There is evidence that the renin-angiotensin system plays an important role in ovulation in cattle. Using an in vivo model, we investigated the role of angiotensin (Ang) II in bovine ovulation by injecting Ang II receptor antagonists into ovulatory follicles. Animals (nZ102) were pre-synchronized and, when the follicles reached 12 mm, they were given the respective treatment and the cows received GnRH agonist (i.m.) to induce ovulation. The ovulation rate was significantly lower when 100 mM saralasin (Ang II receptor antagonist) was intrafollicularly injected (14.3%) in comparison with saline solution (83.3%). Based on these results, a second experiment was carried out to determine the timing of Ang II's critical role in ovulation. Saralasin inhibited ovulation only when applied at 0 and 6 h (16.7 and 42.9% ovulation rate in the 0-and 6-h groups respectively), but not at 12 h (100%) following GnRH agonist treatment. To investigate the subtypes of Ang II receptors implicated in the LH-induced ovulation, losartan (LO; AT 1 -Ang II receptor antagonist), PD123 319 (AT 2 -Ang II receptor antagonist), LOCPD123 319, or saline were intrafollicularly injected when the cows were challenged with GnRH agonist. Ovulation was inhibited by PD123 319 and LOCPD123 319 (50.0 and 33.3% on ovulation rate respectively), but not by LO or saline solution (100% ovulation in both groups). From these results, we suggest that Ang II plays a pivotal role in the early mechanism of bovine ovulation via the AT 2 receptor subtype.

Angiotensin II (AngII) prevents the inhibitory effect of follicular cells on oocyte maturation, but its involvement in LH-induced meiotic resumption remains unknown. The aim of this study was to assess the involvement of AngII in LH-induced meiotic resumption and of prostaglandins (PGs) in the action of AngII. In the experiment I, seven cows were superovulated, intrafollicularly injected with 10 mM saralasin (a competitive AngII antagonist) or saline when the follicles reached a diameter larger than 12 mm, and challenged with a GnRH agonist to induce an LH surge. Fifteen hours after GnRH, the animals were ovariectomized and the oocytes were recovered to determine the stage of meiosis. The oocytes from follicles that received saline were in germinal vesicle (GV) breakdown (30.8%) or metaphase I (MI; 69.2%) stage while those that received saralasin were in the GV stage (100%; P!0.001) 15 h after GnRH agonist. In another experiment, oocytes were co-cultured with follicular hemisections for 15 h to determine whether PGs mediate the effect of AngII on meiotic resumption. Indomethacin (10 mM) inhibited AngII-induced meiotic resumption (13.4 vs 77.5% MI without indomethacin; P!0.001). Furthermore, the GV oocytes progressed to MI at a similar rate when PGE 2 , PGF 2a or AngII was present in the co-culture system with follicular cells (PGE 2 77.4%, PGF 2a 70.0%, and AngII 75.0% MI). In conclusion, our results provide strong evidence that AngII mediates the resumption of meiosis induced by an LH surge in bovine oocytes and that this event is dependent on PGE 2 or PGF 2a produced by follicular cells. Reproduction (2008) 136 733-740

Angiotensin II (AngII) has a role in ovarian follicle development, ovulation, and oocyte meiotic resumption. The objective of the present study was to characterise the AngII profile and the mRNA encoding RAS proteins in a bovine follicular wave. Cows were ovariectomised when the size between the largest (F1) and the second largest follicle (F2) was not statistically different (day 2), slightly different (day 3), or markedly different (day 4). AngII was measured in the follicular fluid and the mRNA abundance of genes encoding angiotensin-converting enzyme (ACE), (pro)renin receptor, and reninbinding protein (RnBP) was evaluated in the follicular cells from F1 and F2. The AngII levels increased at the expected time of the follicular deviation in F1 but did not change in F2. However, the expression of the genes encoding ACE, (pro)renin receptor, and RnBP was not regulated in F1 but was upregulated during or after the follicular deviation in F2. Moreover, RnBP gene expression increased when the F1 was treated with the oestrogen receptor-antagonist in vivo. In conclusion, the AngII concentration increased in the follicular fluid of the dominant follicle during and after deviation and further supports our finding that RAS is present in the ovary regulating follicular dominance.

Angiotensin (Ang) II is widely known for its role in the control of systemic blood vessels. Moreover, Ang II acts on the vascular control of ovarian function, corpus luteum formation, and luteolysis. Over the past 10 years, our research group has been studying the new concept of the renin-angiotensin system (RAS) as an autocrine/paracrine factor regulating steroidogenesis and promoting different cellular responses in the ovary, beyond vascular function. We have developed and used different in vivo and in vitro experimental models to study the role of RAS in the ovary and a brief overview of our findings is presented here. It is widely accepted that there are marked species differences in RAS function in follicle development. Examples of species-specific functions of the RAS in the ovary include the involvement of Ang II in the regulation of follicle atresia in rats vs the requirement of this peptide for the dominant follicle development and ovulation in rabbits and cattle. More recently, Ang-(1-7), its receptor, and enzymes for its synthesis (ACE2, NEP, and PEP) were identified in bovine follicles, implying that Ang-(1-7) has an ovarian function. Other novel RAS components (e.g. (pro)renin receptor and renin-binding protein) recently identified in the bovine ovary show that ovarian RAS is poorly understood and more complex than previously thought. In the present review, we have highlighted the progress toward understanding the paracrine and autocrine control of ovarian antral follicle development and ovulation by ovarian tissue RAS, focusing on in vivo studies using cattle as a model.

It is generally understood that angiotensin II (AngII) promotes follicle atresia in rats, although recent data suggested that this may not be true in cattle. In this study, we aimed to determine in vivo whether AngII alters follicle development in cattle, using intrafollicular injection of AngII or antagonist into the growing dominant follicle or the second largest subordinate follicle. Injection of saralasin, an AngII antagonist, into the growing dominant follicle inhibited follicular growth, and this inhibitory effect was overcome by systemic FSH supplementation. Injection of AngII into the dominant follicle did not affect follicular growth, whereas injection of AngII into the second largest follicle prevented the expected atresia of this subordinate follicle, and the treated follicle grew at the same rate as the dominant follicle for the next 24 h. Inhibition of AngII action in the dominant follicle decreased estradiol concentrations in follicular fluid and the abundance of mRNA encoding aromatase, 3β-hydroxysteroid dehydrogenase, LH receptor, and cyclinD2 in granulosa cells, with minimal effects on theca cells. The effect of AngII on aromatase mRNA levels was confirmed using an in vitro granulosa cell culture system. In conclusion, these data suggest that AngII signaling promotes follicle growth in cattle and does so by regulating genes involved in estradiol secretion and granulosa cell proliferation and differentiation.