Effects of GnRH antagonist treatment on follicular development and angiogenesis in the primate ovary

Abstract

Angiogenesis is required for normal follicular development but the role of gonadotrophins in the control of follicular angiogenesis remains to be elucidated. This study investigated the effects of treatment with GnRH antagonist in vivo on follicular development and angiogenesis in the marmoset. GnRH antagonist was administered on either follicular day 0 or day 5 of the 10-day follicular phase with ovaries collected on day 10. Ovaries from control marmosets were studied at day 5 (mid follicular phase) and day 10 (periovulatory period). Ovaries were fixed, serial sectioned and subjected to morphological analysis and immunocytochemistry to determine cell proliferation and follicular endothelial cell area and in situ hybridization to assess changes in expression of vascular endothelial growth factor (VEGF). Treatment with GnRH antagonist from day 0-10 resulted in an absence of dominant preovulatory follicles seen in controls. In the remaining tertiary follicles granulosa, theca and endothelial cell proliferation was reduced, resulting in a minor reduction in vascular density. However, VEGF mRNA expression was unaffected by treatment. Treatment from day 5-10 did not prevent development of ovulatory size follicles, but they were atretic and lacked VEGF mRNA. These results suggest that while VEGF expression in the preovulatory follicle is under gonadotrophic control it is not dependent on normal gonadotrophin secretion in tertiary follicles, indicating that there are other paracrine factors regulating VEGF expression in the developing ovarian follicle.

Theca: the forgotten cell of the ovarian follicle

“…There are many potential factors involved in controlling angiogenesis in the developing follicle but vascular endothelial growth factor (VEGF) has a central role and has been studied extensively. VEGF, a potent mitogen for endothelial cells (Ferrara & Davis-Smyth 1997), stimulates vascular permeability (Connolly 1991, Theca cells Senger et al 1993), and is highly expressed in granulosa cells and at lower levels in the thecal layer of follicles from the secondary stage onwards in primate ovaries (Taylor et al 2004). In rodents, cows, and pigs, VEGF expression is weak in early follicle development but increases as the follicle progresses to ovulation (Maisonpierre et al 1997, Barboni et al 2000, Greenaway et al 2005, although levels of VEGF decrease in granulosa cells in sheep and marmoset follicles just prior to ovulation (Ravindranath et al 1992, Redmer et al 2001, Taylor et al 2004.…”

Oestrogen and progesterone receptors in the marmoset endometrium: changes during the ovulatory cycle, early pregnancy and after inhibition of vascular endothelial growth factor, GnRH or ovariectomy

“…Marmosets were treated with a GnRH antagonist or VEGF Trap starting between luteal days 14-16 (Wulff et al 2002, Taylor et al 2004. To block the action of VEGF, we employed the VEGF Trap, a recombinant chimeric protein comprising portions of the extracellular domains of the human VEGF receptors VEGFR1 and VEGFR2 expressed in sequence with the Fc portion of human immunoglobulin (Holash et al 2002) (Taylor et al 2004).…”

“…Marmosets were treated with a GnRH antagonist or VEGF Trap starting between luteal days 14-16 (Wulff et al 2002, Taylor et al 2004. To block the action of VEGF, we employed the VEGF Trap, a recombinant chimeric protein comprising portions of the extracellular domains of the human VEGF receptors VEGFR1 and VEGFR2 expressed in sequence with the Fc portion of human immunoglobulin (Holash et al 2002) (Taylor et al 2004). In addition, to determine the effects of total withdrawal of ovarian steroids, four marmosets were ovariectomised following sedation with ketamine hydrochloride (Parke-Davis Veterinary, Pontypool, Gwent, UK), anaesthesia by Saffan (Alphaxalone/Alpadalone, Schering-Plough Animal Health, Welwyn Garden City, UK) and maintenance of analgesia by buprenorphine (Alstoe Animal Health, Melton Mowbray, UK) i.m.…”

“…Oestradiol concentrations in serum collected by exsanguination were determined by an ELISA assay described previously (Taylor et al 2004), all samples being run in a single assay. Plasma progesterone concentrations in routine samples to determine cycle stage were determined by an ELISA assay as previously described (Wulff et al 2001).…”

“…Although some information on the localisation of these receptors in marmoset endometrium has been reported, these observations have been limited to a small number of animals (Niklaus et al 2001, Sierens et al 2004) and disagree with earlier results (Kholkute et al 2000), suggesting an absence of expression in epithelial cells. The detailed evaluations in the current study was made possible as a result of collecting tissue from experiments primarily aimed at elucidating the changes in angiogenesis occurring in the ovary during the normal cycle (Dickson & Fraser 2000), early pregnancy (Rowe et al 2002) and after inhibiting gonadotrophin releasing hormone (GnRH; Taylor et al 2004) or vascular endothelial growth factor (VEGF; Wulff et al 2001Wulff et al , 2002. To compare these treatments, which suppress plasma oestradiol and prevent the ovulatory rise in progesterone, with the complete removal of ovarian steroids, marmosets were ovariectomised at the same stage of the cycle as the GnRH and VEGF antagonist treatments were initiated.…”