Hypoxia up-regulates VEGF ligand and downregulates VEGF soluble receptor mRNA expression in bovine granulosa cells in vitro.

Oxygen concentration (02) in antral ovarian follicles is below that found in most tissues, which is important for adequate granulosa cell function. The VEGF system is linked to angiogenesis and responds to changing 02 by stimulating neovascularization when levels are low. However, in the avascular granulosa cell layer of the follicle, VEGF action is directed to stimulating cell viability and steroidogenesis. The aim of this study was to examine the effect of 02 concentration on granulosa cell expression of the VEGF-system components. Bovine granulosa cells were isolated from medium-sized follicles (4-7 mm in diameter), placed in McCoy 5a medium supplemented with 10 ng/mL of insulin, 1 ng/mL of IGF-I, and 1 ng/mL of FSH, and cultured in four well plates (500 thousand cells per well), on three separate occasions. Culture plates were placed in gas-impermeable jars with a gas mixture containing either 2%, or 5% of O2, or under atmospheric air condition inside an incubator (20% of 02). Media was replaced at 48 h of culture and cells from the plate in each oxygen concentration were pooled for RNA extraction after 96 h. The number of mRNA copies for the VEGF-system components - including ligands (VEGF120, VEGF120b, VEGF165 and VEGF165b), enzymes (cyclin-dependent like kinases-1, CLK1 and serine-arginine protein kinase 1, SRPK1), splicing factors (serine-arginine-rich splicing factors, SRSF1 and SRSF6), and the membrane-bound (VEGFR1, VEGFR2) and soluble forms of the receptors (sVEGFR1 and sVEGFR2) were quantified by qPCR. Granulosa cells cultured with low 02 (2%) had a higher expression of VEGF ligands (P < 0.05) when compared to cells cultured at 20% 02. VEGF164b mRNA was absent in granulosa cells from all culture conditions. The 2 and 5% 02 levels, which coincide with physiological concentrations, in the ovarian follicle, induced higher SRSF6 expression than atmospheric 02 concentrations (20%, P < 0.05). In contrast, mRNA copies for SRPK1, CLK1, SRSF1, VEGFR1 or VEGFR2 did not differ between 02 culture conditions. (P > 0.05). Nonetheless, mRNA copies for the soluble receptors, sVEGFR1 and sVEGFR2, linearly increased (P < 0.05) with 02 concentration. These results suggest that when cultured under hypoxic conditions, granulosa cells may develop an autocrine milieu that favors VEGF's biological effects on their survival and function.

Sphingosine-1-phosphate (S1P) in ovarian physiology and disease.

Sphingosine-1-phoshate (S1P) is a membrane sphingolipid involved in several physiological processes, including cell proliferation, tissue growth, cell survival and migration, inflammation, vasculogenesis, and angiogenesis. Herein, we review the most critical effects of S1P on ovarian function, including its physiological and pathophysiological effects. Based on the available evidence, S1P plays an important role in ovarian physiology, participating as an essential stimulator of follicular development in both the preantral and antral phases, as well as in ovulation and corpus luteum development. Moreover, S1P may be a good cytoprotective agent against cancer treatment side-effects (chemotherapy with or without radiation therapy). In the future, this compound may be given for fertility preservation to women undergoing cancer treatment. However, further studies are required to confirm its efficacy in ovarian protection and also its safety in terms of cancer prognosis, given the biological action of the compound. Under- or over-production of S1P may be related to ovarian pathologies.

Co-ordinated expression of the VEGF system components in granulosa cells to develop a proangiogenic autocrine milieu during ovarian follicle development.

In the present study, we investigated the temporal relationship between angiogenic and antiangiogenic vascular endothelial growth factor isoforms (VEGFxxxa and VEGFxxxb, respectively), the receptors VEGFR1 and VEGFR2, their soluble forms, and the kinases and the splicing factors regulating the synthesis of VEGF isoforms in healthy and atretic antral follicles. The results show a higher (p < 0.05) messenger RNA (mRNA) expression of VEGF120a, VEGF164a, and VEGF120b in healthy than in atretic follicles, but the mRNA expression of VEGF164b was not detected. The mRNA of serine-arginine protein kinase 1 ( SRPK1) was higher ( p < 0.05) in large healthy follicles than in large atretic follicles. In contrast, atretic follicles had higher mRNA expression of a soluble form of the receptor 2 of VEGF ( sVEGFR2) than healthy follicles ( p < 0.05). Additionally, we observed a positive relationship ( p < 0.05) between SRPK1 and serine-arginine-rich splicing factor 1 ( SRSF1) with the angiogenic isoforms VEGF120a and VEGF164a and between CDC-like kinases-1 ( CLK1) and SRSF6 with the antiangiogenic VEGF120b isoform. Principal components analysis (PCA) resulted in two PC explaining 71% of the variation, which was formed by the VEGF isoforms, the kinases and the splicing factor (PC1) and by the VEGF receptors (PC2). When PC analysis was carried out within follicular health status, there were no differences for PC1 between follicular status, whereas PC2 differed between healthy and atretic follicles. In conclusion, the higher mRNA expression for VEGF120a and VEGF164a, the low expression of sVEGFR2, and absent expression of mRNA for VEGF164b provide evidence of a proangiogenic autocrine milieu to support granulosa cells during follicle development.

Supplementation with rumen-protected L-arginine-HCl increased fertility in sheep with synchronized estrus.

The aim of the present study was to evaluate the effects of L-arginine-HCl supplementation on ovulation rate, fertility, prolificacy, and serum VEGF concentrations in ewes with synchronized oestrus. Thirty Suffolk ewes with a mean body weight of 45 ± 3 kg and a mean body condition score (BCS) of 2.4 ± 0.28 were synchronized for estrus presentation with a progestin-containing sponge (20 mg Chronogest® CR) for 9 days plus PGF2-α (Lutalyse; Pfizer, USA) on day 7 after the insertion of the sponge. The ewes were divided into two groups; i.e., a control group (n = 15) that was fed on the native pasture (basal diet) and an L-arginine-HCl group (n = 15) that received 7.8 g of rumen-protected L-arginine-HCl from day 5 of the sponge insertion until day 25 after mating plus the basal diet. The L-arginine-HCl was administered daily via an esophageal probe between days 5 and 9 of the synchronization protocol and every third day subsequently. Blood samples were drawn from the jugular vein every 6 days throughout the entire experimental period. The results revealed that the L-arginine-HCl supplementation increased fertility during the synchronized estrus (P = 0.05). However, no effects were observed on the final BCS (P = 0.78), estrus presentation (P = 0.33), multiple ovulations (P = 0.24), prolificacy (P = 0.63), or serum VEGF concentration. In conclusion, L-arginine-HCl supplementation during the period used in this study increased fertility in sheep with synchronized estrus possibly due to improved embryo-fetal survival during early pregnancy.