Anti-Müllerian hormone and progesterone levels produced by granulosa cells are higher when derived from natural cycle IVF than from conventional gonadotropin-stimulated IVF.

BACKGROUND The study was designed to compare the effect of in vitro FSH stimulation on the hormone production and gene expression profile of granulosa cells (GCs) isolated from single naturally matured follicles obtained from natural cycle in vitro fertilization (NC-IVF) with granulosa cells obtained from conventional gonadotropin-stimulated IVF (c-IVF). METHODS Lutein granulosa cells from the dominant follicle were isolated and cultured in absence or presence of recombinant FSH. The cultures were run for 48 h and six days. Messenger RNA (mRNA) expressions of anti-Müllerian hormone (AMH) and FSH receptor were measured by quantitative polymerase chain reaction (qPCR). AMH protein and progesterone concentration (P4) in cultured supernatant were measured by ELISA and RIA. RESULTS Our results showed that the mRNA expression of AMH was significantly higher in GCs from NC- than from c-IVF on day 6 after treatment with FSH (1 IU/mL). The FSH stimulation increased the concentration of AMH in the culture supernatant of GCs from NC-IVF compared with cells from c-IVF. In the culture medium, the AMH level was correlated significantly and positively to progesterone concentration. CONCLUSIONS Differences in the levels of AMH and progesterone released into the medium by cultured GC as well as in AMH gene expression were observed between GCs obtained under natural and stimulated IVF protocols. The results suggest that artificial gonadotropin stimulation may have an effect on the intra-follicular metabolism. A significant positive correlation between AMH and progesterone may suggest progesterone as a factor influencing AMH secretion.

Growth differentiation factor-9 stimulates progesterone synthesis in granulosa cells via a prostaglandin E2/EP2 receptor pathway

Growth differentiation factor-9 (GDF-9), an oocyte-secreted member of the transforming growth factor β superfamily, progesterone receptor, cyclooxygenase 2 (Cox2; Ptgs2), and the EP2 prostaglandin E2 (PGE2) receptor (EP2; Ptgerep2) are required for fertility in female but not male mice. To define the interrelationship of these factors, we used a preovulatory granulosa cell culture system in which we added recombinant GDF-9, prostaglandins, prostaglandin receptor agonists, or cyclooxygenase inhibitors. GDF-9 stimulated Cox2 mRNA within 2 h, and PGE2 within 6 h; however, progesterone was not increased until 12 h after addition of GDF-9. This suggested that Cox2 is a direct downstream target of GDF-9 but that progesterone synthesis required an intermediate. To determine whether prostaglandin synthesis was required for progesterone production, we analyzed the effects of PGE2 and cyclooxygenase inhibitors on this process. PGE2 can stimulate progesterone synthesis by itself, although less effectively than GDF-9 (3-fold vs. 6-fold increase over 24 h, respectively). Furthermore, indomethacin or NS-398, inhibitors of Cox2, block basal and GDF-9-stimulated progesterone synthesis. However, addition of PGE2 to cultures containing both GDF-9 and NS-398 overrides the NS-398 block in progesterone synthesis. To further define the PGE2-dependent pathway, we show that butaprost, a specific EP2 agonist, stimulates progesterone synthesis and overrides the NS-398 block. In addition, GDF-9 stimulates EP2 mRNA synthesis by a prostaglandin- and progesterone-independent pathway. Thus, GDF-9 induces an EP2 signal transduction pathway which appears to be required for progesterone synthesis in cumulus granulosa cells. These studies further demonstrate the importance of oocyte–somatic cell interactions in female reproduction.

Primary sex determination of placental mammals: a modelling study uncovers dynamical developmental constraints in the formation of Sertoli and granulosa cells

Primary sex determination in placental mammals is a very well studied developmental process. Here, we aim to investigate the currently established scenario and to assess its adequacy to fully recover the observed phenotypes, in the wild type and perturbed situations. Computational modelling allows clarifying network dynamics, elucidating crucial temporal constrains as well as interplay between core regulatory modules.

Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells in vitro

Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2-6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0-10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids.Oocyte secreted factors were shown to stimulate both granulosa cell proliferation (P < 0.001) and oestradiol production (P < 0.001) by granulosa cells throughout culture. In contrast, oocyte secreted factors suppressed granulosa cell progesterone production after both 48 and 144 hours (P < 0.001). Thecal cell numbers were increased by oocyte secreted factors (P = 0.02), together with a suppression in progesterone and androstenedione synthesis after 48 hours (P < 0.001) and after 144 hours (P = 0.02), respectively. Oocyte secreted factors also increased viable cell numbers (P < 0.001) in co-cultures together with suppression of progesterone (P < 0.001) and oestradiol (P < 0.001). In granulosa cell only cultures, SCF increased progesterone production in a dose dependent manner (P < 0.001), whereas progesterone synthesis by theca cells was reduced in a dose dependent manner (P = 0.002). Co-cultured cells demonstrated an increase in progesterone production with increasing SCF dose (P < 0.001) and an increase in oestradiol synthesis at the highest dose of SCF (100 ng/ml). In summary, these findings demonstrate the presence of a co-ordinated paracrine interaction between somatic cells and germ cells, whereby oocyte derived signals interact locally to mediate granulosa and theca cell function. SCF has a role in modulating this local interaction. In conclusion, the oocyte is an effective modulator of granulosa-theca interactions, one role being the inhibition of luteinization

Studies of granulosa cell maturation in dominant and subordinate bovine follicles : novel extracellular matrix focimatrix is co-ordinately regulated with cholesterol side-chain cleavage CYP11A1

During growth of antral ovarian follicles granulosa cells first become associated with a novel type of extracellular matrix, focimatrix, and at larger sizes follicles become either subordinate or dominant. To examine this, bovine subordinate (9.0±s.e.m. 0.4 mm; n=16), partially dominant (12.0±0.6 mm; n=18) and fully dominant (15.0±0.4 mm; n=14) follicles were examined by real time RT-PCR analyses of granulosa cells and by immunohistochemistry of focimatrix. Changes in the expression of FSH receptor, LH receptor, cholesterol side-chain cleavage (CYP11A1), 3β-hydroxysteroid dehydrogenase, aromatase (CYP19A1) and inhibin-α and β-B were observed as expected for follicle sizes examined. After adjusting for size differences, only CYP11A1 was significantly different between the groups, and elevated in dominant follicles. Also after adjusting for differences in size there were no significant differences in expression of focimatrix components collagen type IV α-1 (COL4A1), laminin β-2, nidogen 1 (NID1), and perlecan (HSPG2) or the volume density of NID1 and -2 and HSPG2. The volume density of focimatrix components in laminin 111 was significantly elevated in dominant follicles. Adjusting for analysis of more than one follicle per animal and for multiple correlations, CYP11A1 mRNA levels were highly correlated with the focimatrix genes COL4A1, NID1 and -2 and HSPG2. Thus, focimatrix may potentially regulate CYP11A1 expression, and the regulation of both could be important in follicular dominance.

Effect of pregnant mare serum gonadotropin on the induction and degradation of FSH and LH receptors in the granulosa cell of the immature rat

The relative induction of FSH and LH receptors in the granulosa cells of immature rat ovary by pregnant mare serum gonadotropin (PMSG) has been studied. A single injection of PMSG (15 IU) brought about a 3- and 12-fold increase in FSH and LH receptor concentration,respectively, in the granulosa cells. Maximal concentration was reached by 72 h but the receptor levels showed a sharp decline during the next 24–48 h. The kinetic properties of the newly formed FSH receptors were indistinguishable from the pre-existing ones. The induced FSH receptors were functional as demonstrated by an increase in the in vitro responsiveness of the cells to exogenous FSH in terms of progesterone production. Treatment of immature rats with cyanoketone, an inhibitor of Δ5,3β-hydroxysteroid dehydrogenase, prior to PMSG injection effectively reduced the PMSG-stimulated increase in the serum estradiol, uterine weight and LH receptors but had no effect on the FSH receptor induction. The ability of PMSG to induce gonadotropin receptors can be arrested at any given time by injecting its antibody, thereby suggesting a continuous need for the hormonal inducer. Estrogen in the absence of the primary inducer was unable to maintain the induced LH and FSH receptor concentration. Inhibition of prostaglandin synthesis using indomethacin also had no effect on either the induction or degradation of gonadotropin receptors. Administration of PMSG antiserum, 48 h after PMSG injection, brought about a rapid decline in the induced receptors over the next 24 h, with a rate constant and \iota 1/2 of 0.078 h−1 and 8.9 h for FSH receptors and 0.086 h−1 and 8.0 h for the LH receptors, respectively.

La dynamique chromatinienne induite par le pic de LH dans les cellules de granulosa chez la souris

La régulation transcriptionnelle des gènes est un processus indispensable sans lequel la diversité phénotypique des cellules ainsi que l’adaptation à leur environnement serait inexistant. L’identification des éléments de régulation dans le génome est d’une importance capitale afin de comprendre les mécanismes gouvernant l’expression des gènes spécifiques à un type cellulaire donné. Ainsi, suite au pic de LH, le follicule ovarien entre dans un programme intensif de différentiation cellulaire, orchestré par des modifications majeures du profile transcriptionnel des cellules de granulosa, déclenchant ultimement l’ovulation et la lutéinisation, processus indispensables à la fertilité femelle. L’hypothèse supportée par cette étude stipule qu’une réorganisation de la structure chromatinienne survient aux régions régulatrices d’une panoplie de gènes dans les heures suivant le pic de LH et qu’en isolant et identifiant ces régions, il serait possible de retrouver des éléments essentiels aux processus d’ovulation et de lutéinisation. Ainsi, en utilisant un protocole standard de superovulation chez la souris, les éléments de régulation se modifiant 4h suivant l’administration de hCG ont été isolés et identifiés dans les cellules de granulosa en utilisant la méthode FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) combinée à un séquençage haut débit. Cette étude a démontré que suite au stimulus ovulatoire, les cellules de granulosa subissent une reprogrammation majeure des éléments de régulation, qui est corrélée avec une modification drastique de leurs fonctions biologiques. De plus, cette étude a mis en évidence une association majoritaire des éléments de régulation à des régions intergéniques distales et à des introns, indiquant que ces régions ont une importance capitale dans la régulation transcriptionnelle dans les cellules de granulosa. Cette étude a également permis d’identifier une panoplie de régulateurs transcriptionnels reconnus pour être essentiels à la fonction ovarienne, ainsi que leur sites de liaison dans le génome, démontrant que la méthode FAIRE est une méthode assez puissante pour permettre la prédiction d’événements moléculaires précis ayant un sens physiologique réel.

La régulation du gène CYP19A1 dans les cellules de granulosa bovine in vitro

L’oestradiol joue un rôle important dans la reproduction en général, particulièrement dans la croissance folliculaire chez la vache. La production de l’œstradiol nécessite l’expression du gène CYP19A1 suite à la stimulation des cellules de granulosa par l’hormone folliculostimulante (FSH) ou le facteur de croissance insulinique de type 1 (IGF-1). Chez la vache, il existe six promoteurs (1.1 ; 1.2 ; 1.3 ; 1.4 ; 1.5 et 2) qui dirigent la transcription du gène CYP19A1 dans les cellules de la granulosa. Le principal promoteur qui dirige la transcription au niveau de l’ovaire (cellules de granulosa) est le promoteur 2 (P2). Cependant, l’effet de la FSH et de l’IGF-1 sur l’activation de ces promoteurs d’aromatase demeure mal connu. De plus, la demi-vie du transcrit CYP19A1 est très courte avec une région 3’UTR relativement longue. L’analyse de la séquence 3’UTR montre la présence des motifs ARE (séquence riche en AU), des études antérieur montrent que ces séquences impliquent dans la régulation de la stabilité ou la dégradation de l’ARNm, ce qui est fort probable que la courte demi-vie de l’ARNm CYP19A1 est sous le contrôle post-transcriptionel. L’objectif de la thèse visait à étudier la régulation de l’expression du gène CYP19A1 chez la vache. Il y a deux thèmes soit étude de la régulation transcriptionnelle ciblant le promoteur et soit étude de la régulation post-transcriptionnelle impliquant la région 3’non traduite (3’UTR). Le premier objectif vise à étudier la régulation transcriptionnelle du gène CYP19A1. Nous avons étudié l'activité du promoteur ovarien bovin dans deux modèles de cellules de la granulosa, les cellules lutéinisées et nonlutéinisées in vitro, suite à une stimulation des cellules par la FSH ou IGF-1. Nous avons également évalué la voie de signalisation impliquée dans la régulation des différents promoteurs en utilisant un RT-PCR et un gène rapporteur (les différents promoteurs d’aromatase ont été insérés dans le vecteur pGL3promoter en amont du gène exprimant la luciférase). Les résultats de RT-PCR démontrent que la FSH et l’IGF-1 augmentent les concentrations d’ARNm provenant des deux promoteurs 2 et 1.1 dans les cellules de la granulosa non lutéinisées. Des expériences subséquentes ont montré que la FSH stimule le promoteur 2 via la voie PKA tandis que l'IGF-1 stimule le promoteur 2 via la voie PKC. La FSH et l’IGF-1 stimulent l’expression du promoteur 1.1 via la voie PI3K. L’analyse de l’activité luciférase démontre que dans les cellules de granulosa lutéinisées, la FSH stimule le promoteur 1.1 de façon dose dépendante et ne semble y avoir aucun effet significatif sur le promoteur 2. Nous avons donc comparé l’activité du promoteur PII/P2 humain, du rat, de la chèvre et de la vache dans les cellules de granulosa bovine lutéinisées. Le résultat le plus significatif est que le promoteur 2 bovine (et caprine) dépend de plusieurs facteurs de transcription (NR5A2, FOXL2) comparé au promoteur PII humain et celui du promoteur proximal du rat qui dépendent principalement de l'AMPc. En effet, nos résultats ont démontré une expression raisonnablement robuste du P2 bovine lorsque les cellules sont traitées à la forskoline, NR5A2 et FOXL2. Le facteur FOXL2 semble déterminer l'activité du promoteur 2 chez le ruminant. Le deuxième objectif vise à étudier la régulation post-transcriptionnelle du gène CYP19A1. Pour ce faire, nous avons déterminé la séquence minimale de l'ARNm CYP19A1 requise pour la régulation de sa demi-vie. Différents séquences de la région 3’UTR ont été insérés dans le vecteur pGL3promoter en aval du gène exprimant la luciférase ou soit dans le vecteur pGEMTeasy. Le vecteur pGL3promoter a été transfecté dans les cellules de granulosa lutéinisées pour évaluer l'impact de la séquence 3'UTR sur l'expression du gène rapporteur de la luciférase, alors que le vecteur pGEMTeasy a été utilisé pour la transcription in vitro afin de générer de l’ARNm. Ce dernier sera utilisé en réaction croisée au UV avec des extraits protéiques pour démontrer l’association du complexe ARNm/protéine. L’analyse de l’activité luciférase a permis d’identifier une séquence de 200 pb située entre 926 et 1134 pb de la région 3'UTR de l’ARNm CYP19A1 qui a réduit significativement l’activité de la luciférase. Selon les analyses de la réaction croisée au UV, une ou plusieurs protéines de 66 et 80 kDA se lient spécifiquement à la séquence de 200 pb qui réduit l’activité de luciférase. Cette protéine s'exprime dans les cellules de granulosa, mais n’a pas été détectée dans d'autres tissus comme le foie et le cœur. Par ailleurs, l’utilisation du gène rapporteur sensible à la FSH a suscité l’intérêt d'une compagnie pharmaceutique qui vend de l’equine chorionic gonadotropin (eCG) pour lui permettre de distinguer facilement l’eCG ayant une forte activité FSH et donc, avoir un produit commercial plus efficace et de meilleure qualité. Dans cette étude, nous avons développé un système de bioessai à la FSH basé sur la transfection des cellules avec un récepteur à la FSH et un gène rapporteur colorimètrique qui permet d’estimer l’activité de la FSH dans le sérum de la jument et qui pourrait être applicable au niveau de la ferme/industrie.

Bone morphogenetic proteins (BMP)-4,-6, and-7 potently suppress basal and luteinizing hormone-induced androgen production by bovine theca interna cells in primary culture: Could ovarian hyperandrogenic dysfunction be caused by a defect in thecal BMP signaling?

We reported recently that bovine theca interna cells in primary culture express several type-I and type-II receptors for bone morphogenetic proteins (BMPs). The same cells express at least two potential ligands for these receptors (BMP-4 and - 7), whereas bovine granulosa cells and oocytes express BMP-6. Therefore, BMPs of intrafollicular origin may exert autocrine/paracrine actions to modulate theca cell function. Here we report that BMP-4, - 6, and - 7 potently suppress both basal ( P < 0.0001; respective IC50 values, 0.78, 0.30, and 1.50 ng/ml) and LH-induced ( P < 0.0001; respective IC50 values, 5.00, 0.55, and 4.55 ng/ml) androgen production by bovine theca cells while having only a moderate effect on progesterone production and cell number. Semiquantitative RT-PCR showed that all three BMPs markedly reduced steady-state levels of mRNA for P450c17. Levels of mRNA encoding steroidogenic acute regulatory protein, P450scc, and 3 beta-hydroxysteroid dehydrogenase were also reduced but to a much lesser extent. Immunocytochemistry confirmed a marked reduction in cellular content of P450c17 protein after BMP treatment ( P < 0.001). Exposure to BMPs led to cellular accumulation of phosphorylated Smad1, but not Smad2, confirming that the receptors signal via a Smad1 pathway. The specificity of the BMP response was further explored by coincubating cells with BMPs and several potential BMP antagonists, chordin, gremlin, and follistatin. Gremlin and chordin were found to be effective antagonists of BMP-4 and - 7, respectively, and the observation that both antagonists enhanced ( P < 0.01) androgen production in the absence of exogenous BMP suggests an autocrine/paracrine role for theca-derived BMP- 4 and - 7 in modulating androgen production. Collectively, these data indicate that an intrafollicular BMP signaling pathway contributes to the negative regulation of thecal androgen production and that ovarian hyperandrogenic dysfunction could be a result of a defective autoregulatory pathway involving thecal BMP signaling.

Theca cells and the regulation of ovarian androgen production

Theca cells are essential for female reproduction being the source of androgens that are precursors for follicular oestrogen synthesis and also signal through androgen receptors (AR) in the ovary and elsewhere. Theca cells arise from mesenchymal cells around the secondary follicle stage. Their recruitment, proliferation and cytodifferentiation are influenced, directly or indirectly, by paracrine signals from granulosa cells and oocyte although uncertainty remains over which are the critically important signals at particular stages. In a reciprocal manner, theca cells secrete factors that influence granulosa cell proliferation and differentiation at different follicle stages. Differentiated theca interna cells acquire responsiveness to luteinizing hormone (LH) and other endocrine signals and express components of the steroidogenic machinery required for androgen biosynthesis. They also express insulin-like peptide 3 (INSL3) and its receptor (RXFP2), levels of which increase during bovine antral follicle development. INSL3 signaling may play a role in promoting androgen biosynthesis since knockdown of either INSL3 or its receptor (RXFP2) in bovine theca cells inhibits androgen biosynthesis while exogenous INSL3 can raise androgen secretion. Bone morphogenetic proteins (BMPs) of thecal or granulosal origin suppress thecal production of both INSL3 and androgen. Inhibin, produced in greatest amounts by granulosa cells of preovulatory follicles, reverses these BMP actions. Thus, BMP-induced inhibition of thecal androgen production may be mediated by reduced INSL3-RXFP2 signaling. Activins also inhibit androgen production in an inhibin-reversible manner and recent evidence in sheep indicates that theca cells synthesize and secrete activin, implying an autocrine role in suppressing androgen biosynthesis in smaller follicles, akin to that envisaged for BMPs.

Testosterona e gonadotrofina coriônica humana estimulam a esteroidogênese em células da granulosa de folículo pré-ovulatório de égua?

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expressão gênica das isoformas do receptor do hormônio luteinizante(LHR) em células células da granulosa antes, durante e após o desvio folicular em novilhas Nelore

Pós-graduação em Medicina Veterinária - FMVZ

Expressão gênica do receptor do hormônio luteinizante (LHR), em células da teca e da granulosa de folículos antrais bovinos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expressão dos transcritos do receptor de LH em células da granulosa de fêmeas nelore submetidas ou não à superestimulação ovariana

Pós-graduação em Medicina Veterinária - FMVZ

Efeito da progesterona na expressão do RNAm do receptor de LH e enzimas esteroidogênicas reguladoras do desenvolvimento folicular em células da granulosa de novilhas nelore

Pós-graduação em Zootecnia - FMVZ