic disease caused by activating LH receptor gene mutations, clinically expressed only in males. In preliminary studies, in addition to the expected testosterone increase, we found high inhibin B levels before the age of normal puberty. The objective of the study was to assess the cellular origin of serum inhibin thanks to testis section immunostaining. Serum testosterone, gonadotropin, inhibin B, pan-alphaC-inhibin, and anti-Mullerian hormone levels were measured. Immunostaining was performed using specific anti-alpha- and anti-beta-subunit antibodies. Five boys from three families (mutation M398T or I542L) were investigated at onset (2-6 yr), on ketoconazole treatment, and at adolescence. Testis biopsies were performed in three subjects before the disease was fully characterized. The high testosterone levels were suppressed by ketoconazole. Anti-Mullerian hormone levels were inversely related to testosterone: low at diagnosis, elevated after testosterone suppression. Despite FSH suppression, inhibin B and pan-alphaC-inhibin levels were high from clinical onset to adolescence. Biopsy specimens showed normal Sertoli cell complement and germ cell maturation until the spermatocyte II stage. Sertoli and Leydig cells displayed positive inhibin alpha-subunit immunostaining. Only Leydig cells and spermatogonia stained positively for the inhibin betaB-subunit. Familial male-limited precocious puberty is a unique model of inhibin B secretion, demonstrating that Leydig cells can produce significant amounts of the dimeric molecule. Our results also suggest that the pubertal FSH rise is not required for full expression of the two inhibin B genes and for the initiation of germ cell maturation.

PI3K/PKCzeta‐induced Phosphorylation of Sp1 and p107 Inhibitor Release Has a Critical Role in HDAC Inhibitor‐Induced Depression of Transcription of the Luteinizing Hormone Receptor (LHR) Gene

PI3K/PKCzeta‐induced Phosphorylation of Sp1 and p107 Inhibitor Release Has a Critical Role in HDAC Inhibitor‐Induced Depression of Transcription of the Luteinizing Hormone Receptor (LHR) Gene

A Common Polymorphism Renders the Luteinizing Hormone Receptor Protein More Active by Improving Signal Peptide Function and Predicts Adverse Outcome in Breast Cancer Patients

Epidemiological and animal studies indicate a carcinogenic role of estrogens in breast tissue. The pituitary gonadotropin LH is an important regulator of estrogen production in premenopausal women, whereas even in women after menopause, 10-25% of ovarian steroid hormone production is LH dependent. We hypothesized that an LH receptor (LHR) gene variant may affect LHR function and thereby influence disease outcome in breast cancer patients. The association of a polymorphic CTCCAG (Leu-Gln) insertion (insLQ), in the signal peptide encoded by exon 1 of the LHR gene with breast cancer risk, (disease-free) survival, and clinicopathological features was studied in a large cohort of 751 breast cancer patients with complete follow-up. Functional analysis of the insLQ-LHR and non-LQ-LHR (no LQ insertion) was carried out using transfection studies. We found a significant association between the insLQ-LHR and a shorter disease-free survival (hazard ratio, 1.34; confidence interval, 1.11-1.63; P = 0.003). The mechanism of the effect of insLQ on LHR function involves increased receptor sensitivity (insLQ-LHR has a 1.9 times lower EC(50) than non-LQ-LHR; P = 0.02) and plasma membrane expression (insLQ-LHR has 1.4 times higher B(max); P = 0.0006) rendering the insLQ-LHR allele more active. The insLQ polymorphism increases LHR activity, thereby shortening breast cancer disease-free survival, probably by increasing estrogen exposure in female carriers.

Coordinated Changes in DNA Methylation and Histone Modifications Regulate Silencing/Derepression of Luteinizing Hormone Receptor Gene Transcription

We have previously demonstrated that transcription of the luteinizing hormone receptor (LHR) gene is subject to repression by histone deacetylation at its promoter region, where a histone deacetylase (HDAC)/mSin3A complex is anchored at a proximal Sp1 site. The present studies have shown that epigenetic silencing and activation of the LHR gene is achieved through coordinated regulation at both the histone and DNA levels. The HDAC inhibitor trichostatin A (TSA) evoked robust but significantly lower activation of the LHR gene in JAR than in MCF-7 cells. This effect was localized to the 176-bp promoter region, which is highly methylated in JAR and lightly methylated in MCF-7 cells. Consequently, TSA and the DNA demethylating reagent 5-azacytidine (5-AzaC) caused marked synergistic activation of the LHR gene in JAR but not in MCF-7 cells. Multiple site-specific lysine acetylation of H3/H4 is associated with such LHR gene activation. Methylation or acetylation of H3 at K9 is present at the silenced and derepressed LHR promoter, respectively. While DNA methylation levels did not affect the histone code of the LHR gene promoter, demethylation of the promoter CpG sites was necessary for maximal stimulation of this gene. Mechanistically, the combined actions of TSA and 5-AzaC, but not either 5-AzaC or TSA alone, resulted in complete demethylation of the LHR gene promoter in JAR cells. Release of the repressive HDAC/mSin3A complex from the LHR gene promoter in both cell types required both TSA-induced changes of histone modifications and, concurrently, a demethylated promoter. Also, Dnmt1 was largely dissociated from the LHR gene promoter in the presence of TSA or TSA plus 5-AzaC, and binding of MBD2 in JAR cells was diminished upon conversion of the promoter to a demethylated state. Such changes induced a more permissive chromatin where recruitment of polymerase II and TFIIB to the promoter was significantly increased. The activated state of the LHR gene induced by TSA and 5-AzaC in JAR and MCF-7 cells was observed basally in LHR-expressing PLC cells, in which the promoter is unmethylated and associated with hyperacetylated histones. Consequently, PLC cells are unresponsive to drug treatment. These findings have elucidated a regulatory mechanism whereby concurrent dissociation of repressors and association of activators and basal transcriptional components, resulting from coordinated histone hyperacetylation and DNA demethylation, lead to derepression of the LHR gene expression.

Regulation of Expression of the Chorionic Gonadotropin/Luteinizing Hormone Receptor Gene in the Human Myometrium: Involvement of Specificity Protein-1 (Sp1), Sp3, Sp4, Sp-Like Proteins, and Histone Deacetylases

At present there is little information on the regulatory processes by which the chorionic gonadotropin (CG)/LH receptor gene is regulated in the human myometrium during pregnancy and labor. Employing human primary myometrial cell cultures in conjunction with DNA affinity purification assays/Western analysis, DNA binding studies, CG/LH promoter luciferase reporter gene deletion constructs in transfection assays, and measurement of endogenous mRNA levels in vivo by duplex RT-PCR, we have determined the role that the major transcriptional regulatory sequences from the +1 ATG codon to -2678 bp play in modulating expression of the CG/LH receptor gene in the myometrium. We report that the distal -180 to -2678 bp region of the promoter, although capable of binding members of the Jun family via the multiple activator protein-1 sites within this region, has no significant role in regulating the expression of the CG/LH receptor gene in myometrial cells. In contrast, the two specificity protein-1 to -4 (Sp1-4) GC boxes within the +1 to -180 bp proximal promoter are central to expression of the gene in the myometrium. However, not only are Sp1/Sp3 proteins involved in this process, but Sp4 and a novel Sp-like factor(s) also have an intimate part in transcriptional regulation of the gene. It would appear that Sp1/Sp3/Sp4 and Sp-like proteins are involved in recruiting histone deacetylase complexes to the proximal promoter, preventing chromatin remodeling resulting in transcriptional repression of the gene. Our data suggest that administration of the histone deacetylase inhibitor trichostatin A to human myometrial cells in vitro and in vivo substantially removes this silencing effect on expression of the gene and may implicate the use of this and similar agents in increasing myometrial CG/LH receptor levels and subsequent maintenance of uterine relaxation during fetal maturation.

Can heterozygous intron mutations in the Luteinizing Hormone receptor gene cause delayed puberty?

Loss-of-function mutations of the LH receptor gene (Leydig cell hypoplasia) inactivate the receptor and give rise to primary amenorrhea and infertility in women and intersexual genitalia, hypospadias, micropenis and infertility in men. To evaluate if delayed puberty in boys and primary amenorrhoea in 46,XX girls can be due to partial Leydig cell hypoplasia we have studied the LH receptor gene in 35 patients. 8 girls were referred to pediatric endocrine outpatient clinics because of unexplained primary amenorrhoea and 27 boys because of extremely delayed puberty. The girls were 15.1–19.5 years old (mean 16.3 years) and showed normal breast development. The boys were 14,3–19,2 years old (mean 15.7 years) and presented with puberty Tanner stage 1 or 2 (testis volume below 5ml). In all male patients underlying diseases like chronic diseases or endocrine disorders especially hypogonadotropic hypogonadism by GnRH or Buserelin stimulation test were excluded. In all patients the protein-encoding part of all exons and the intron-exon boundaries were analyzed resulting in the identification of 7 new heterozygous LH receptor gene changes in 3 girls (+2 bp in Intron 5 (A to G); +18 bp in Intron 7 (C to T)) and in 5 boys (insertion of 18 AS, Exon 1; +3 bp in intron 1 (G to C); +5 bp Intron 5 (T to G); -8 bp Intron 8 (C to G); V553L in exon 11 (GTT to CTT). With exception of the alteration in intron 8 (-8 bp), all changes were not found in 300 control persons. On the other allele of the patients DNA no changes in the LH receptor gene were identified, indicating that the alterations identified in these patients cannot be linked free from doubt to LCH. Due to the fact that we have found in 2 not related girls presenting with primary amenorrhea the same heterozygous transition in intron 5 of the donor splicing site one may speculate that an aberrant splicing of one allele might affect the second allele. Further studies of more patients with delayed puberty and in vitro experiments with the mutant receptors will follow.

The role of luteinizing hormone activity in controlled ovarian stimulation

The role of LH in the natural menstrual cycle is undisputed. The active participation of LH in both steroidogenesis and ovulation is well established, but its potential effect on oocyte maturation in the issue of assisted reproduction protocols remains a topic of debate. Although several studies have added to our understanding of the specific actions of androgens in human follicular development, some discrepancies persist regarding their role in oocyte atresia. Clinical situations, where LH is either decreased or absent (e.g. in women with hypogonadotrophic hypogonadism or LH-receptor gene mutations), provide important data supporting the necessity for a minimal amount of LH to evoke ovulation. Recent use of GnRH antagonists, which results in profound suppression of LH concentration, in combination with the pharmacological production of recombinant gonadotrophins, has attracted the attention of investigators. Identification of sub-fertilized women, in whom LH administration could be beneficial and should be indicated, is arousing ever more interest. Based on the available data in the literature, the aims of this review are to assess the role of both endogenous and exogenous LH activity in stimulated cycles, and to evaluate the effects of recombinant human LH supplementation on the ovarian hormonal milieu and on the main outcomes of controlled stimulated cycles.

Targeted disruption of LH receptor gene revealed the importance of uterine LH signaling

Numerous previous studies have demonstrated that LH and hCG can directly regulate several uterine functions. We investigated in the present study, whether uterine phenotype in LH receptor knockout animals resulted also from the absence of direct actions of LH in the uterus. The phenotype consisted of marked growth reduction of uterus, decreased thickness of endometrial and myometrial layers, number of endometrial glands, height of luminal epithelium and vascular space. Analysis of uterine gene expression by mouse genome U74Av2 Affymetrix genechips revealed a differential expression of 155 genes by more than 3-fold (range 3–53-fold) between null and wild-type animals. Of these, 89 genes decreased and 66 increased in uterus of null animals. Semi-quantitative RT-PCR confirmed the differential expression of several selected genes. The decreased genes can be clustered into 18 functional families and the increased into 15 functional families. Semi-quantitative RT-PCR, Western blotting and immunocytochemistry demonstrated a decreased expression of ERβ, PR-A, PR-B and AR in uterus of null animals as compared with wild-type siblings. Twenty-one-day estradiol and progesterone replacement therapy did not normalize the decrease in the number of endometrial glands and several genes that either decreased or increased in expression. The partial success of therapy suggests that direct LH actions could be required to completely normalize the uterus. In summary, findings on the knockout model reaffirm that LH and hCG control uterine functions directly as well as indirectly through increasing ovarian synthesis of steroid hormones and both actions are required for normal uterine biology.

Novel insertion frameshift mutation of the LH receptor gene: problematic clinical distinction of Leydig cell hypoplasia from enzyme defects primarily affecting testosterone biosynthesis

Leydig cell hypoplasia (LCH) is a rare autosomal recessive condition that interferes with normal development of male external genitalia in 46,XY individuals and is caused by inactivating mutations of the LH receptor gene. The clinical and biochemical diagnostic parameters of LCH are not always specific and may therefore show significant overlap with other causes of insufficient testicular steroid biosynthesis. We have studied a 46,XY newborn with completely female external genitalia and palpable testes. Due to an increased basal serum ratio of androstenedione/testosterone, 17 beta-hydroxysteroid dehydrogenase type 3 (17 beta-HSD 3) deficiency was initially suspected. DNA analysis of the corresponding HSD17B3 gene, however, showed no abnormalities in the entire coding region. In contrast, direct sequencing of the LH receptor gene revealed a novel homozygous single nucleotide insertion in exon 11 (codon A589fs) producing a frame shift in the open reading frame predicting for premature termination of translation 17 amino acids downstream. From the genetic perspective, this mutation represents the first frame shift mutation in the LH receptor gene ever reported to date. From the clinical standpoint, LCH should always be considered in the differential diagnosis as steroid profiles may not be informative. Therefore, molecular genetic analysis should be warranted for androgen biosynthesis defects in all cases.

163 DOES LH PLUS hCG, USED TO INDUCE OVULATION IN SUPERSTIMULATED COWS, IMPROVE PREGNANCY RATES AFTER TRANSFER OF EMBRYOS THAT ARE NOT EXCELLENT?

The LH receptor (LHr) present in ovarian follicle cells is essential for the biological response due to its binding to the ligand (LH). The events following the LH surge are related to the presence of LHr and its affinity to LH. Studies on LHr gene expression in theca and granulosa cells from bovine follicles have demonstrated the presence of at least four isoforms of LHr mRNA by alternative splicing. Two of the four detected isoforms have an open reading frame (ORF) and can be translated to functional proteins (receptors coupled with G protein) with different affinities to their ligands (Robert C et al. 2003 Reproduction 125, 437–446). From those two isoforms with ORF, one (full) has affinity to both LH and hCG molecules, whereas the second isoform (with deletion of exon 10) has affinity to hCG only. Recently we have detected the same four isoforms described by Robert et al. in both granulosa and theca cells from abattoir. Based on this information, the present study tested the hypothesis that, in cows superstimulated with FSH, administration of both LH and hCG, as an attempt to stimulate any variety of LH receptor present in the follicles, would result in improvement of oocyte quality and/or increase in ovulation rate. Nelore cows (Bos taurus indicus) were superstimulated according to the protocol termed P-36 (Barros CM et al. 2003 Theriogenology 59, 524 abst), and embryos were flushed 7 to 8 days after inducing ovulation. Ovulation was induced with LH (pLH, 12.5 mg, i.m.; Lutropin, Vetrepharm, London, Ontario, Canada; Group 1) or both LH (12.5 mg) and hCG (1500 IU, i.m.; Choragon, Ferring GmbH, Kiel, Germany; Group 2). Superstimulation protocol and embryo transfer were performed simultaneously on both groups. Mean (± SEM) of total structures, viable embryos, and viability rate were: 12.4 ± 2.36, 10.0 ± 2.38 and 80.8% (Group 1, n = 8 flushings) and 12.2 ± 2.03, 8.9 ± 1.66, and 73.1% (Group 2, n = 14); there was no significant difference between groups (P = 0.96; P = 0.71 (ANOVA), and P = 0.18 (Fisher's Exact Test), respectively). In a subset of embryos (excellent, good, fair, and poor qualities) transferred without freezing, the pregnancy rates for groups 1 and 2 were 41.7% (25/60) and 56.9% (37/65), respectively (P = 0.11, Fisher's Exact Test). The overall pregnancy rates from embryos of excellent, good, and fair qualities were 46.3% (25/54) and 58.9% (33/56), respectively for Groups 1 and 2 (P = 0.25). When only embryos of good, fair, and poor qualities were pooled, the pregnancy rates were 18.8% (06/32) and 60.0% (30/50), respectively for Groups 1 and 2 (P = 0.0003). It is concluded that simultaneous administration of LH and hCG to induce ovulation in superstimulated animals did not alter production of viable embryos or viability rate. However, the use of hCG in addition to LH administration could be beneficial for embryos of good, fair, and poor qualities. Nogueira has a fellowship from FAPESP (São Paulo).

237 EXPRESSION OF THE FULL-LENGTH AND ALTERNATIVELY SPLICED BOVINE LH RECEPTOR mRNAs IN GRANULOSA CELLS FROM FOLLICLES ≥ 7 mm IN DIAMETER

The objective of this study was to characterize the pattern of gene expression of LH receptor (LHR) transcripts from bovine antral follicles from 5 to 14 mm in diameter (theca and granulosa cells, TC and GC, respectively) in crossbreed cattle and from cultured GC. From ovaries collected in abattoir (Bos indicus � B. taurus cattle), antral follicles were dissected, and samples of TC and GC were obtained for total RNA extraction (Trizol protocol). Steroid concentrations in the follicular fluid were determined by RIA. Samples of GC cultured for 6 days (obtained from B. taurus follicles), were treated with FSH (0 (control), 1, or 10 ng) and processed for total RNA extraction. Total RNA (1 �g) was utilized in the RT reaction (SuperScript III; Invitrogen, Brasil Ltd., S�o Paulo, Brazil). The PCR conditions were 29 cycles of 95�C, 60�C, and 70�C (1 min each) for denaturation, annealing, and extension (PTC200 thermocycler; MJ Research, Biozym, Landgraaf, The Netherlands). Gene expression of LHR was measured by semiquantitative RT-PCR with specific primers to amplify the fragment from exon 9 to exon 11 (LHRBC; full-length amplicon with 1240 bp). To investigate a putative site of alternative splicing on exon 3 specific primers were utilized to amplify a fragment containing the exons 2 to 9 (LHRA). As an internal control of the PCR, GAPDH expression was used. After sequencing, four isoforms were detected from the LHRBC fragment two with deletion of exon 10 (M2 and M4) and two with deletion of part of exon 11 (M3 and M4) as well as the fragment without deletions (M1). There was no correlation (P &gt; 0.05; Spearman correlation) between LHRBC isoform expression and steroid hormones or follicular diameter (TC). However, estradiol and progesterone concentrations (r &gt; 0.51 and P &lt; 0.01) and follicular diameter (r &gt; 0.82 and P &lt; 0.01) were correlated with expression of the four LHRBC isoforms (GC). Expression of isoforms from fragment LHRBC was observed in GC from follicles with a diameter of 7 mm. In six follicles with a diameter of 7 mm, only one (16.7%) expressed LHR in GC, whereas most of the follicles e 8 mm expressed LHR (87.5%, 21/24). No LHR isoforms were detected in GC from follicles (n = 7) d 6 mm in diameter. From LHRA fragment amplification, an alternative transcript with a deletion of 75 bp, homologous to the rat exon 3, was detected by sequencing. In cultured GC (without FSH treatment), only a weak LHR expression (in vitro control) was observed when compared with the in vivo control (TC sample). The treatment with FSH (1 or 10 ng) was effective to induce LHR expression in cultured GC, however, with a different pattern of expression (lower M1/M2 ratio) when compared to in vivo GC samples (0.8 � 0.14 vs. 3.5 � 0.66; mean � SEM, P &lt; 0.01, unpaired t-test). It is concluded that, in GC from follicles with a diameter e 7 mm, the gene expression of the LHR was positively correlated with follicular diameter and with estradiol and progesterone concentration in the follicular fluid. Treatment with FSH was effective in inducing LHR expression in cultured GC, however, with a different pattern than in the in vivo control. Additionally, sites of alternative splicing were detected in exons 3, 10, and 11 from the bovine LHR gene. This work was supported by a Fellowship from FAPESP.