AMH Regulation by Steroids in the Mammalian Testis: Underlying Mechanisms and Clinical Implications.

Anti-Müllerian hormone (AMH) is a distinctive biomarker of the immature Sertoli cell. AMH expression, triggered by specific transcription factors upon fetal Sertoli cells differentiation independently of gonadotropins or sex steroids, drives Müllerian duct regression in the male, preventing the development of the uterus and Fallopian tubes. AMH continues to be highly expressed by Sertoli until the onset of puberty, when it is downregulated to low adult levels. FSH increases testicular AMH output by promoting immature Sertoli cell proliferation and individual cell expression. AMH secretion also showcases a differential regulation exerted by intratesticular levels of androgens and estrogens. In the fetus and the newborn, Sertoli cells do not express the androgen receptor, and the high androgen concentrations do not affect AMH expression. Conversely, estrogens can stimulate AMH production because estrogen receptors are present in Sertoli cells and aromatase is stimulated by FSH. During childhood, sex steroids levels are very low and do not play a physiological role on AMH production. However, hyperestrogenic states upregulate AMH expression. During puberty, testosterone inhibition of AMH expression overrides stimulation by estrogens and FSH. The direct effects of sex steroids on AMH transcription are mediated by androgen receptor and estrogen receptor α action on AMH promoter sequences. A modest estrogen action is also mediated by the membrane G-coupled estrogen receptor GPER. The understanding of these complex regulatory mechanisms helps in the interpretation of serum AMH levels found in physiological or pathological conditions, which underscores the importance of serum AMH as a biomarker of intratesticular steroid concentrations.

Morphology and Biochemistry of Ovulation.

The process of ovulation involves multiple and iterrelated genetic, biochemical, and morphological events: cessation of the proliferation of granulosa cells, resumption of oocyte meiosis, expansion of cumulus cell-oocyte complexes, digestion of the follicle wall, and extrusion of the metaphase-II oocyte. The present narrative review examines these interrelated steps in detail. The combined or isolated roles of the follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are highlighted. Genes indiced by the FSH genes are relevant in the cumulus expansion, and LH-induced genes are critical for the resumption of meiosis and digestion of the follicle wall. A non-human model for follicle-wall digestion and oocyte release was provided.

O processo de ovulação envolve modificações genéticas, bioquímicas e morfológicas múltiplas e interrelacionadas: suspensão da proliferação das células da granulosa, reinício da meiose do oócito, expansão das células do complexo cumulus-oócito, digestão da parede folicular, e extrusão do oócito. Esta revisão narrativa examina em detalhes cada um desses eventos e os principais genes e proteínas envolvidos. Mais importante, a ação combinada ou isolada do hormônio folículo-estimulante (HFE) e do hormônio luteinizante (HL) é destacada. Detalha-se o papel do HFE na expansão do cumulus e do HL na digestão da parede folicular, permitindo a extrusão do oócito na superfície ovariana. Proveu-se um modelo não humano para explicar a digestão da parede folicular.

Morphology and Biochemistry of Ovulation Morfologia e bioquímica da ovulação

Abstract The process of ovulation involves multiple and iterrelated genetic, biochemical, and morphological events: cessation of the proliferation of granulosa cells, resumption of oocyte meiosis, expansion of cumulus cell-oocyte complexes, digestion of the follicle wall, and extrusion of the metaphase-II oocyte. The present narrative review examines these interrelated steps in detail. The combined or isolated roles of the folliclestimulating hormone (FSH) and luteinizing hormone (LH) are highlighted. Genes indiced by the FSH genes are relevant in the cumulus expansion, and LH-induced genes are critical for the resumption ofmeiosis and digestion of the follicle wall. A nonhuman model for follicle-wall digestion and oocyte release was provided.

Resumo O processo de ovulação envolve modificações genéticas, bioquímicas e morfológicas múltiplas e interrelacionadas: suspensão da proliferação das células da granulosa, reinício da meiose do oócito, expansão das células do complexo cumulus-oócito, digestão da parede folicular, e extrusão do oócito. Esta revisão narrativa examina em detalhes cada um desses eventos e os principais genes e proteínas envolvidos. Mais importante, a ação combinada ou isolada do hormônio folículo-estimulante (HFE) e do hormônio luteinizante (HL) é destacada. Detalha-se o papel do HFE na expansão do cumulus e do HL na digestão da parede folicular, permitindo a extrusão do oócito na superfície ovariana. Proveu-se um modelo não humano para explicar a digestão da parede folicular.

Electrophysiological Responses to Different Follicle-Stimulating Hormone Isoforms on Human Cumulus Oophorus Cells: Preliminary Results.

OBJECTIVE: The aim of the present study was to provide a better understanding of the specific action of two follicle-stimulating hormone (FSH) isoforms (ß-follitropin and sheep FSH) on the membrane potential of human cumulus cells. METHODS: Electrophysiological data were associated with the characteristics of the patient, such as age and cause of infertility. The membrane potential of cumulus cells was recorded with borosilicate microelectrodes filled with KCl (3 M) with tip resistance of 15 to 25 MΩ. Sheep FSH and ß-follitropin were topically administered onto the cells after stabilization of the resting potential for at least 5 minutes. RESULTS: In cumulus cells, the mean resting membrane potential was - 34.02 ± 2.04 mV (n = 14). The mean membrane resistance was 16.5 ± 1.8 MΩ (n = 14). Sheep FSH (4 mUI/mL) and ß-follitropin (4 mUI/mL) produced depolarization in the membrane potential 180 and 120 seconds after the administration of the hormone, respectively. CONCLUSION: Both FSH isoforms induced similar depolarization patterns, but ß-follitropin presented a faster response. A better understanding of the differences of the effects of FSH isoforms on cell membrane potential shall contribute to improve the use of gonadotrophins in fertility treatments.

OBJETIVO: O objetivo do presente estudo foi fornecer uma melhor compreensão da ação específica de duas isoformas de hormônio folículo estimulante (FSH, sigla em inglês) (ß-folitropina e FSH ovino) no potencial de membrana de células do cumulus oophorus humanas. MéTODOS: Dados eletrofisiológicos foram associados às características da paciente, como idade e causa da infertilidade. O potencial de membrana das células do cumulus foi registrado com microeletrodos de borossilicato preenchidos com KCl (3 M) com uma resistência de 15 a 25 MΩ. O FSH ovino e a ß-folitropina foram administrados topicamente nas células após a estabilização do potencial de repouso durante pelo menos 5 minutos. RESULTADOS: Nas células do cumulus, o potencial médio de membrana em repouso foi de -34,02 ± 2,04 mV (n = 14). A resistência média da membrana foi de 16,5 ± 1,8 MΩ (n = 14). O FSH ovino (4 mUI/mL) e a ß-folitropina (4 mUI/mL) produziram despolarização no potencial de membrana 180 e 120 segundos após a aplicação do hormônio, respectivamente. CONCLUSãO: Ambas as isoformas de FSH induzem padrões de despolarização semelhantes, mas a ß-folitropina apresentou uma resposta mais rápida. Uma melhor compreensão das diferenças dos efeitos das isoformas do FSH no potencial da membrana celular contribuirá para aprimorar o uso das gonadotrofinas no estímulo ovariano controlado e em protocolos de maturação oocitária in vitro.

Electrophysiological responses to different follicle-stimulating hormone isoforms on human cumulus oophorus cells: preliminary results / Respostas eletrofisiológicas a diferentes isoformas de FSH em células humanas do cumulus oophorus: resultados preliminares

Abstract Objective The aim of the present study was to provide a better understanding of the specific action of two follicle-stimulating hormone (FSH) isoforms (β-follitropin and sheep FSH) on the membrane potential of human cumulus cells. Methods Electrophysiological data were associated with the characteristics of the patient, such as age and cause of infertility. The membrane potential of cumulus cells was recorded with borosilicate microelectrodes filled with KCl (3 M) with tip resistance of 15 to 25 MΩ. Sheep FSH and β-follitropin were topically administered onto the cells after stabilization of the resting potential for at least 5 minutes. Results In cumulus cells, the mean resting membrane potential was - 34.02 ± 2.04 mV (n = 14). The mean membrane resistance was 16.5 ± 1.8 MΩ (n = 14). Sheep FSH (4 mUI/mL) and β-follitropin (4 mUI/mL) produced depolarization in the membrane potential 180 and 120 seconds after the administration of the hormone, respectively. Conclusion Both FSH isoforms induced similar depolarization patterns, but β-follitropin presented a faster response. A better understanding of the differences of the effects of FSH isoforms on cell membrane potential shall contribute to improve the use of gonadotrophins in fertility treatments.

Resumo Objetivo O objetivo do presente estudo foi fornecer uma melhor compreensão da ação específica de duas isoformas de hormônio folículo estimulante (FSH, sigla em inglês) (β-folitropina e FSH ovino) no potencial de membrana de células do cumulus oophorus humanas. Métodos Dados eletrofisiológicos foram associados às características da paciente, como idade e causa da infertilidade. O potencial de membrana das células do cumulus foi registrado com microeletrodos de borossilicato preenchidos com KCl (3 M) com uma resistência de 15 a 25 MΩ. O FSH ovino e a β-folitropina foram administrados topicamente nas células após a estabilização do potencial de repouso durante pelo menos 5 minutos. Resultados Nas células do cumulus, o potencial médio de membrana em repouso foi de -34,02 ± 2,04 mV (n = 14). A resistência média da membrana foi de 16,5 ± 1,8 MΩ (n = 14). O FSH ovino (4 mUI/mL) e a β-folitropina (4 mUI/mL) produziram despolarização no potencial de membrana 180 e 120 segundos após a aplicação do hormônio, respectivamente. Conclusão Ambas as isoformas de FSH induzem padrões de despolarização semelhantes, mas a β-folitropina apresentou uma resposta mais rápida. Uma melhor compreensão das diferenças dos efeitos das isoformas do FSH no potencial da membrana celular contribuirá para aprimorar o uso das gonadotrofinas no estímulo ovariano controlado e em protocolos de maturação oocitária in vitro.

The involvement of gonadotropins and gonadal steroids in the ovulatory dysfunction of the potamodromous Salminus hilarii (Teleostei: Characidae) in captivity.

Potamodromous teleosts that require migration to reproduce show dysfunctions that block ovulation and spawning while in captivity. To understand the physiological basis of these reproductive dysfunctions, follicle-stimulating hormone b subunit (fshb) and luteinizing hormone b subunit (lhb) gene expression analyses by real-time quantitative PCR, together with measurements of estradiol (E 2), 17α-hydroxyprogesterone (17α-OHP) and 17α,20ß-dihydroxy-4-pregnen-3-one (17α,20ß-DHP) levels, were carried out throughout the reproductive cycle of the potamodromous Salminus hilarii. The following reproductive stages were evaluated in captive and wild females: previtellogenic (PV), advanced maturation/mature (AM) and regression/spent (REG/SPENT). In the wild females, fshb expression decreased from the PV to the AM stage, and the opposite pattern was detected for E 2, which increased from the PV to the AM stage. fshb was expressed at lower levels in captive than in wild females, and this difference did not change during the reproductive cycle. lhb expression also increased from the PV to the AM stage in both groups, but the wild females at the AM and REG/SPENT stages showed higher lhb expression levels than the captive females. The concentrations of 17α-OHP did not change during the reproductive cycle, and the levels were higher in the captive than in the wild females at all reproductive stages. 17α,20ß-DHP levels did not change between wild and captive females. However, in captive females, the transition from PV to AM stage was followed by an increase in 17α,20ß-DHP levels. These data indicate that dysfunctions in the gonadotropins and steroids synthesis pathways cause the ovulation failure in captive S. hilarii.

Non-classical effects of androgens on testes from neonatal rats.

The intratesticular testosterone concentration is high during the early postnatal period although the intracellular androgen receptor expression (iAR) is still absent in Sertoli cells (SCs). This study aimed to evaluate the non-classical effects of testosterone and epitestosterone on calcium uptake and the electrophysiological effects of testosterone (1µM) on SCs from rats on postnatal day (pnd) 3 and 4 with lack of expression of the iAR. In addition, crosstalk on the electrophysiological effects of testosterone and epitestosterone with follicle stimulating hormone (FSH) in SCs from 15-day-old rats was evaluated. The isotope (45)Ca(2+) was utilized to evaluate the effects of testosterone and epitestosterone in calcium uptake. The membrane potential of SCs was recorded using a standard single microelectrode technique. No immunoreaction concerning the iAR was observed in SCs on pnd 3 and 4. At this age, both testosterone and epitestosterone increased the (45)Ca(2+) uptake. Testosterone promoted membrane potential depolarization of SCs on pnd 4. FSH application followed by testosterone and epitestosterone reduced the depolarization of the two hormones. Application of epitestosterone 5 min after FSH resulted in a delay of epitestosterone-promoted depolarization. The cell resistance was also reduced. Thus, in SCs from neonatal Wistar rats, both testosterone and epitestosterone act through a non-classical mechanism stimulating calcium uptake in whole testes, and testosterone produces a depolarizing effect on SC membranes. Testosterone and epitestosterone stimulates non-classical actions via a membrane mechanism, which is independent of iAR. FSH and testosterone/epitestosterone affect each other's electrophysiological responses suggesting crosstalk between the intracellular signaling pathways.

Caracterização estrutural do hormônio folículo estimulante e seu papel na fisiologia de células ovarianas / Structural Characterization of the Follicle Stimulating Hormone and its role in the physiology of ovarian cells

The follicle stimulating hormone (FSH) is a heterodimeric glycoprotein synthesized and secreted in the pituitary anterior portion (adenohypophysis). The FSH performs important actions in the later stages of follicular development and maturation. The FSH exerts multiple functions such as the activation of more than 100 genes encoding different answers. The action of FSH is restricted to the granulosa cells. This gonadotropin acts through the activation of the enzyme adenylate cyclase in the plasma membrane, which forms AMPc from ATP. The AMPc promotes metabolic changes that characterize the gonadotropic action, usually through the phosphorylation of protein kinases. During the physiological process of ovulation, estrogens enhance the stimulatory effect of FSH and cause the release of FSH/LH required for ovulation. The mature follicle itself is responsible for ovulation, as it secrets estradiol, hormone responsible for the pre-ovulatory peak of LH, resulting in ovulation. Thus, the addition of FSH to the culture media characterizes important actions, as the follicular development and the ovulation process. Thus, the revision of the objective was to describe the role of FSH and its mechanism of action and receptor in ovarian follicular development. And as well as the effect of this on survival, activation and follicular growth.

O hormônio folículo estimulante (FSH) é uma glicoproteína heterodímera sintetizada e secretada na porção anterior da hipófise (adenohipófise). O FSH desempenha ações relevantes nos estádios mais avançados do desenvolvimento e maturação folicular. O FSH exerce múltiplas funções como a ativação de mais de 100 genes que codificam diferentes respostas. A ação do FSH está restrita as células da granulosa. Esta gonadotrofina atua por meio da ativação da enzima adenilciclase na membrana plasmática, a qual forma AMPc a partir de ATP. O AMPc promove as mudanças metabólicas que caracterizam a ação gonadotrópica, normalmente por meio da fosforilação de proteínas-quinases. Durante o processo fisiológico da ovulação, os estrógenos melhoram o efeito estimulador do FSH e provocam a liberação de FSH/LH necessária para a ovulação. O próprio folículo maduro é responsável pela ovulação, pois secreta estradiol, hormônio responsável pelo pico pré-ovulatório de LH, resultando na ovulação. Diante disso, a adição de FSH aos meios de cultivo caracteriza ações importantes, como, o desenvolvimento folicular e o processo de ovulação. Desta forma, o objetivo da revisão foi descrever o papel do FSH e seu mecanismo de ação e receptores no desenvolvimento folicular ovariano. E bem como, o efeito deste sobre a sobrevivência, a ativação e o crescimento folicular.

Caracterização estrutural do hormônio folículo estimulante e seu papel na fisiologia de células ovarianas / Structural Characterization of the Follicle Stimulating Hormone and its role in the physiology of ovarian cells

The follicle stimulating hormone (FSH) is a heterodimeric glycoprotein synthesized and secreted in the pituitary anterior portion (adenohypophysis). The FSH performs important actions in the later stages of follicular development and maturation. The FSH exerts multiple functions such as the activation of more than 100 genes encoding different answers. The action of FSH is restricted to the granulosa cells. This gonadotropin acts through the activation of the enzyme adenylate cyclase in the plasma membrane, which forms AMPc from ATP. The AMPc promotes metabolic changes that characterize the gonadotropic action, usually through the phosphorylation of protein kinases. During the physiological process of ovulation, estrogens enhance the stimulatory effect of FSH and cause the release of FSH/LH required for ovulation. The mature follicle itself is responsible for ovulation, as it secrets estradiol, hormone responsible for the pre-ovulatory peak of LH, resulting in ovulation. Thus, the addition of FSH to the culture media characterizes important actions, as the follicular development and the ovulation process. Thus, the revision of the objective was to describe the role of FSH and its mechanism of action and receptor in ovarian follicular development. And as well as the effect of this on survival, activation and follicular growth.(AU)

O hormônio folículo estimulante (FSH) é uma glicoproteína heterodímera sintetizada e secretada na porção anterior da hipófise (adenohipófise). O FSH desempenha ações relevantes nos estádios mais avançados do desenvolvimento e maturação folicular. O FSH exerce múltiplas funções como a ativação de mais de 100 genes que codificam diferentes respostas. A ação do FSH está restrita as células da granulosa. Esta gonadotrofina atua por meio da ativação da enzima adenilciclase na membrana plasmática, a qual forma AMPc a partir de ATP. O AMPc promove as mudanças metabólicas que caracterizam a ação gonadotrópica, normalmente por meio da fosforilação de proteínas-quinases. Durante o processo fisiológico da ovulação, os estrógenos melhoram o efeito estimulador do FSH e provocam a liberação de FSH/LH necessária para a ovulação. O próprio folículo maduro é responsável pela ovulação, pois secreta estradiol, hormônio responsável pelo pico pré-ovulatório de LH, resultando na ovulação. Diante disso, a adição de FSH aos meios de cultivo caracteriza ações importantes, como, o desenvolvimento folicular e o processo de ovulação. Desta forma, o objetivo da revisão foi descrever o papel do FSH e seu mecanismo de ação e receptores no desenvolvimento folicular ovariano. E bem como, o efeito deste sobre a sobrevivência, a ativação e o crescimento folicular.(AU)

Exploring the cyclooxygenase 2 (COX2)/15d-Δ(12,14)PGJ(2) system in hamster Sertoli cells: regulation by FSH/testosterone and relevance to glucose uptake.

We have previously described a stimulatory effect of testosterone on cyclooxygenase 2 (COX2) expression and prostaglandin (PG) synthesis, and the involvement of PGs in the modulation of testosterone production in Leydig cells of the seasonal breeder Syrian hamster. In this study, we investigated the existence of a COX2/PGs system in hamster Sertoli cells, its regulation by testosterone and FSH, and its effect on glucose uptake. COX2 expression was observed in Sertoli cells of both reproductively active and inactive adult hamsters. Testosterone and the plasma membrane-impermeable testosterone-BSA significantly induced COX2 expression, mitogen activated protein kinases 1/2 (MAPK1/2) phosphorylation and 15d-Δ(12,14)PGJ(2) production in Sertoli cells purified from photoperiodically regressed hamsters. These actions were abolished by the antiandrogen bicalutamide and by the inhibitor of MAPK kinase (MEK1/2) U0126, suggesting that testosterone exerts its stimulatory effect on COX2/PGs through a non-classical mechanism that involves the presence of androgen receptors and MAPK1/2 activation. FSH also stimulated COX2/PGs via MAPK1/2 phosphorylation. FSH and testosterone stimulate, whereas 15d-Δ(12,14)PGJ(2) via PPARγ inhibits, [2,6-(3)H]-2-deoxy-d-glucose ([(3)H]-2-DOG) uptake. Meloxicam, a selective COX2 inhibitor, further increases [(3)H]-2-DOG uptake in the presence of FSH or testosterone. Thus, in addition to their positive effect, FSH and testosterone may also exert an indirect negative regulation on glucose uptake which involves the COX2/15d-Δ(12,14)PGJ(2)/PPARγ system. Overall, these results demonstrate the presence of a COX2/PG system in hamster Sertoli cells which might act as a local modulator of FSH and testosterone actions.

Anti-müllerian hormone and sertoli cell function in paediatric male hypogonadism.

In the prepubertal male, Sertoli cells are the most active testicular cell population. Without stimulation tests, prepubertal hypogonadism can only be evidenced if Sertoli cell function is assessed. Anti-müllerian hormone (AMH) is a distinctive marker of the prepubertal Sertoli cell. Serum AMH is high from fetal life until puberty. In postnatal life, AMH testicular production is stimulated by FSH and potently inhibited by androgens. In anorchid patients, AMH is undetectable. In prepubertal males with fetal- or childhood-onset primary or central hypogonadism affecting the whole gonad, serum AMH is low. Conversely, when hypogonadism only affects Leydig cells (i.e., LH/human chorionic gonadotrophin receptor or steroidogenic enzyme defects), serum AMH is normal/high. AMH is also normal/high in patients with androgen insensitivity. In patients of pubertal age with central hypogonadism, AMH is low for Tanner stage - reflecting lack of FSH stimulus, - but high for age - reflecting lack of testosterone inhibitory effect. FSH treatment results in serum AMH rise, whereas human chorionic gonadotrophin treatment increases testosterone levels which inhibit AMH production. In conclusion, AMH determination is helpful in assessing gonadal function, without need for stimulation tests, and orientates the aetiological diagnosis of paediatric male hypogonadism. Furthermore, serum AMH is an excellent marker of FSH and androgen action in the testis.

Coordinated regulation of follicle development by germ and somatic cells.

The continuum of folliculogenesis begins in the fetal ovary with the differentiation of the oogonia and their isolation within the primordial follicles. Primordial follicle activation is an enigmatic process, whereby some follicles enter the growing pool to become primary follicles, thereby embarking on an irreversible progression towards ovulation or atresia. This process is under the coordinated regulation of factors from the oocyte itself, as well as from the somatic cells of the ovary, in particular the theca and granulosa cells, which are structural components of the follicle. These two influences provide the principal stimuli for the growth of the follicle to the late preantral or early antral stage of development. The endocrine effects of the gonadotrophins FSH and LH are essential to the continued progression of the follicle and most atresia can be attributed to the failure to receive or process the gonadotrophin signals. The peri-ovulatory state has received intensive investigation recently, demonstrating a coordinated role for gonadotrophins, steroids, epidermal growth factor family proteins and prostaglandins. Thus, a complex programme of coordinated interaction of governing elements from both germ and somatic cell sources is required for successful follicle development.

Regulation of expression of Sertoli cell glucose transporters 1 and 3 by FSH, IL1 beta, and bFGF at two different time-points in pubertal development.

Sertoli cells are necessary to provide adequate levels of lactate for germ cell development. Lactate production is hormonally regulated by follicle-stimulating hormone (FSH) and by a large set of intratesticular regulators such as interleukin-1 beta (IL1 beta) and basic fibroblast growth factor (bFGF). Little is known regarding the critical step in the production of this metabolite, viz., the entrance of glucose into the cell as mediated by GLUTs. The aim of the present study was to investigate the expression of the glucose transporters GLUT1 and GLUT3 and its possible regulation by FSH, IL1 beta, and bFGF in Sertoli cells at two different time-points in sexual development. Sertoli cells retaining the ability to undergo mitosis (obtained from 8-day-old rats) and in the process of terminal differentiation (obtained from 20-day-old rats) were examined. Testicular tissue sections and Sertoli cell monolayers obtained from 8- and 20-day-old rats showed positive immunostaining for GLUT1 and GLUT3 proteins. GLUT1 and GLUT3 mRNA levels were detected at the two ages analyzed. Treatment of Sertoli cells obtained from 8- and 20-day-old rats with FSH, IL1 beta, and bFGF for various periods of time (12, 24, and 48 h) increased GLUT1 without changing GLUT3 mRNA levels. Our results thus show that Sertoli cells express GLUT1 and GLUT3 throughout pubertal development, and that, in Sertoli cells, only GLUT1 is regulated by hormones during pubertal development. Hormonal regulation of GLUT1 expression and consequently glucose uptake and lactate production may be a key molecular event in the regulation of spermatogenesis by hormones.

Diverse FSH and testosterone signaling pathways in the Sertoli cell.

FSH and testosterone exert different regulatory effects on the seminiferous epithelium; they act through multiple and complex signaling routes to direct the development of the germ cells into mature spermatozoa. In addition to their well-known pathways of action, both hormones have recently been recognized to have new signaling routes that are linked to the Ca(2+) ion, including, among others, the regulation of cell proliferation by FSH and the regulation of cell migration by testosterone.

In vitro fertilization and development of OPU derived goat and sheep oocytes.

The recent upgrade in IVP technology seen in cattle can be adapted to embryo production in small ruminants to overcome limitations exhibited by surgical procedures on preserving the reproductive potential of donors and the efficiency of embryo production. The aim of the present study was to assess the current procedures used in cattle for the production of IVP embryos in goats and sheep based on laparoscopic-aided ovum pick-up (LOPU) supplied oocytes. Sexually matured goat and sheep donors were treated during the breeding season with FSH and subjected to laparoscopic-guided follicular puncture under general anaesthesia. The collected cumulus-oocyte complexes were matured in medium 199 and fertilized by frozen-thawed spermatozoa using Talp medium supplemented with heparin and oestrus-sheep serum. Cleaved ova were either cultured in sheep in vitro fertilization medium plus amino acids or transferred to sheep oviducts. Blastocyst rate, hatching rate and development rate up to term were used as markers of embryo function. The results obtained for goat and sheep involving 30 and 35 donors respectively (10 and 9 LOPU sessions) were 81.2% and 85.2% of oocyte collection rate; 88.3% and 98.6% oocyte incubation rate; 85.6% and 76.0% fertilization rate; 82.4% and 93.4% of cleavage rate; 50.0% and 61.5% IVP blastocyst rate; 42.1% and 45.5% blastocyst rate in oviducts; 73.0% and 66.7% embryo survival up to term, respectively. The results are comparable to those obtained in small ruminants and in bovines suggesting that requirements for embryo production and development are similar.

Multiple facets of follicle-stimulating hormone receptor function.

Follicle-stimulating hormone (FSH) is a glycoprotein hormone produced by the anterior pituitary gland. This gonadotropin plays an essential role in reproduction. Its receptor (FSHR) belongs to the superfamily of G protein-coupled receptors (GPCR), specifically the family of rhodopsin-like receptors. Agonist binding to the FSHR triggers the rapid activation of multiple signaling cascades, mainly the cAMP-adenylyl cyclase-protein kinase A cascade, that impact diverse biological effects of FSH in the gonads. As in other G protein-coupled receptors, the several cytoplasmic domains of the FSHR are involved in signal transduction and termination of the FSH signal. Here we summarize some recent information on the signaling cascades activated by FSH as well as on the role of the intracytoplasmic domains of the FSHR in coupling to membrane and cytosolic proteins linked to key biological functions regulated by the FSH-FSHR system.

Effect of human gonadotropins on spermiation and androgen biosynthesis in the testis of the toad Bufo arenarum (Amphibia, Anura).

This paper analyzes, in the toad Bufo arenarum, the effect on spermiation and androgen secretion of two human recombinant gonadotropins, human recombinant LH (hrLH) and human recombinant FSH (hrFSH) as well as the well-known spermiation-inducing hormone, human chorionic gonadotropin (hCG). For this purpose, testes were incubated with different concentrations of hrLH (0.01-2.5 microg/ml) and hrFSH (0.05-5 microg/ml), and results were compared with those obtained with 2.5 microg/ml hCG. Spermiation was most efficiently stimulated by hrFSH, which elicited a higher response than either hrLH or hCG. Both hrFSH and hrLH produced a bell-shaped dose-response curve, with a 50% inhibition on spermiation at a concentration twice higher than that necessary to get the highest response. However, none of the gonadotropins yielded a biphasic response on androgen secretion, hrLH producing the highest response at a concentration that evoked a 70% inhibition in the spermiation test. Regarding steroidogenesis, hrLH and hrFSH were more active than hCG. Taken together, the results described in this paper suggest that, in B. arenarum, spermiation and androgen secretion are mediated by different receptors. After comparing the effects of recombinant hormones, we conclude that hrFSH has a greater effect on spermiation than hCG or hrLH.

Functional significance of the BBXXB motif reversed present in the cytoplasmic domains of the human follicle-stimulating hormone receptor.

The minimal structural motif, BBXXB (where B represents a basic amino acid residue and X a non-basic residue), located in particular regions of the intracellular domains of cell surface membrane receptors is involved in the G protein-activating activity of a number of G protein-coupled receptors. The human FSH receptor (hFSHR) exhibits a reversed BBXXB motif (BXXBB) in the juxtamembrane region of the third intracellular loop (IL3) and the carboxyl terminus (Ctail) of the receptor; however the importance of this sequence on receptor function remains unclear. In the present study, we analyzed the effects of mutations in this structural motif on hFSHR expression, receptor-mediated effector activation and agonist-provoked receptor internalization. Human embryonic kidney 293 cells were transiently transfected with plasmids containing the cDNA of the wild-type (Wt) hFSHR or several hFSHR mutants in which basic amino acids of the minimal structural motif at the IL3 and Ctail were replaced with alanine (i.e. AXXAA, AXXBB, BXXAB and BXXBA mutants). Alanine substitution of the three basic residues present in the IL3-BXXBB (IL3-AXXAA mutant) yielded a < or =60 kDa possibly under-glycosylated form of the FSHR, whereas the same substitutions in the Ctail resulted in the immature >62 kDa form of the receptor; both AXXAA hFSHR mutants completely failed to bind agonist and activate effector. Individual substitutions resulted in different cAMP responses to agonist stimulation: the IL3-AXXBB and IL3-BXXBA mutant hFSHRs failed to evoke Gs protein activation, whereas agonist-stimulated cAMP production was completely normal when the IL3-BXXAB mutant was expressed. All three IL3 mutants bound [125I]-labelled FSH in a similar fashion to the Wt hFSHR. Ligand-binding, cell surface membrane receptor expression and agonist-provoked effector activation were significantly affected by the individual substitutions at the Ctail-BXXBB motif: the Ctail-AXXBB variant exhibited reduced (approximately 50%) maximal cAMP response and ability to bind ligand, whereas both ligand binding and effector activation was severely reduced or abolished by expression of the Ctail-BXXBA and -BXXAB hFSHR mutants; the expression levels of the 80 kDa form of the receptor correlated with the magnitude of ligand-provoked cAMP production and binding capability of the mutant receptors. Upon stimulation by agonist, all mutants with detectable ligand-binding activity internalized following the pattern exhibited by the Wt hFSHR species. These results indicate that the BXXBB motif at the IL3 of the hFSHR is essential for coupling the activated receptor to the Gs protein, whereas the same motif in the Ctail is apparently more important for membrane expression.

Relative contribution of estrogen withdrawal and gonadotropins increase secondary to ovariectomy on prostaglandin generation in mesenteric microvessels.

Recent studies have established that ovariectomy impairs endothelial function, partially by increasing vasoconstrictor prostaglandins generation. Because ovariectomy causes concomitant lack of estrogen and increase of gonadotropins (ie, LH and FSH), in this study we explored the relative role of estrogen and LH/FSH in modulating vasoconstrictor prostaglandins generation in mesenteric arteriolar bed of SHR. Endothelium-dependent relaxation to acetylcholine (ACh) and bradykinin (Bk) was markedly reduced in ovariectomized (OVX) compared with SHR in physiological estrus (OE). Estrogen replacement (OVX + E), but not the decrease in LH/FSH levels with leuprolide (OVX + Leu), corrected the altered vasorelaxation response in OVX. Treatment of mesenteries with diclofenac, prostaglandin-H synthase (PGHS) inhibitor, significantly enhanced the relaxing response in arteries from OVX and OVX + Leu, but not those from OE, indicating that a PGHS-derived vasoconstrictor has modified the endothelium-dependent response during estrogen but not LH/FSH deprivation. Confirming these data, in response to exogenous arachidonic acid, whereas arteries from OVX and OVX + Leu exhibited a marked and similar vasoconstrictor response, the arteries from OE and OVX + E rats exhibited a slight vasodilation. We also demonstrated by RT-PCR that ovariectomy significantly increased PGHS-2 but not PGHS-1 mRNA expression in comparison to OE. The PGHS-2 overexpression in OVX was corrected by estrogen replacement, but not by the reduction of LH/FSH levels. Altogether these data strongly support a role for hypoestrogenism rather than LH/FSH enhancement, associated with the removal of ovaries, in the increase of vasoconstrictor prostaglandins, possibly by a mechanism involving PGHS-2 overexpression.

AMH/MIS: what we know already about the gene, the protein and its regulation.

(AMH/MIS) was first suggested by Jost, more than Four decades before this gonadal glycoprotein was purified and its gene and promoter sequenced. In mammals, AMH expression is triggered by SOX9 in Sertoli cells at the onset of testicular differentiation, and regulated by SF1, GATA factors, WT1, DAX1 and FSH. Ovarian granulosa cells also secrete AMH from late foetal life. In males, AMH is secreted into the bloodstream at high levels until puberty when it is down-regulated by androgens and meiotic germ cells and its directional secretion switches from the basal compartment to the seminiferous tubule lumen. In birds and reptiles, AMH expression shows particular features. Serum AMH determination is useful to study testicular function in boys and in patients with gonadal tumours. AMH levels in seminal and follicular fluid may also be of clinical use.