GnRH agonist versus GnRH antagonist in IVF/ICSI cycles with recombinant LH supplementation: DNA fragmentation and apoptosis in granulosa cells

Objective: To compare the level of apoptosis and DNA fragmentation in the human granulosa cell (GC) layer exposed to an agonist or antagonist of GnRH in intracytoplasmic sperm injection (ICSI) cycles supplemented with recombinant LH (rLH).Study design: Patients without ovulatory dysfunction, aged <= 37 years and in their first ICSI cycle were prospectively randomised to receive either a long GnRH agonist protocol or a multi-dose antagonist protocol. In both groups, recombinant FSH supplemented with rLH was used for ovarian stimulation, and the GCs were collected during oocyte denudation. The GCs were then analysed for DNA fragmentation by TUNEL assay and for apoptosis using the annexin-V assay. The outcomes were given as the percentage of GCs with DNA fragmentation and apoptosis out of the total number of GCs analysed. Comparison of the agonist versus the antagonist group was performed using the Mann-Whitney test.Results: DNA fragmentation: 32 patients were included in either the GnRH agonist group (n = 16) or the antagonist group (n = 16). The percentage of GCs with positive DNA fragmentation did not differ significantly (P = 0.76) between the agonist group (15.5 +/- 9.4%) and the antagonist group (18.8 +/- 13.3%). Apoptosis: 28 patients were included in either the GnRH agonist group (n = 14) or the antagonist group (n = 14). The percentage of GCs positive for apoptosis did not differ significantly (P = 0.78) between the agonist group (34.6 +/- 14.7%) and the antagonist group (36.5 +/- 22%).Conclusions: The results suggest that therapy with either an agonist or antagonist of GnRH is associated with comparable levels of DNA fragmentation and apoptosis in granulosa cells in ICSI cycles supplemented with rLH. (C) 2012 Elsevier B.V. All rights reserved.

Regulation of angiotensin type 2 receptor in bovine granulosa cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

THE EFFECT OF SERUM ON IN-VITRO MATURATION, IN-VITRO FERTILIZATION AND STEROIDOGENESIS OF BOVINE OOCYTES COCULTURED WITH GRANULOSA-CELLS

The influence of fetal calf serum alone (FCS) or associated with proestrous (FCS+PCS), estrous (FCS+ECS) or metaestrous (FCS+MCS) cow serum added to the culture medium and of the steroids produced by co-cultured granulosa cells were evaluated in terms of the in vitro maturation (TVM) and fertilization (IVF) of bovine oocytes. Supplementation of the medium with FCS+ECS and FCS+MCS resulted in higher proportions of oocytes that reached metaphase II (96.0% and 93.3%, respectively) and in higher proportions of embryos that reached the four- and eight-cell/morula stages (51.9% and 65.6%, respectively), whereas the supplementation with FCS and FCS+PCS resulted in only 79.2% and 67.5%, respectively, of matured oocytes and 26.7% and 34.3%, respectively, of cleaved embryos. These findings show that the best IVM and IVF were obtained at lower concentrations of estradiol produced by co-cultured granulosa cells (supplementation with FCS+ECS: 10.3 ng/ml and FCS+MCS: 2.1 ng/ml), whereas the worst-results in IVM and IVF occurred at higher concentrations of estradiol that were obtained with FCS (33.1 ng/ml) and FCS+PCS (19.9 ng/ml) supplementation. These data suggest an inhibitory effect of estradiol on resumption of oocyte meiosis in vitro.

Expression of LH receptor mRNA splice variants in bovine granulosa cells: Changes with follicle size and regulation by FSH in vitro

In cattle, most evidence suggests that granulosa cells express LH receptors (LHR) after (or as) the follicle becomes dominant, however there is some suggestion that granulosa cells from smaller pre-dominant follicles may express several LHR mRNA splice variants. The objective of this study was to measure LHR expression in bovine follicles of defined size and steroiclogenic ability, and in granulosa cells from small follicles (< 6 mm diameter) undergoing differentiation in vitro. Serniquantitative RT-PCR demonstrated that LHR mRNA was undetectable in granulosa cells of follicles < 7 mm diameter (nondominant follicles), and increased with follicle diameter in follicles > 7 mm diameter. Splice variants with deletions of exon 10 and part of exon 11 were detected as previously described, and we detected a novel splice variant with a deletion of exon 3. Cultured granulosa cells contained LHR mRNA, but with significantly greater amounts of variants with deletions of exon 10 and/or exon 11 compared with cells from dominant follicles. FSH increased the abundance of some but not all LHR mRNA splice variants in cultured granulosa cells. The addition of LH to cultured cells did not increase progesterone secretion, despite the presence of LHR mRNA. Collectively, these data suggest that granulosa cells do not acquire functional LHR until follicle dominance occurs.

Fibroblast Growth Factor-2 Regulation of Sprouty and NR4A Genes in Bovine Ovarian Granulosa Cells

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

The effect of serum on in vitro maturation, in vitro fertilization and steroidogenesis of bovine oocytes co-cultured with granulosa cells.

The influence of fetal calf serum alone (FCS) or associated with proestrous (FCS+PCS), estrous (FCS+ECS) or metaestrous (FCS+MCS) cow serum added to the culture medium and of the steroids produced by co-cultured granulosa cells were evaluated in terms of the in vitro maturation (IVM) and fertilization (IVF) of bovine oocytes. Supplementation of the medium with FCS+ECS and FCS+MCS resulted in higher proportions of oocytes that reached metaphase II (96.0% and 93.3%, respectively) and in higher proportions of embryos that reached the four- and eight-cell/morula stages (51.9% and 65.6%, respectively), whereas the supplementation with FCS and FCS+PCS resulted in only 79.2% and 67.5%, respectively, of matured oocytes and 26.7% and 34.3%, respectively, of cleaved embryos. These findings show that the best IVM and IVF were obtained at lower concentrations of estradiol produced by co-cultured granulosa cells (supplementation with FCS+ECS: 10.3 ng/ml and FCS+MCS: 2.1 ng/ml), whereas the worst results in IVM and IVF occurred at higher concentrations of estradiol that were obtained with FCS (33.1 ng/ml) and FCS+PCS (19.9 ng/ml) supplementation. These data suggest an inhibitory effect of estradiol on resumption of oocyte meiosis in vitro.

Ovarian superstimulation using FSH combined with equine chorionic gonadotropin (eCG) upregulates mRNA encoding proteins involved with LH receptor intracellular signaling in granulosa cells from Nelore cows

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

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)