Effects of PI3K and FSH on steroidogenesis, viability and embryo development of the cumulus-oocyte complex after in vitro culture.

The purpose of this study was to evaluate the effects of FSH and PI3K on the nuclear maturation, viability, steroidogenesis and embryo development of bovine cumulus-oocyte complexes (COCs). Oocyte maturation was achieved with MIV B, MIV B+100 µM LY294002, MIV B+10 ng/mL follicle stimulating hormone (FSH), or MIV B+10 ng/mL FSH+100 µM LY294002 treatments for 22-24 h. After the cultured COCs were denuded, oocytes were separated into those that extruded polar bodies (mature) and those that did not, and real-time polymerase chain reaction (PCR) for BAX, BCL2, LHR, FSHR, CYP11A1, CYP19A1 and HSD17B1 genes was performed. The culture medium was collected to determine the levels of 17ß-estradiol (E2) and progesterone (P4). The trypan blue test was used to study COC viability, and embryo development was evaluated. FSH increased nuclear maturation and PI3K blocked the maturation but did not influence oocyte viability. BAX and BCL2 expression levels in the cumulus cells were only affected by FSH, and the BAX levels decreased after treatment with LY294002. FSH increased the levels of E2 and P4, however inhibition of PI3K decreased E2 levels. MIV B enhanced levels of LHR, FSHR, CYP11A1, CYP19A1 and HSD17B1, whereas LY294002 inhibited the expression levels of all genes. MIV B+FSH decreased the expression levels of all genes except CYP11A1. LY294002 did not demonstrate any effects in the presence of FSH. Embryo development was significantly decreased when the MIV B+FSH medium was used. In conclusion, FSH controls the steroidogenesis, viability and gene expression in COCs. PI3K plays essential roles in nuclear maturation, steroidogenesis and embryo development.

Insulin influences developmental competence of bovine oocytes cultured in α-MEM plus follicle-simulating hormone.

The aim of this study was to evaluate the dose-response effect of insulin, plus follicle-simulating hormone (FSH) at a fixed concentration, in a serum-free defined culture medium (DCM) on the in vitro maturation of bovine cumulus-oocyte complexes (COCs). For oocyte nuclear maturation, the expression levels of GDF9, GLUT1, PRDX1 and HSP70.1 transcripts related to oocyte and embryo developmental competence were analysed. For in vitro maturation (IVM), cumulus-oocyte complexes from slaughterhouse ovaries were distributed into four groups based on insulin concentration added to serum-free DCM, which was composed of alpha minimum essential medium (α-MEM), as basal medium: (1) DCM control: 0 ng/ml; (2) DCM1: 1 ng/ml; (3) DCM10: 10 ng/ml; and (4) DCM100: 100 ng/ml. After IVM, the nuclear status of a sample of oocytes was analysed and the other oocytes were submitted for in vitro fertilization (IVF) and in vitro culture (IVC). Different concentrations of insulin did not affect significantly the nuclear maturation and cleavage rate (72 h post-insemination) across all groups. Blastocyst rate (192 h post-insemination) did not differ in DCM control (24.3%), DCM1 (27.0%) and DCM10 (26.3%) groups, but the DCM100 (36.1%) group showed a greater blastocyst rate (P 0.05) was observed at the different insulin concentrations. The results indicated that insulin added to DCM influenced levels of transcripts related to cellular stress (HSP70-1 and PRDX1) and oocyte competence (GDF9) in bovine oocytes and at higher concentrations enhanced blastocyst production.

Regulation of steroidogenesis by atrial natriuretic peptide (ANP) in the rat testis: differential involvement of GC-A and C receptors.

Previous studies have established a stimulatory effect of natriuretic peptides (NP) on testosterone production in mouse Leydig cells as intense as that of LH. Chronic administration of ANP in mice, on the other side, reduced testosterone levels. So, the understanding of the role of ANP on testicular steroidogenesis has been impaired by discrepant findings. The aim of the present study was to clarify the physiological role of ANP in the rat testis steroidogenesis using a model that preserves the interactions between testis cells and a medium devoid of any circulating factors that could interfere with testosterone production. First, ANP was immunolocalized in the interstitial compartment of the rat testis, mainly in Leydig cells. We also determined the presence of ANP and both GC-A (guanylyl cyclase A) and C receptors by real-time PCR in testis. Perfusion in vitro of testis with ANP (1 and 3x10(-7)M) stimulated testosterone production in a time- and dose-dependent manner. On the other side, testosterone secretion induced by LH was blunted by ANP. Similar effect was obtained using the specific C receptor ligand, cANF, indicating the involvement of C receptor in such response. In conclusion, ANP stimulated testosterone production in the rat testis perfused in vitro but decreased testosterone production LH-induced, effect that seems to involve C receptor. To this extent, our results suggest the existence of a local and complex peptidergic system in the rat testis, involving ANP and its receptors that could importantly modulate the androgen biosynthesis.

Efeitos da endotelina-1 sobre a esteroidogênese e o crescimento celular em cultura de células da granulosa de bovinos / Endothelin-1 effects on steroidogenesis and cellular growth of bovine granulosa cells culture

Além do controle neuroendócrino do eixo hipotalâmico-hipofisáriogonadal existem fatores autócrinos e parácrinos que controlam o crescimento, seleção e ovulação nos mamíferos. Estes fatores ainda não estão bem estabelecidos. Objetivando analisar as variações nas concentrações plasmáticas da endotelina-1 em um ciclo estral bovino e seus efeitos sobre o cultivo de células da granulosa, foram analisadas amostras plasmáticas de 5 dias (0, 1, 3, 7 e 13) de um ciclo estral, tendo como base o dia da ovulação (dia 0) e 3 horas (8, 13 e 16) do 14º dia do ciclo. As variações nas concentrações plasmáticas de endotelina-1 não foram significativas para os dias assim como para as horas do dia analisado. Células da granulosa obtidas de folículos antrais foram cultivadas durante 48 horas com diferentes concentrações de endotelina. Os resultados demonstraram que a endotelina atua de maneira dosedependente estimulando a produção de progesterona e inibindo o crescimento celular.

Besides the neuroendocrine control of the hypotalamic-hypophisarygonodal axis there are autocrines and paracrines factors that control the growth, selection and ovulation in the mammals. These factors are not well established yet. Aiming to analyze the variations in the endotelin-1plasmatic concentrations in a bovine estral cycle and their effects on the culture of granulosa cells, plasma samples of 5 days (0,1,3,7 and 13) from an estral cycle have been analyzed, having as basis the ovulation day (day 0) and 3 hours (8,13,16) of the fourteenth day of the cycle. The variations in the endothelin-1 concentrations were not significant for the days as well as for the hours of the analyzed day. Granulosa cells obtained from antral follicles were cultivated during 24 hours with different concentrations of endothelin. The results demonstrated that the endothelin-1 acts in a dose-dependent manner stimulating the production of progesterone and inhibiting the cellular growth.

Concentrações de FSH, LH, estradiol, progesterona e histamina no soro, no fluido peritoneal e no fluido folicular de mulheres com e sem endometriose / FSH, LH, estradiol, progesterone, and histamine concentrations in serum, peritoneal fluid and follicular fluid of women with and without endometriosis

OBJETIVOS: relatos da literatura mostram que não há dados conclusivos sobre a associação entre a endometriose e as concentrações de hormônios envolvidos no controle da reprodução. Este estudo foi realizado para determinar as concentrações de FSH, LH, estradiol (E), progesterona (P) e histamina (Hi) no soro, no fluido peritoneal e no fluido folicular de mulheres com e sem endometriose. MÉTODOS: a extensão da doença foi estadiada de acordo com a American Fertility Society classification (1997). Para a coleta de soro e de fluido peritoneal foram selecionadas 28 mulheres com endometriose submetidas à laparoscopia diagnóstica (18 mulheres inférteis com endometriose I-II e dez mulheres inférteis com endometriose III-IV). Para o grupo controle, foram selecionadas 21 mulheres férteis submetidas à laparoscopia para esterilização tubárea. O fluido folicular foi obtido de 39 mulheres inférteis submetidas a fertilização in vitro (21 mulheres com endometriose e 18 mulheres sem endometriose). RESULTADOS: as concentrações de FSH e LH no soro, no fluido peritoneal e no fluido folicular não diferiram significativamente entre os grupos. As concentrações de E e P no fluido peritoneal foram significativamente mais baixas em mulheres inférteis com endometriose (E: 154,2±15,3 para estágios I-II e 89,3±9,8 ng/mL para estágios III-IV; P: 11,2±1,5 para estágios I-II e 7,6±0,8 ng/mL para estágios III-IV) em comparação com mulheres controle (E: 289,1±30,1; P: 32,8±4,1 ng/mL) (Testes de Kruskal-Wallis/Dunn; p<0,05). No soro, as concentrações de E e P seguiram o mesmo padrão. No fluido folicular, as concentrações de E e Hi foram significativamente mais baixas em mulheres com endometriose (E: 97,4±11,1 ng/mL; Hi: 6,6±0,9 ng/mL) em comparação com mulheres sem endometriose (E: 237,5±28,5 pg/mL; Hi: 13,8±1,3 ng/mL), enquanto os níveis de P não revelaram diferença significativa entre os grupos (teste t de Student; p<0,05). CONCLUSÕES: nossos resultados indicam disfunção...

PURPOSE: literature reports show that there are no conclusive data about the association between endometriosis and the concentrations of hormones involved in the control of reproduction. Thus, the present study was undertaken to determine FSH, LH, estradiol (E), progesterone (P), and histamine (Hi) concentrations in serum, peritoneal fluid and follicular fluid of women with and without endometriosis. METHODS: the extent of the disease was staged according to the revised American Fertility Society classification (1997). For the collection of serum and peritoneal fluid, 28 women with endometriosis undergoing diagnostic laparoscopy were selected (18 infertile women with endometriosis I-II and ten infertile women with endometriosis III-IV). For the control group, 21 fertile women undergoing laparoscopy for tubal sterilization were selected. Follicular fluid was obtained from 39 infertile women undergoing in vitro fertilization (21 women with endometriosis and 18 women without endometriosis). RESULTS: FSH and LH levels in serum, peritoneal fluid and follicular fluid did not differ significantly between groups. On the other hand, E and P concentrations in the peritoneal fluid were significantly lower in infertile women with endometriosis (E: 154.2±15.3 for stages I-II and 89.3 ng/mL±9.8 ng/mL for stages III-IV; P: 11.2±1.5 for stages I-II and 7.6 ng/mL±0.8 for stages III-IV) in comparison with control women (E: 289.1 ng/mL±30.1; P: 32.8±4.1 ng/mL) (Kruskal-Wallis/Dunn tests; p<0.05). In serum, estradiol and progesterone concentrations followed the same pattern. In the follicular fluid, E and Hi concentrations were significantly lower in women with endometriosis (E: 97.4±11.1 pg/mL; Hi: 6.6±0.9 ng/mL) in comparison to women without endometriosis (E: 237.5±28.5 pg/mL; Hi: 13.8±1.3 ng/mL) (Student t-test; p<0.05), while progesterone levels revealed no significant difference between groups. CONCLUSIONS: our results indicate ovary dysfunction in...

Imunorreatividade para endotelina-1 no plasma e fluido folicular de eqüinos e bovinos / Endothelin-1 immunoreactive in the plasma and follicular fluid of horses and cattle

endotelina-1, um peptídeo isolado recentemente, tem sido observada em diferentes tecidos e está envolvida em várias funçöes reprodutivas. Propusemo-nos a determinar as concentraçöes da endotelina-1 por radioimunoensaio no plasma de eqüinos e bovinos e no fluido folicular de eqüinos. Foram testados 2 tipos de inibidores de degradaçäo protéica no plasma e fluido folicular. A imunorreatividade média foi de 47,5 por cento, a dose mínima detectável foi de 1 pg/tubo e o coeficiente de variaçäo inter e intra-ensaio foi de 2,36 por cento e 7,26 por cento, respectivamente. O paralelismo entre a curva padräo e as curvas determinadas com diferentes volumes de plasma e fluido folicular valida o radioimunoensaio de endotelina-1 para estas espécies. Segundo nosso conhecimento, este é o primeiro relato de literatura que detecta por radioimunoensaio a endotelina-1 no plasma e fluido folicular de eqüinos e plasma de bovinos. Os resultados do presente trabalho sugerem a utilizaçäo do método de radioimunoensaio utilizando-se kits comerciais para a determinaçäo das concentraçöes da endotelina-1 no plasma de eqüinos e bovinos e fluido folicular de eqüinos, pelo fato de este apresentar alta sensibilidade, precisäo e reprodutibilidade, podendo assim ser utilizado no estudo de múltiplas funçöes da endotelina-1 nestas espécies animais e, em especial, no sistema endócrino-reprodutivo