Gonadotropin releasing hormone antagonist use in controlled ovarian stimulation and intrauterine insemination cycles in women with polycystic ovary syndrome.

OBJECTIVE: To evaluate the effect of the GnRH antagonist on gonadotropin ovulation induction in women with PCOS. MATERIALS AND METHODS: A total of 175 intrauterine insemination (IUI) cycles in women with polycystic ovary syndrome (PCOS) were included in the study. Women in the control group (n = 87) underwent controlled ovarian stimulation (COS) with recombinant follicle stimulating hormone (r-FSH) only, while women in the study group (n = 88) were administered r-FSH plus cetrorelix. RESULTS: As expected, the mean value of luteinizing hormone and progesterone, on the day of human chorionic gonadotropin administration were statistically significantly lower in patients receiving GnRH antagonist than the control group (p = 0.002). Premature luteinization occurred in only one of the patients in the GnRH antagonist group (1.1%) and in 15 of the 88 cycles in the control group (17.2%), showing a significant difference between the two groups (P = 0.001). The clinical pregnancy rate per cycle was higher in GnRH-antagonist group compared to the control group but the difference did not reach to a statistical significance (25% vs 14.9%, P = 0.096). CONCLUSIONS: Adding GnRH-antagonist in COS/IUI cycles in women with PCOS resulted in a lower incidence of premature luteinization but did not improve pregnancy rates. However, owing to some benefits, antagonist therapy could be considered as a reasonable alternative to IVF in order to reduce PCOS patients'emotional distress.

'Carriers of variant luteinizing hormone (V-LH) among 1593 Baltic men have significantly higher serum LH'.

Luteinizing hormone (LH) is a pituitary heterodimeric glycoprotein essential in male and female reproduction. Its functional polymorphic variant (V-LH) is determined by two missense mutations (rs1800447, A/G, Trp8Arg; rs34349826, A/G, Ile15Thr) in the LH ß-subunit encoding gene (LHB; 19q13.3; 1111 bp; 3 exons). Among women, V-LH has been associated with higher circulating LH and reduced fertility, but the knowledge of its effect on male reproductive parameters has been inconclusive. The objective of this study was to assess the effect of V-LH on hormonal, seminal and testicular parameters in the Baltic young men cohort (n = 986; age: 20.1 ± 2.1 years) and Estonian idiopathic infertility patients (n = 607; 35.1 ± 5.9 years). V-LH was detected by genotyping of the underlying DNA polymorphisms using PCR-RFLP combined with resequencing of a random subset of subjects. Genetic associations were tested using linear regression under additive model and results were combined in meta-analysis. No significant difference was detected between young men and infertility patients for the V-LH allele frequency (11.0 vs. 9.3%, respectively). V-LH was associated with higher serum LH in both, the young men cohort (p = 0.022, allelic effect = 0.26 IU/L) and the idiopathic infertility group (p = 0.008, effect = 0.59 IU/L). In meta-analysis, the statistical significance was enhanced (p = 0.0007, resistant to Bonferroni correction for multiple testing; effect = 0.33 IU/L). The detected significant association of V-LH with increased serum LH remained unchanged after additional adjustment for the SNPs previously demonstrated to affect LH levels (FSHB -211G/T, FSHR Asn680Ser, FSHR -29A/G). Additionally, a suggestive trend for association with reduced testicular volume was observed among young men, and with lower serum FSH among infertility patients. The V-LH carrier status did not affect sperm parameters and other circulating reproductive hormones. For the first time, we show a conclusive contribution of V-LH to the natural variance in male serum LH levels. Its downstream clinical consequences are still to be learned.

Molecular characterization of three gonadotropin subunits and their expression patterns during ovarian maturation in Cynoglossus semilaevis.

The endocrine regulation of reproduction in a multiple spawning flatfish with an ovary of asynchronous development remains largely unknown. The objectives of this study were to monitor changes in mRNA expression patterns of three gonadotropin hormone (GTH) subunits (FSHß, LHß and CGα) and plasma GTH levels during ovarian maturation of half-smooth tongue sole Cynoglossus semilaevis. Cloning and sequence analysis revealed that the cDNAs of FSHß, LHß and CGα were 541, 670 and 685 bp in length, and encode for peptides of 130, 158 and 127 amino acids, respectively. The number of cysteine residues and potential N-linked glycosylation sites of the flatfish GTHs were conserved among teleosts. However, the primary structure of GTHs in Pleuronectiformes appeared to be highly divergent. The FSHß transcriptional level in the pituitary remained high during the vitellogenic stage while plasma levels of FSH peaked and oocyte development was stimulated. The LHß expression in the pituitary and ovary reached the maximum level during oocyte maturation stages when the plasma levels of LH peaked. The brain GTHs were expressed at the different ovarian stages. These results suggested that FSH and LH may simultaneously regulate ovarian development and maturation through the brain-pituitary-ovary axis endocrine system in tongue sole.

Trp28Arg/Ile35Thr LHB gene variants are associated with elevated testosterone levels in women with polycystic ovary syndrome.

INTRODUCTION: Polycystic Ovary Syndrome (PCOS) is a complex endocrine disorder, of multifactorial etiology, which affects 6-10% of women of reproductive age. It is considered the leading cause of anovulatory infertility, menstrual disorders and hyperandrogenism in this population. The genetic basis of PCOS is still largely unknown despite significant family clustering; determining its mode of inheritance is particularly difficult given the heterogenic presentation of the disease. MATERIALS AND METHODS: 130 Brazilian women, aged 14-42 years, who met the 2003 Rotterdam criteria for PCOS diagnosis, were included, and 96 healthy women constituted the control group. Presence of hirsutism was classified using the modified Ferriman-Gallwey score (F-G score) as absent (≤7), mild (8-14), and severe (≥15). Blood levels of luteinizing hormone (LH), total testosterone (TT), dehydroepiandrosterone sulfate (DHEA-S) and androstenedione were determined. The coding region of the luteinizing hormone beta-subunit (LHB) gene was amplified and sequenced. Differences in allelic and genotypic frequency distribution of each polymorphism across controls and cases were estimated by the Mantel-Haenszel chi-square or Fisher's exact test (p<0.05), and the probability of an association between the detection of a polymorphism and presence of a diagnosis of PCOS, by logistic regression. RESULT(S): Sequencing detected 8 polymorphisms in the LHB gene coding region. Two polymorphisms in linkage disequilibrium were significantly more prevalent in the presence of hyperandrogenemia: rs1800447/rs34349826 (Trp28Arg/Ile35Thr) (p=0.02). CONCLUSION(S): In this series, a modulatory effect of LHB polymorphisms on hyperandrogenemia phenotype of PCOS was observed; however, this finding needs to be replicated in other populations.

Variant-beta luteinizing hormone is not associated with poor ovarian response to controlled ovarian hyperstimulation.

BACKGROUND: The most common genetic variant of luteinizing hormone (LH), variant-betaLH, has a different bioactivity than the wildtype. Carrying the variant allele was associated with an increased consumption of exogenous gonadotropin to achieve optimal ovarian response for in vitro fertilization procedures (IVF). The aim of this study was to examine if variant-betaLH was also more common in patients with a poor ovarian response to exogenous gonadotropin which negatively influenced treatment outcome. FINDINGS: 36 patients with poor ovarian response to ovarian stimulation for IVF and 98 controls with a normal response were genotyped for variant-betaLH using DNA sequencing. The carrier frequency in the control group was 17%. No association was found between poor ovarian response and variant-betaLH. CONCLUSIONS: Testing patients for variant-betaLH prior to IVF is unlikely to predict poor ovarian response.

Free fatty acids induce Lhb mRNA but suppress Fshb mRNA in pituitary LßT2 gonadotropes and diet-induced obesity reduces FSH levels in male mice and disrupts the proestrous LH/FSH surge in female mice.

Female obesity is associated with insulin resistance, hyperandrogenemia, and reproductive dysfunction. We hypothesized that elevated free fatty acids (FFAs) might directly modulate pituitary gonadotropin production. FFAs caused a time- and dose-dependent increase in phosphorylation of the MAPKs p38MAPK, c-Jun N-terminal kinase (JNK)-1/2, and ERK1/2 in LßT2 gonadotrope cells. Furthermore, FFAs up-regulated Lhb mRNA expression acutely, an effect that was blocked by JNK inhibition, but suppressed Fshb mRNA expression, an effect that was independent of MAPK signaling. FFAs enhanced the activation of the MAPKs in the presence of GnRH, although the cotreatment did not alter Lhb induction but did eliminate the GnRH induction of Fshb. FFAs also suppressed activin-induced Fshb expression. Knockdown experiments showed that the FFA effect on the inflammatory kinases p38MAPK and JNK and on Lhb, but not Fshb, mRNA expression is mediated via toll-like receptor-2 and toll-like receptor-4 and was mimicked by lipopolysaccharide stimulation. In vivo, male C57BL/6 mice on a high-fat diet showed reduced FSH levels consistent with the suppression of Fshb seen in vitro. Histological analysis of the testes showed an increased number of abnormal seminiferous tubules. Female mice on a high-fat diet lacked the expected proestrus LH and FSH surge and exhibited an increase in the number of days at estrus and a reduced number of days at proestrus, and ovaries had significantly fewer corpora lutea. Taken together, our findings suggest that lipid excess can lead to reproductive defects in both male and female mice.

Evidence for cross-talk between the LH receptor and LH during implantation in mice.

The present study investigated the first interaction that occurs between the blastocyst and endometrium during implantation. Given the ethical objections to studying implantation in humans, a mouse model was used to study the dialogue between luteinising hormone (LH) and luteinising hormone receptor (LHCGR). Several studies performed on LHCGR-knockout mice have generated controversy regarding the importance of the dialogue between LH and LHCGR during implantation. There has been no demonstration of a bioactive LH-like signal produced by the murine blastocyst. The first aim of the present study was to examine and quantify, using radioimmunoassay, the generation of a bioactive LH signal by the murine blastocyst. We went on to examine and quantify endometrial Lhcgr expression to validate the mouse model. Expression of LHCGR in mouse uteri was demonstrated using immunohistochemistry and western blot analysis. To quantify the expression of Lh in the mouse blastocyst and Lhcgr in the endometrium, reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative (q) RT-PCR were performed. The results demonstrate that Lhcgr expression in BALB/c mouse endometrial epithelium is increased at the time of implantation and indicate that LHCGR may contribute to the implantation process. In support of this hypothesis, we identified a bioactive LH signal at the time of murine blastocyst implantation.

Hypogonadism in a patient with two novel mutations of the luteinizing hormone ß-subunit gene expressed in a compound heterozygous form.

CONTEXT: LH gene mutations are rare; only four mutations have been described. The affected individuals are hypogonadal. PATIENT: We describe the clinical features of a 31-yr-old man who presented with delayed puberty and azoospermia and was found to have hypogonadism associated with an absence of circulating LH. MAIN OUTCOME MEASURES AND RESULTS: The patient had a 12-bp deletion in exon 2 in the LH ß-subunit gene and a mutation of the 5' splice site IVS2+1G→T in the same gene present in a compound heterozygous state. The first mutation predicts a deletion of four leucines of the hydrophobic core of the signal peptide. The second mutation disrupts the splicing of mRNA, generating a gross abnormality in the processing. The patient's heterozygous parents were clinically normal. The phenotype of a 16-yr-old sister of the proband, carrying the same mutations, was characterized by normal pubertal development and oligomenorrhea. CONCLUSION: This report unravels two novel mutations of the LH gene critical for synthesis and activity of the LH molecule. The insight gained from the study is that normal pubertal maturation in women can occur in a state of LH deficiency, whereas LH is essential for maturation of Leydig cells and thus steroidogenesis, puberty, and spermatogenesis in man. These mutations should be considered in girls and boys with selective deficiency of LH.

Microarray screening for novel preeclampsia biomarker candidates.

INTRODUCTION: Our aim was to identify novel biomarker candidates for the near-term prediction of preeclampsia in a homogenous collective. In this study, we screened at the genome-wide level for gene expression in placental villous tissue from patients with severe preeclampsia in comparison to normal healthy pregnancies. MATERIAL AND METHODS: Total RNA was extracted from placental villous tissue from 9 preeclamptic patients and 7 normotensive controls after scheduled cesarean sections. After sample pooling, gene expression analysis was performed using six Affymetrix Human Gene 1.0 ST arrays, followed by quantitative RT-PCR and validation of selected markers in the serum of patients at the protein level. RESULTS: In total, 896 significantly differentially expressed genes were identified (p ≤ 0.05). After restricting these to molecules present in the circulation, 9 upregulated and 5 downregulated genes were selected. Four of them (ß-hCG, HTRA4, LHB1, all upregulated; and NOX4, downregulated) were validated by quantitative real-time RT-PCR. Finally, the maternal plasma protein levels of 2 of these genes (LHB and ß-hCG) were confirmed to be significantly different between preeclampsia cases and controls. DISCUSSION: We identified 14 potential new biomarker candidates for preeclampsia and validated 4 of them by quantitative RT-PCR and 2 of them with subsequent serum protein analyses. Further studies will assess the optimal marker combination for the imminent prediction of impending preeclampsia.

Seasonal effect of GnIH on gonadotrope functions in the pituitary of goldfish.

Gonadotropin-inhibitory hormone (GnIH) inhibits gonadotropin release in birds and mammals. To investigate its role in teleosts, we examined the effects of synthetic goldfish (g)GnIH on pituitary LH-ß and FSH-ß subunit, and gGnIH receptor (gGnIH-R) mRNA levels and LH secretion in goldfish. Intraperitoneal injections of gGnIH increased pituitary LH-ß and FSH-ß mRNA levels at early to late gonadal recrudescence, but reduced serum LH and pituitary gGnIH-R mRNA levels, respectively, at early to mid-recrudescence and later stages of recrudescence. Static incubation with gGnIH elevated LH secretion from dispersed pituitary cell cultures from prespawning fish, but not at other recrudescent stages; suppressed LH-ß mRNA levels at early recrudescence and prespawning but elevated LH-ß at mid-recrudescence; and consistently attenuated FSH-ß mRNA in a dose-specific manner. Results indicate that in goldfish, regulation of LH secretion and gonadotropin subunit mRNA levels are dissociated in the presence of gGnIH and dependent on maturational status and administration route.

Determination of Fsh quantity and bioactivity during sex differentiation and oogenesis in European sea bass.

Follicle-stimulating hormone (FSH) is a glycoprotein hormone that plays a key role in the regulation of gonadal functions in vertebrates. The present study reports the monitoring of pituitary and plasma Fsh levels during sex differentiation and oogenesis in European sea bass (Dicentrarchus labrax) using a homologous immunoassay and an in vitro bioassay. Both assays were used complementarily for the first time in a fish species. High levels of Fsh bioactivity in plasma were found during the initial phases of sexual differentiation. Plasma and pituitary Fsh (quantity and bioactivity) levels and biological to immunological (B:I) ratios were higher in females than in males, suggesting sexual dimorphism in the synthesis and potency of Fsh. In females, the B:I ratios in adult were lower than during sex differentiation indicating that Fsh would be less biopotent in the adult stage. Plasma Fsh bioactivity levels increased during vitellogenesis, suggesting that Fsh would be involved in the regulation of the midphases of oogenesis, whereas luteinizing hormone would be responsible for the final events.

Defining global gene expression changes of the hypothalamic-pituitary-gonadal axis in female sGnRH-antisense transgenic common carp (Cyprinus carpio).

BACKGROUND: The hypothalamic-pituitary-gonadal (HPG) axis is critical in the development and regulation of reproduction in fish. The inhibition of neuropeptide gonadotropin-releasing hormone (GnRH) expression may diminish or severely hamper gonadal development due to it being the key regulator of the axis, and then provide a model for the comprehensive study of the expression patterns of genes with respect to the fish reproductive system. METHODOLOGY/PRINCIPAL FINDINGS: In a previous study we injected 342 fertilized eggs from the common carp (Cyprinus carpio) with a gene construct that expressed antisense sGnRH. Four years later, we found a total of 38 transgenic fish with abnormal or missing gonads. From this group we selected the 12 sterile females with abnormal ovaries in which we combined suppression subtractive hybridization (SSH) and cDNA microarray analysis to define changes in gene expression of the HPG axis in the present study. As a result, nine, 28, and 212 genes were separately identified as being differentially expressed in hypothalamus, pituitary, and ovary, of which 87 genes were novel. The number of down- and up-regulated genes was five and four (hypothalamus), 16 and 12 (pituitary), 119 and 93 (ovary), respectively. Functional analyses showed that these genes involved in several biological processes, such as biosynthesis, organogenesis, metabolism pathways, immune systems, transport links, and apoptosis. Within these categories, significant genes for neuropeptides, gonadotropins, metabolic, oogenesis and inflammatory factors were identified. CONCLUSIONS/SIGNIFICANCE: This study indicated the progressive scaling-up effect of hypothalamic sGnRH antisense on the pituitary and ovary receptors of female carp and provided comprehensive data with respect to global changes in gene expression throughout the HPG signaling pathway, contributing towards improving our understanding of the molecular mechanisms and regulative pathways in the reproductive system of teleost fish.

Components of the porcine anterior pituitary insulin-like growth factor system throughout the estrous cycle.

Components of the circulating and anterior pituitary insulin-like growth factor (IGF) system vary in response to steroids in pigs. However, whether serum and anterior pituitary concentrations of the IGF system vary throughout the estrous cycle has not been determined. To further examine this relationship, estrus was synchronized in 40 gilts of similar age and weight (180 d; 120 kg) by feeding 15 mg altrenogest for 15 d to synchronize estrus. Gilts were checked twice daily for expression of estrus beginning 3 d after the end of altrenogest treatment and continuing for 7 d. The first day each gilt exhibited estrus was designated as day 1 of the estrous cycle. Blood samples were obtained by jugular venipuncture on days 1, 4, 7, 10, 13, 16, 19, and 22 of the estrous cycle. On days 7, 13, 19, and 22 of the estrous cycle 10 pigs were killed and anterior pituitary glands (AP) were collected. Serum concentrations of IGF-I and AP concentrations of IGF-I were determined by radioimmunoassay. Relative amounts of AP IGF binding protein (IGFBP) were determined by western ligand blot analysis. Relative expression of AP IGF-I, IGF-I receptor (IGF-I-R), gonadotropin-releasing hormone receptor (GnRHR), and luteinizing hormone (LH)-ß subunit were determined by real-time reverse transcription polymerase chain reaction. Serum concentrations of IGF-I fluctuated throughout the estrous cycle. Mean serum concentrations of IGF-I decreased (P < 0.02) from day 1 through day 10, increased (P < 0.02) on days 13 through 16, and then decreased (P < 0.02) from days 19 through 22. Mean AP concentrations of IGF-I were greater (P < 0.03) on day 19 than on all other days, whereas no difference was detected (P > 0.05) in mean AP concentrations of IGF-I on days 7, 13, and 22. Mean relative amounts of AP IGFBP-2 and -5 were each greater (P < 0.02) in gilts on day 19 than on all other days, whereas no difference was detected (P > 0.05) in mean relative amounts of AP IGFBP-2 and -5 among pigs on days 7, 13, and 22 of the estrous cycle. Relative expression AP IGF-I was greater (P < 0.05) on days 13, 19, and 22 than on day 7 of the estrous cycle. Similarly, the relative expression of AP IGF-IR was increased (P < 0.05) in gilts on days 13, 19, and 22 compared with day 7. The relative expression of GnRHR was greater (P < 0.05) on days 13 and 22 of the estrous cycle than on day 7. The relative expression of LHß subunit was greater (P < 0.05) on day 19 of the estrous cycle than on days 7, 13, and 22. Anterior pituitary release of LH throughout the porcine estrous cycle may be modulated by changes in the intrapituitary IGF system.

Plasticity of the reproductive axis caused by social status change in an african cichlid fish: I. Pituitary gonadotropins.

Social position in a dominance hierarchy is often tightly coupled with fertility. Consequently, an animal that can recognize and rapidly take advantage of an opportunity to rise in rank will have a reproductive advantage. Reproduction in all vertebrates is controlled by the brain-pituitary-gonad axis, and in males of the African cichlid fish Astatotilapia burtoni, GnRH1 neurons at the apex of this axis are under social control. However, little is known about how quickly social information is transformed into functional reproductive change, or about how socially controlled changes in GnRH1 neurons influence downstream actions of the brain-pituitary-gonad axis. We created an opportunity for reproductively suppressed males to ascend in status and then measured how quickly the perception of this opportunity caused changes in mRNA and protein levels of the pituitary gonadotropins. mRNA levels of the ß-subunits of LH and FSH rose rapidly in the pituitary 30 min after suppressed males perceived an opportunity to ascend. In contrast, mRNA levels of GnRH receptor-1 remained unchanged during social transition but were higher in stable dominant compared with subordinate males. In the circulation, levels of both LH and FSH were also quickly elevated. There was a positive correlation between mRNA in the pituitary and circulating protein levels for LH and FSH, and both gonadotropins were positively correlated with plasma 11-ketotestosterone. Our results show that the pituitary is stimulated extremely rapidly after perception of social opportunity, probably to allow suppressed males to quickly achieve reproductive success in a dynamic social environment.

Función hormonal y flujo sanguíneo útero-ovárico en pacientes sometidos a salpingectomía / Hormonal function and uterine and ovary blood flow in patients under salpingectomy

El objetivo de la investigación fue comparar los efectos de la salpingectomía sobre la función hormonal y el flujo sanguíneo útero-ovárico. Se incluyeron 33 mujeres sanas con ciclos menstruales regulares. La salpingectomía fue realizada inmediatamente en los 5 días siguientes a la menstruación. Se tomaron muestras de sangre venosa para determinar las concentraciones de las hormonas folículo estimulante y luteinizante, estradiol y progesterona antes y luego de 3 meses de la cirugía. El flujo sanguíneo fue evaluado usando Doppler color y de pulso para medir el índice de resistencia, índice de pulsatilidad y velocidad sistólica pico en la vasculatura ovárica y las arterias uterinas. Se observó un aumento estadísticamente significativo en las concentraciones de hormonas folículo estimulante y luteinizante después de 3 meses de la salpingectomía (P<0,05). No se encontraron diferencias estadísticas significativas en las concentraciones de estradiol y progesterona (P=ns). Se observaron variaciones estadísticas significativas en los valores de velocidad sistólica pico, índice de resistencia e índice de pulsatilidad de la arteria ovárica al comparar los valores antes y 3 meses después de la salpingectomia (P<0,05). También se observó un aumento estadísticamente significativo en el volumen de los ovarios después de 3 meses de la cirugía (P<0,05). Se concluye que luego de 3 meses, la salpingectomía produce cambios en las concentraciones de gonadotropinas, tamaño de los ovarios y en los parámetros ecográficos Doppler de las arterias ováricas

The objetive of research was to compare the effects of salpingectomy over hormonal function and uterine and ovary blood flow. Thirty-three healthy women with normal menstrual cycles were included. Salpingectomy was performed immediately in five days following menstruation. A venous blood sample was taken to determine folicular stimulant and luteinizant hormones, estradiol and progesterone before and after three months of surgery. Bood flow was evaluated using color and pulse Doppler to measure resistance index, pulsatility index and peak systolic flow in ovary vasculare and uterine artery. There were statically significant increased in folicular stimulant and luteinizant hormones concentrations after three months of salpingectomy (P<0.05). There were not significant differences in estradiol and progesterone concentrations (P=ns). There were statistically significant variations in values of peak systolic flow, resistance index and pulsatility index of ovarian arteries when values before and three moths after salpingectomy were compared (P<0.05). There was also a statistically significant increase in ovary volume after three months of surgery (P=0.05). It is concluded that after three months, salpingectomy produces changes in gonadotropins concentrations, ovary size and ultrasound Doppler parameters of ovarian artery

The intrapituitary endocrine events during maturation and timing of puberty in the female sheep.

The aim of this study was to determine the maturational activity of gonadotroph cells, the site of synthesis, storage and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in Polish Merino female sheep born after the summer solstice. The actual time of puberty of these lambs was delayed until the following breeding season, when they were 14 months old. Changes were examined in 12 peripubertal (30-, 52-week-old) and pubertal (Days 15 and 17 of the second ovarian cycle) females. Histomorphological and functional changes in the gonadotroph population were assayed with hybridohistochemistry, immunohistochemistry, computer-assisted image analysis and radioimmunoassay. The percentage of the adenohypophyseal area (PAA) occupied by gonadotrophs containing LHbeta-mRNA was higher and the LH plasma concentration and pulse frequency were lower in the 52-week-old sheep in comparison with the 30-week-old sheep (P<0.05). The PAA occupied by immunoreactive (ir)-LHbeta-cells remained stable at the 30th and 52nd weeks of age and then increased at the pubertal follicular phase. The PAA occupied by ir-FSHbeta-cells was higher in the 52-week-old sheep compared with the 30-week-old sheep and then lower at the pubertal follicular phase (P<0.05). The PAA occupied by gonadotrophs containing LHbeta-mRNA or FSHbeta-mRNA was lower at the pubertal follicular phase in comparison with the 52nd week of age (P<0.05). In pubertal sheep, the PAA occupied by gonadotrophs containing LHbeta-mRNA or FSHbeta-mRNA was higher and the PAA occupied by ir-LHbeta or ir-FSHbeta-cells was lower at the preovulatory phase in comparison with the follicular phase of the cycle (P<0.05). In conclusion, the photoperiodic suspension of gonadotroph population's maturational functions has been observed at the level of LH storage and release but not at the level of LH synthesis during the expected time of puberty in female sheep of an aseasonal breed such as Merino. The findings show the heterogeneity in the patterns of LH and FSH post-transcriptional processing during the period of peripubertal/pubertal transition, explained by the different intrapituitary regulation at the level of post-transcriptional synthesis and storage rather, than at the level of release. Altogether, intrapituitary mechanisms of ovine maturation could have the histomorphological feature. Our observations prompt the hypothesis that the female lamb may be able to transduce changes in day length into the appropriate endocrine cues for sexual maturation after attainment by the pituitary gonadotroph population the full peripubertal efficiency, manifested by the sufficient storage of LH.

Effects of a depot formulation of the GnRH agonist leuprorelin on the ultrastructure of male rat pituitary gonadotropes.

To clarify the acute and chronic effects of GnRH agonists on pituitary gonadotropes, changes in the ultrastructure of male rat gonadotropes were examined immunocytochemically and morphometrically after the administration of a one-month depot formulation of the GnRH agonist, leuprorelin. Immediately after the depot administration, the relative amounts of secretory granules drastically decreased in gonadotropes concomitantly with a marked increase in the plasma LH level. After the acute hyperstimulated phase, secretory granules in gonadotropes were gradually restored although the newly synthesized granules were less densely immunolabeled for LHbeta; their relative amounts and sizes were still significantly smaller than the controls after depot treatment for 28 days. Eighty-four days after the leuprorelin depot administration, however, the ultrastructural characteristics of pituitary gonadotropes appeared to recover as observed in controls: there were no significant differences in the relative amounts, sizes, and labeling densities for LHbeta of secretory granules, and the amounts of chromogranin A (CgA) and secretogranin II (SgII) were restored in secretory granules to control levels. When the rats were repeatedly treated with the leuprorelin depot at intervals of 4 weeks, the expression and intracellular storage levels of gonadotropins remained highly suppressed, judging from the labeling density for LHbeta. These findings suggest that the depot formulation of the GnRH agonist could suppress both the biosynthesis and release of gonadotropins for a month by synergistically depleting the intracellular storage of secretory granules at the onset of the treatment and by inducing the subsequent desensitization of the GnRH receptor signaling.

Urinary beta-luteinizing hormone and beta-follicle stimulating hormone immunoenzymometric assays for population research.

OBJECTIVE: We developed assays for measurement of urinary betaLH and betaFSH under collection and storage conditions typical of non-clinical research settings. DESIGN AND METHODS: IEMAs for free betaLH and total betaFSH were validated by standard methods. Stability of urinary betaLH and betaFSH was tested across freeze-thaws and stored long term at 4 degrees C or -20 degrees C, or short term at room temperature, and with heating to dissociate the subunits. RESULTS: The IEMAs exhibited acceptable parallelism, specificity, recovery (averaging 100% for betaLH, 97% for betaFSH), imprecision (maximum within-run and between run CVs, respectively, 4.8% and 25.7% for betaLH, 5.6% and 17.0% for betaFSH), and minimum detectable dose (2.5 pmol/L for betaLH, 6.8 pmol/L for betaFSH). Urine and serum measures were highly correlated (r=0.95 for LH, 0.86 for FSH). There was no consistent decline with any storage type. Dissociation of subunits by heating was needed for betaLH, but not betaFSH. CONCLUSION: These IEMAs measure free betaLH and total betaFSH, overcoming inter-individual variability in, and collection and storage effects on, subunit dissociation, without the need for urine preservatives.

Effects of long-term treatment with resveratrol and subcutaneous and oral estradiol administration on pituitary function in rats.

Hormone replacement therapy (HRT) has been used for several decades to treat menopausal discomforts. However, in the light of recent studies that draw attention to the potential hazards of conventional HRT, various attempts have been undertaken to search for alternatives to classical HRT. Phytoestrogens are claimed to be capable of positively influencing menopausal symptoms, including hot flushes. We designed a long-term study of 3 months to assess the effects of subcutaneous and orally fed 17beta-estradiol (E2), as well as the actions of resveratrol (RES) on pituitary function in female rats. Our results have demonstrated that RES binds with a 10-fold lower affinity to estrogen receptor (ER)-alpha than to ERbeta. The data from the in vivo study revealed that a dosage of 5 microg and 50 microg RES/kg bodyweight per day given to ovariectomized (OVX) rats achieved serum levels of 1.0 and 8.1 microM respectively. Long-term treatment of OVX rats with RES revealed no estrogenic potential on pituitary function in vivo as assessed by LH and prolactin secretion and by regulation of mRNAs for LHalpha, LHbeta, and GnRH receptor. Subcutaneous treatment with E2 in silastic capsules exerted stronger effects on LH and prolactin secretion, as well as on LHbeta, LHalpha, GnRH receptor, and ERbeta mRNA regulation compared with orally applied estradiol benzoate despite comparable serum levels. Levels of aryl hydrocarbon receptor (AhR) mRNA in the pituitary were increased following OVX and attenuated by long-term E2 treatment, whereas RES did not modulate AhR mRNA expression.

Frequency of the allelic variant (Trp8Arg/Ile15Thr) of the luteinizing hormone gene in a Brazilian cohort of healthy subjects and in patients with hypogonadotropic hypogonadism.

PURPOSE: To evaluate the frequency of allelic variant Trp8Arg/Ile15Thr in the luteinizing hormone beta-subunit gene in a Brazilian population of healthy subjects and in patients with hypogonadotropic hypogonadism. SUBJECTS AND METHODS: Two hundred and two adults (115 women) with normal sexual function and 48 patients (24 women) with hypogonadotropic hypogonadism underwent a molecular study of the the luteinizing hormone beta-subunit gene using a polymerase chain reaction technique followed by enzymatic digestion with the restriction enzymes Nco I (for detection of the Trp8Arg point mutation) and Fok I (for detection of the Ile15Thr point mutation). Basal luteinizing hormone and FSH, testosterone, or estradiol levels were measured in 37 healthy subjects (21 women) and in 27 hypogonadotropic hypogonadism patients (13 women) by immunofluorometric methods (hLH-Spec and hFSH-Spec, AutoDELFIA, Wallac Oy, Turku, Finland). RESULTS: The genetic variant of the luteinizing hormone beta-subunit gene was present at a similar frequency in healthy subjects (14.4%) compared to patients with hypogonadotropic hypogonadism (16.6%). There was no effect of the allelic variant of the luteinizing hormone beta-subunit gene on luteinizing hormone levels in patients with hypogonadotropic hypogonadism as compared to healthy subjects. CONCLUSION: This study indicates that the allelic variant Trp8Arg/Ile15Thr of the luteinizing hormone beta-subunit gene is a common polymorphism in the Brazilian population (14.4%). The same frequency of this luteinizing hormone variant in the groups with hypogonadotropic hypogonadism and in the healthy subjects excludes a relationship between this variant and hypogonadotropic hypogonadism.