A polymorphism in the growth hormone-releasing hormone receptor gene: clinical significance?

Two forms of the growth hormone-releasing hormone (GHRH) receptor (GHRH-R) exist in terms of a polymorphism at codon 57. The most common allele possesses GCG, coding for Ala. This codon can also be ACG, replacing the Ala with Thr. The present study demonstrates that the latter occurs in about 20% of pituitary somatotrophinomas, removed from patients with acromegaly. Somatotrophinomas possessing the alternative allele respond, on average, more strongly to GHRH in terms of GH secretion in vitro than tumors which are homozygous for the more common allele. The distribution of the two allelic forms of the GHRH-R did not significantly differ between acromegalic and non-acromegalic subjects. Thus, while the alternative allelic forms may, at least partially, contribute to the variable response of serum GH levels to i.v. GHRH observed in acromegalic and normal subjects, it is unlikely that subjects possessing the rarer form containing Thr in place of Ala at residue 57 are at increased risk of developing acromegaly.

The frequency of follicle stimulating hormone receptor gene polymorphisms in Iranian infertile men with azoospermia

Background: Azoospermia is the medical condition of a man not having any measurable level of sperm in his semen. Follicle stimulating hormone (FSH) is a member of the glycoprotein hormone family that plays an important role in human reproduction because of its essential role in normal spermatogenesis. Various Single Nucleotide Polymorphisms (SNPs) have been reported within FSH receptor (FSHR) gene that may affect the receptor function. Objective: The present study aimed to investigate the correlation between two FSHR SNPs at positions A919G, A2039G, and susceptibility to azoospermia in a group of Iranian azoospermic men. The association between FSH levels within the sera and A919G and A2039G alleles and genotypes were also investigated. Materials and Methods: This case control study was performed on 212 men with azoospermia (126 non-obstructive and 86 obstructive) and 200 healthy Iranian men. Two FSHR gene SNPs were genotyped using PCR-RFLP method. The relationship between FSH levels within the sera and A919G and A2039G alleles and genotypes were also investigated. Results: Statistical analysis indicated that at A919G position, AA genotype and A allele were more frequent in obstructive azoospermia cases compared to non- obstructive or normal men (p=0.001). Regarding A2039G polymorphisms, no significant difference was observed between both azoospermia groups and the controls. The mean level of serum FSH was higher in the non-obstructive men compared to the obstructive patients (23.8 versus 13.8, respectively, p= 0.04). Conclusion: The results of the present study indicated that the genetic polymorphisms in the FSHR gene might increase the susceptibility to azoospermia in Iranian men.

Association of follicle stimulating hormone receptor promoter with ovarian response in IVF-ET patients

Background: Poor ovarian response phenomenon has been observed in some of the in vitro fertilization-embryo transfer patients. Some investigations found that follicle stimulating hormone receptor (FSHR) gene plays a role in the process, but no direct evidence shows the correlation between genotypes of FSHR and ovarian response. Objective: Exploring the molecular mechanism behind the mutation of FSHR promoter association with ovarian granulosa cells and poor ovarian response. Materials and Methods: This cross sectional study was performed using 158 women undergoing the controlled short program ovarian stimulation for IVF treatment. The 263 bp DNA fragments before the follicle stimulating hormone (FSH) receptor 5' initiation site were sequenced in the patients under IVF cycle, 70 of which had poor ovarian response and 88 showed normal ovarian responses. Results: With a mutation rate of 40%, 63 in 158 cases showed a 29th site G→A point mutation; among the mutated cases, the mutation rate of the poor ovarian responders was significantly higher than the normal group (60% versus 23.9%; χ2=21.450, p<0.001). Besides, the variability was also obvious in antral follicle count, and ovum pick-ups. The estradiol peak values and the number of mature eggs between the two groups had significant difference. However, there was no obvious variability (t=0.457, p=0.324) in the basic FSH values between the two groups (normal group, 7.2±2.3 U/L; mutation group, 7.1±2.0 U/L). Conclusion: The activity of FSHR promoter is significantly affected by the 29th site G→A mutation that will weaken promoter activity and result in poor response to FSH.

Regulation and Function of GATA Transcription Factors in Adrenocortical Tumors and Granulosa Cells

Transcription factors play a key role in tumor development, in which dysfunction of genes regulating tissue growth and differentiation is a central phenomenon. The GATA family of transcription factors consists of six members that bind to a consensus DNA sequence (A/T)GATA(A/G) in gene promoters and enhancers. The two GATA factors expressed in the adrenal cortex are GATA-4 and GATA-6. In both mice and humans, GATA-4 can be detected only during the fetal period, whereas GATA-6 expression is abundant both throughout development and in the adult. It is already established that GATA factors are important in both normal development and tumorigenesis of several endocrine organs, and expression of GATA-4 and GATA-6 is detected in adrenocortical tumors. The aim of this study was to elucidate the function of these factors in adrenocortical tumor growth. In embryonal development, the adrenocortical cells arise and differentiate from a common pool with gonadal steroidogenic cells, the urogenital ridge. As the adult adrenal cortex undergoes constant renewal, it is hypothesized that undifferentiated adrenocortical progenitor cells reside adjacent to the adrenal capsule and give rise to daughter cells that differentiate and migrate centripetally. A diverse array of hormones controls the differentiation, growth and survival of steroidogenic cells in the adrenal gland and the gonads. Factors such as luteinizing hormone and inhibins, traditionally associated with gonadal steroidogenic cells, can also influence the function of adrenocortical cells in physiological and pathophysiological states. Certain inbred strains of mice develop subcapsular adrenocortical tumors in response to gonadectomy. In this study, we found that these tumors express GATA-4, normally absent from the adult adrenal cortex, while GATA-6 expression is downregulated. Gonadal markers such as luteinizing hormone receptor, anti-Müllerian hormone and P450c17 are also expressed in the neoplastic cells, and the tumors produce gonadal hormones. The tumor cells have lost the expression of melanocortin-2 receptor and the CYP enzymes necessary for the synthesis of corticosterone and aldosterone. By way of xenograft studies utilizing NU/J nude mice, we confirmed that chronic gonadotropin elevation is sufficient to induce adrenocortical tumorigenesis in susceptible inbred strains. Collectively, these studies suggest that subcapsular adrenocortical progenitor cells can, under certain conditions, adopt a gonadal fate. We studied the molecular mechanisms involved in gene regulation in endocrine cells in order to elucidate the role of GATA factors in endocrine tissues. Ovarian granulosa cells express both GATA-4 and GATA-6, and the TGF-β signaling pathway is active in these cells. Inhibin-α is both a target gene for, and an atypical or antagonistic member of the TGF-β growth factor superfamily. In this study, we show that GATA-4 is required for TGF-β-mediated inhibin-α promoter activation in granulosa cells, and that GATA-4 physically interacts with Smad3, a TGF-β downstream protein. Apart from the regulation of steroidogenesis and other events in normal tissues, TGF-β signaling is implicated in tumors of multiple organs, including the adrenal cortex. Another signaling pathway found often to be aberrantly active in adrenocortical tumors is the Wnt pathway. As both of these pathways regulate the expression of inhibin-α, a transcriptional target for GATA-4 and GATA-6, we wanted to investigate whether GATA factors are associated with the components of these signaling cascades in human adrenocortical tumors. We found that the expression of Wnt co-receptors LRP5 and LRP6, Smad3, GATA-6 and SF-1 was diminished in adrenocortical carcinomas with poor outcome. All of these factors drive inhibin-α expression, and their expression in adrenocortical tumors correlated with that of inhibin-α. The results support a tumor suppressor role previously suggested for inhibin-α in the mouse adrenal cortex, and offer putative pathways associated with adrenocortical tumor aggressiveness. Unraveling the role of GATA factors and associated molecules in human and mouse adrenocortical tumors could ultimately contribute to the development of diagnostic tools and future therapies for these diseases.

Administração de cafeína durante a gestação altera parâmetros testiculares na prole de camundongos C57/BL6 na vida adulta

Metabólitos ativos da cafeína apresentam toxicidade, podendo causar efeitos deletérios em muitos órgãos no período fetal pelo consumo materno. O estudo objetivou avaliar o papel da administração de cafeína durante a gestação em vários parâmetros importantes para a função testicular em camundongos adultos C57BL/6. Fêmeas grávidas foram divididas em: grupo controle (C), que receberam injeções de sol. salina (0,9% NaCl) e grupo cafeína (CF), que receberam diariamente injeções subcutâneas de 20 mg / kg de cafeína. Filhotes tiveram livre acesso à ração padrão desde o desmame até três meses de idade, quando foram mortos. O grupo CF apresentou uma redução no consumo alimentar (C = 4,36 0,14; CF = 3,79 0,05/g, p<0,05) e ganho de massa corporal (C = 25,73 0,14; CF = 21,08 0,41/g, p=0,0001). Ainda este grupo, apresentou alterações estruturais nos testículos da prole, como redução no peso relativo (C = 0,078 0,003; CF = 0,063 0,002/g, p=0,002), diâmetro tubular (C = 455,4 2,7; CF = 393,5 3,1/m, p=0,0001), lume tubular (C = 310,6 2,5; CF = 254,8 2,9/m, p=0.0001) e altura do epitélio seminífero (C = 144,8 0,9; CF = 138,7 1,3/m, p=0,0005) observados pela técnica de morfometria. Testosterona sérica avaliada por quimioluminescência foi reduzida (C = 6,6 2,8; CF = 0,5 0,2/ng/ml, p=0.04). Western Blotting foi utilizado para análise de expressão protéica. Houve aumento do receptor de leptina (C = 0,81 0,04; CF = 1,28 0,15/g, p=0,01), do receptor para o hormônio leteinizante (C = 0,92 0,05; CF = 0,74 0,05/g, p=0,01),da aromatase (C = 0,32 0,03; CF = 0,48 0,05/g, p=0,03) e do regulador pró apoptose (C = 0,27 0,02; CF = 0,42 0,03/g, p=0,01) enquanto o receptor para hormônio folículo estimulante (FSHR) (C = 0,76 0,06; CF = 0,56 0,04/g, p=0,02), o receptor para proteína reguladora da esteroidogênese (C = 1,009 0,065; CF = 0,584 0,060/g, p=0,0007), a proteína do fator de crescimento vascular endotelial (C = 0,33 0,08; CF = 0,05 0,01/g, p=0,009), o receptor de androgênio (C = 0,97 0,11; CF = 0,59 0,07/g, p=0,02) e o antígeno nuclear de proliferação celular (C = 0,93 0,11; CF = 0,48 0,07/g, p=0,01) foram reduzidos. Este estudo sugere que o consumo de cafeína durante a gravidez parece ser nocivo à fertilidade de animais machos, caracterizando o fenômeno de programação, o que gera alterações funcionais e estruturais nos testículos da prole adulta.

Efeito da restrição alimentar materna durante a lactação sobre a função ovariana da prole fêmea na puberdade e vida adulta

α.O objetivo deste estudo foi avaliar se a restrição alimentar materna durante a lactação altera a função ovariana da prole na puberdade e na vida adulta. No dia do nascimento da ninhada, as ratas foram separadas aleatoriamente nos seguintes grupos: (C) controle = dieta com 23% de proteína; (PER) restrição protéica calórica = dieta com 8% de proteína. Após o desmame, as proles tiveram livre acesso a dieta com 23% proteína e apenas os animais na fase diestro foram sacrificados com uma dose letal de pentobarbital aos 40 e 90 dias de idade. O sangue foi retirado por punção cardíaca e o soro armazendo para posterior dosagem dos níveis hormonais por radioimunoensaio. O ovário direito foi excisado, pesado e armazenado a 80C para subseqüente avaliação do receptor de androgênio (AR), das isoformas dos receptores de estrogênio (ERα, ERβ1 e ERβ2), do receptor do hormônio folículo estimulante (FSHR), do receptor do hormonio luteinizante (LHR), das isoformas dos receptores de leptina (Ob-R, Ob-Ra and Ob-Rb), e da enzima aromatase pela técnica de RT-PCR. O ovário esquerdo foi incluido em parafina, seccionado em cortes de 5 μm de espessura e processado por análise histológica de rotina, para classificação dos foliculos ovarianos. Na puberdade, o grupo PER apresentou um aumento significativo (p<0,05) no número dos folículos pré-antrais e antrais, enquanto o número de folículos primordiais, folículos de Graaf e corpo lúteo foram reduzidos significativamente (p<0,05). As concentrações sericas de estradiol (pg/ml) foram significativamente aumentadas (p<0,05). Houve aumento significativo na expressão do RNAm dos FSHR (p<0.05) e LHR (p<0.05), e diminuição significativa na expressão do RNAm dos AR (p<0.05), ERα (p<0.05), ERβ1 (p<0.05) e ERβ2 (p<0.05). Na vida adulta, a restrição alimentar causou uma diminuição no número total de folículos ovarianos, entretanto esta redução só foi significativa no número dos folículos primordiais, primários e de Graaf (p<0.05). Houve redução significativa da expressão da enzima aromatase (p<0.01), do FSHR (p<0.05), LHR (p<0.05), Ob-R (p<0.05) e Ob-Rb (p<0.05). Podemos concluir que a restrição alimentar materna durante a lactação causa uma programação metabólica no ovário da prole, alterando a foliculogênese, expressão das diferentes isoformas dos receptores de estrogênio, leptina, androgênio e gonadotropinas e da enzima aromatase. Essa programação, possivelmente decorrente dos baixos níveis de leptina nos primeiros dias de vida, pode contribuir para uma senescência precoce e redução da fertilidade já descrita na literatura.

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. (C) 2007 Published by Elsevier B.V.

Criptorquidia e exposição in utero ao di(n-butil)-ftalato e à acrilamida: avaliação morfológica do dano testicular

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Leydig-cell tumour in children: variable clinical presentation, diagnostic features, follow-up and genetic analysis of four cases

BACKGROUND: Testicular tumours are relatively uncommon in infants and children, accounting for only 1-2% of all paediatric solid tumours. Of these approximately 1.5% are Leydig-cell tumours. Further, activating mutations of the luteinizing hormone receptor gene (LHR), as well as of the G protein genes, such as Gsalpha (gsp) and Gialpha (gip2) subunits, and cyclin-dependent kinase gene 4(CDK4) have been associated with the development of several endocrine neoplasms. AIMS/METHODS: In this report, the clinical variability of Leydig-cell tumours in four children is described. The LHR-, gsp-, gip2- and CDK4 genes were investigated to establish the possible molecular pathogenesis of the variable phenotype of the Leydig-cell tumours. RESULTS: No activating mutations in these genes were found in the four Leydig-cell tumours studied. Therefore, the absence of activating mutations in LHR, as well as in both the 'hot spot' regions for activating mutations within the G-alpha subunits and in the regulatory 'hot spot' on the CDK4 genes in these tumours indicates molecular heterogeneity among Leydig-cell tumours. CONCLUSION: Four children with a variable phenotype caused by Leydig-cell tumours are described. A molecular analysis of all the 'activating' genes and mutational regions known so far was performed, but no abnormalities were found. The lessons learnt from these clinically variable cases are: perform ultrasound early and most importantly, consider discrepancies between testicular swelling, tumour size and androgen production.

Remodeling of extracellular matrix at ovulation of the bovine ovarian follicle

Using immunohistochemistry and RNA analyses we examined the fate of components of a newly identified matrix that develops between granulosa cells (focimatrix, abbreviated from focal intraepithelial matrix) and of the follicular basal lamina in ovulating bovine ovarian follicles. Pre- and postovulatory follicles were generated by treatment with estradiol (Day 1), progesterone (Days 1-10), and prostaglandin analogue (Day 9) with either no further treatment (Group 1, n = 6) and or with 25 mg porcine LH (Day 11, Group 2, n = 8 or Day 10, Group 3, n = 8) and ovariectomy on Day 12 (12-14 hr post LH in Group 2, 38-40.5 hr in Group 3). In the time frame examined no loss of follicular basal lamina laminin chains beta 2 and gamma 1 or nidogen 1 was observed. In the follicular basal lamina collagen type IV alpha 1 and perlecan were present prior to ovulation; after ovulation collagen type IV alpha 1 was discontinuously distributed and perlecan was absent. Versican in the theca interna adjacent to the follicular basal lamina in preovulatory follicles was not observed post ovulation, however, the granulosa cells then showed strong cytoplasmic staining for versican. Expression of versican isoforms V0, V1, and V3 was detected at all stages. Focimatrix was observed in preovulatory follicles. It contained collagen type IV alpha 1, laminins beta 2 and gamma 1, nidogen 1 and perlecan and underwent changes in composition similar to that of the follicular basal lamina. In conclusion focimatrix and the follicular basal lamina are degraded at ovulation. Individual components are lost at different times.

Impairing follicle-stimulating hormone (FSH) signaling in vivo: Targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance

Pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone stimulate the gonads by regulating germ cell proliferation and differentiation. FSH receptors (FSH-Rs) are localized to testicular Sertoli cells and ovarian granulosa cells and are coupled to activation of the adenylyl cyclase and other signaling pathways. Activation of FSH-Rs is considered essential for folliculogenesis in the female and spermatogenesis in the male. We have generated mice lacking FSH-R by homologous recombination. FSH-R-deficient males are fertile but display small testes and partial spermatogenic failure. Thus, although FSH signaling is not essential for initiating spermatogenesis, it appears to be required for adequate viability and motility of the sperms. FSH-R-deficient females display thin uteri and small ovaries and are sterile because of a block in folliculogenesis before antral follicle formation. Although the expression of marker genes is only moderately altered in FSH-R −/− mice, drastic sex-specific changes are observed in the levels of various hormones. The anterior lobe of the pituitary gland in females is enlarged and reveals a larger number of FSH- and thyroid-stimulating hormone (TSH)-positive cells. The phenotype of FSH-R −/− mice is reminiscent of human hypergonadotropic ovarian dysgenesis and infertility.

The negative feedback actions of progesterone on gonadotropinreleasing hormone secretion are transduced by the classical progesterone receptor

Progesterone (P) powerfully inhibits gonadotropin-releasing hormone (GnRH) secretion in ewes, as in other species, but the neural mechanisms underlying this effect remain poorly understood. Using an estrogen (E)-free ovine model, we investigated the immediate GnRH and luteinizing hormone (LH) response to acute manipulations of circulating P concentrations and whether this response was mediated by the nuclear P receptor. Simultaneous hypophyseal portal and jugular blood samples were collected over 36 hr: 0–12 hr, in the presence of exogenous P (P treatment begun 8 days earlier); 12–24 hr, P implant removed; 24–36 hr, P implant reinserted. P removal caused a significant rapid increase in the GnRH pulse frequency, which was detectable within two pulses (175 min). P insertion suppressed the GnRH pulse frequency even faster: the effect detectable within one pulse (49 min). LH pulsatility was modulated identically. The next two experiments demonstrated that these effects of P are mediated by the nuclear P receptor since intracerebroventricularly infused P suppressed LH release but 3α-hydroxy-5α-pregnan-20-one, which operates through the type A γ-aminobutyric acid receptor, was without effect and pretreatment with the P-receptor antagonist RU486 blocked the ability of P to inhibit LH. Our final study showed that P exerts its acute suppression of GnRH through an E-dependent system because the effects of P on LH secretion, lost after long-term E deprivation, are restored after 2 weeks of E treatment. Thus we demonstrate that P acutely inhibits GnRH through an E-dependent nuclear P-receptor system.

Effect of diethylstilbesterol and prolactin on the induction of follicle stimulating hormone receptors in immature and cycling rats

Induction of follicle stimulating hormone receptor in the granulosa cells of intact immature rat ovary by diethylstilbesterol, an estrogen, has been studied. A single injection of 4 mg of diethylstilbesterol produced 72 h later a 3-fold increase in follicle stimulating hormone receptor concentration as monitored by [125I]-oFSH binding to isolated cells. The newly induced receptors were kinetically indistinguishable from the preexisting ones, as determined by Lineweaver-Burk plot of the binding data. The induced receptors were functional as evidenced by increased ability of the granulosa cells to incorporate [3H]-leucine into cellular proteins. Neutralization of endogenous follicle stimulating hormone and luteinizing hormone by administering specific antisera had no effect on the ability of diethylstilbesterol to induce follicle stimulating hormone receptors, whereas blockade of endogenous prolactin secretion by ergobromocryptin administration significantly inhibited (∼ 30 %) the response to diethylstilbesterol; this inhibition could be completely relieved by ovine prolactin treatment. However, ovine prolactin at the dose tried did not by itself enhance follicle stimulating hormone receptor level. Administration of ergobromocryptin to adult cycling rats at noon of proestrus brought about as measured on diestrusII, (a) a reduction of both follicle stimulating hormone (∼ 30 %) and luteinizing hormone (∼ 45 %) receptor concentration in granulosa cells, (b) a drastic reduction in the ovarian tissue estradiol with no change in tissue progesterone and (c) reduction in the ability of isolated granulosa cells to convert testosterone to estradiol in response to follicle stimulating hormone. Ergobromocryptin treatment affected only prolactin and not follicle stimulating hormone or luteinizing hormone surges on the proestrus evening. Treatment of rats with ergobromocryptin at proestrus noon followed by an injection of ovine prolactin (1 mg) at 1700 h of the same day completely reversed the ergobromocryptin induced reduction in ovarian tissue estradiol as well as the aromatase activity of the granulosa cells on diestrus II, thus suggesting a role for proestrus prolactin surge in the follicular maturation process.