Angiogenesis and vascular function in the ovary.

Ovarian function is dependent on the establishment and continual remodelling of a complex vascular system. This enables the follicle and/or corpus luteum (CL) to receive the required supply of nutrients, oxygen and hormonal support as well as facilitating the release of steroids. Moreover, the inhibition of angiogenesis results in the attenuation of follicular growth, disruption of ovulation and drastic effects on the development and function of the CL. It appears that the production and action of vascular endothelial growth factor A (VEGFA) is necessary at all these stages of development. However, the expression of fibroblast growth factor 2 (FGF2) in the cow is more dynamic than that of VEGFA with a dramatic upregulation during the follicular-luteal transition. This upregulation is then likely to initiate intense angiogenesis in the presence of high VEGFA levels. Recently, we have developed a novel ovarian physiological angiogenesis culture system in which highly organised and intricate endothelial cell networks are formed. This system will enable us to elucidate the complex inter-play between FGF2 and VEGFA as well as other angiogenic factors in the regulation of luteal angiogenesis. Furthermore, recent evidence indicates that pericytes might play an active role in driving angiogenesis and highlights the importance of pericyte-endothelial interactions in this process. Finally, the targeted promotion of angiogenesis may lead to the development of novel strategies to alleviate luteal inadequacy and infertility.

Insulin-like growth factors-I and -II and insulin-like growth factor-binding protein-2 in dominant equine follicles during spring transition and the ovulatory season.

The period between seasonal anoestrus and cyclicity is characterized in many mares by cyclical growth and regression of large dominant follicles. The insulin-like growth factor (IGF) system plays a key role in follicular growth and regression; therefore, we hypothesized that changes in the IGF system and its binding proteins would modulate onset of cyclicity in mares. Ovaries were obtained from pony mares on the day after detection of an actively growing 30 mm transitional anovulatory follicle, and also at the second or third oestrus of the breeding season on the day after the preovulatory follicle reached 30 mm in diameter. Size of dominant follicles at the time of removal was similar in transition (32 +/- 0.8 mm) and at oestrus (34 +/- 0.6 mm). IGF-I mRNA was present in granulosa cells, with low thecal expression, whereas IGF-II mRNA was confined to the theca layer. Expression of IGF-I and -II mRNAs, and intrafollicular concentrations of oestradiol, were lower (P < 0.01; paired t test) in transitional anovulatory follicles than in preovulatory follicles. Messenger RNA encoding IGFBP-2 was present in both theca and granulosa layers. Steady-state concentrations of mRNA encoding IGFBP-2 mRNA increased (P < 0.001) in theca in preovulatory follicles. Intrafollicular concentrations of IGFBP-2 were higher (P < 0.001) in transitional than in preovulatory follicles. The similarity in circulating concentrations of IGF-I in transitional and cyclic mares, suggested that the somatotrophic axis is not involved in transition from anovulatory to ovulatory cycles. The results suggest that the increased expression of IGF-I and -II mRNAs in preovulatory follicles, along with the decrease in IGFBP-2 concentrations, could increase the bioavailability of intrafollicular IGF in large follicles during the breeding season, and support our hypothesis that intrafollicular IGF bioavailability must exceed a threshold level before ovulation can occur.

Corpus luteum (CL) function: local control mechanisms.

LH and PGF(2alpha) are the principal luteotrophic and luteolytic hormones in domestic animals, however, it is becoming increasingly apparent that intra-ovarian factors can modulate luteal function. For example, the insulin-like growth factors (IGF-I and -II) can regulate ovarian function, and have direct effects on ovarian cells. An important role for the IGFs in regulating ovarian function is suggested by the multiple effects of IGFs on both follicular and luteal steroidogenesis. Expression of mRNA encoding IGF-I, IGF-II and the type 1 IGF receptor has also been detected in the ruminant CL and is suggestive of autocrine/paracrine roles for both IGF-I and -II in the regulation of luteal function. The actions of the IGFs are further modulated by their association with specific binding proteins (IGFBPs), which regulate the transport of IGFs and their presentation to specific receptors. IGFBPs have been detected in the CL of domestic animals, and inhibitory effects on IGF-I-stimulated progesterone production have been demonstrated. The rapid cyclical changes in luteal growth and regression are associated with rapid changes in vasculature. The principle angiogenic factors include the fibroblast growth factors (FGFs), vascular endothelial growth factor (VEGF) and the angiopoietins (Ang). Other locally produced factors include cytokines such as TNF-alpha and IL-1beta. One such factor is monocyte chemoattractant protein (MCP-1), which increases after exogenous PGF(2alpha). An influx of macrophages takes place in the CL around luteolysis, possibly in response to MCP-1 release, but these changes are not observed in cattle when luteolysis is inhibited. In conclusion locally produced factors are important in the control of luteal function, although their roles have yet to fully elucidated.

Insulin-like growth factor (IGF) system in the oocyte and somatic cells of bovine preantral follicles.

Many studies have highlighted the role of the insulin-like growth factor (IGF) system in the control of antral follicular growth. However, much less is known about the involvement of the IGF system in the regulation of preantral follicular development. In an attempt to address this lack of knowledge, the present study describes the spatial and temporal patterns of expression of mRNA encoding components of the IGF system in bovine follicles during preantral stages of development. mRNA was detected by in situ hybridization using frozen sections (14 microm) of bovine ovarian tissue. Serial sections were probed with 35S-labelled bovine riboprobes. Type 1 IGF receptor mRNA was detected in granulosa cells and in the oocyte of preantral follicles; however, in this study, as in previous studies, it was not possible to detect mRNA encoding either IGF-I or -II. IGF binding protein (IGFBP)-2 mRNA was present in granulosa cells and oocytes of preantral follicles, and immunoreactive IGFBP-2 was detected around granulosa cells during this early stage of development. Occasionally, preantral follicles were identified in which there was no expression of IGFBP-2 in granulosa cells or the oocyte. IGFBP-3 mRNA was detected in the oocyte of preantral follicles and in the surrounding stromal tissue. mRNAs encoding IGFBP-2 and -3, and type 1 IGF receptor were first detected in type 2 follicles. In conclusion, although the IGF ligands are not expressed in preantral follicles, mRNAs encoding the type 1 IGF receptor, and IGFBP-2 and -3 were present and showed unique spatial patterns of expression within preantral follicles.

Effect of dietary energy and protein on bovine follicular dynamics and embryo production in vitro: associations with the ovarian insulin-like growth factor system.

Heifers were assigned either low or high (HE) levels of energy intake and low or high concentrations of dietary crude protein. The effect of these diets on the plasma concentrations of insulin, insulin-like growth factor (IGF)-I, and urea on follicular growth and early embryo development is described. We propose that the observed dietary-induced changes in the ovarian IGF system increase bioavailability of intrafollicular IGF, thus increasing the sensitivity of follicles to FSH. These changes, in combination with increased peripheral concentrations of insulin and IGF-I in heifers offered the HE diet, contribute to the observed increase in growth rate of the dominant follicle. In contrast to follicular growth, increased nutrient supply decreased oocyte quality, due in part to increased plasma urea concentrations. Clearly a number of mechanisms are involved in mediating the effects of dietary energy and protein on ovarian function, and the formulation of diets designed to optimize cattle fertility must consider the divergent effects of nutrient supply on follicular growth and oocyte quality.

Expression of mRNA encoding insulin-like growth factors I and II and the type 1 IGF receptor in the bovine corpus luteum at defined stages of the oestrous cycle.

Previous studies have implicated insulin-like growth factors I and II (IGF-I and -II), in the regulation of ovarian function. The present study investigated the localization of mRNA encoding IGF-I and -II and the type 1 IGF receptor using in situ hybridization to determine further the roles of the IGFs within the bovine corpus luteum at precise stages of the oestrous cycle. Luteal expression of mRNA encoding IGF-I and -II and the type 1 IGF receptor was detected throughout the oestrous cycle. The expression of IGF-I mRNAvaried significantly during the oestrous cycle. IGF-I mRNA concentrations were significantly higher on day 15 than on day 10, and IGF-I mRNA in the regressing corpus luteum at 48 h after administration of exogenous prostaglandin was significantly greater than in the early or mid-luteal phase (days 5 and 10). In contrast, there was no significant effect of day of the oestrous cycle on expression of mRNA for IGF-II and the type 1 IGF receptor in the corpus luteum. Expression of IGF-II mRNA was localized to a subset of steroidogenic luteal cells and was also associated with cells of the luteal vasculature. mRNA encoding the type 1 IGF receptor was widely expressed in a pattern indicative of expression in large and small luteal cells. These data demonstrate that the bovine corpus luteum is a site of IGF production and reception throughout the luteal phase. Furthermore, this study highlights the potential of IGF-II in addition to IGF-I in the autocrine and paracrine regulation of luteal function.

Expression of mRNA encoding IGF-I, IGF-II and type 1 IGF receptor in bovine ovarian follicles.

IGFs regulate gonadotrophin-stimulated proliferation and differentiation of granulosa and theca cells in vitro. However, the detailed pattern of mRNA expression of IGFs in bovine follicles remains controversial. The objectives of this study were therefore to describe the temporal and spatial pattern of expression of mRNA encoding IGF-I, IGF-II and the type 1 IGF receptor in bovine follicles in vivo. The expression of mRNA encoding IGF-II was detected in theca tissue from around the time of antrum formation up to and during the development of dominance. No IGF-II mRNA expression was detected in granulosa cells. In the majority of follicles we were unable to detect mRNA encoding IGF-I in either granulosa or theca tissue from follicles at any stage of development. Occasionally low amounts of mRNA encoding IGF-I were detected in the theca externa and connective tissue surrounding some follicles. Type 1 IGF receptor mRNA was detected in both granulosa and theca cells of preantral and antral follicles. Expression was greater in granulosa tissue compared with theca tissue. We also measured IGF-I and -II mRNA in total RNA isolated from cultured granulosa and theca cells using reverse transcriptase PCR. In contrast to the in vivo results, IGF-II mRNA was detected in both granulosa and theca tissue. IGF-I mRNA was detected in theca tissue and in very low amounts in granulosa cells. Using a specific IGF-I RIA we were unable to detect IGF-I immunoreactivity in granulosa conditioned cell culture media. Using immunohistochemistry we detected IGF-I immunoreactivity in some blood vessels within the ovarian stroma. We conclude from these results that IGF-II is the principal intrafollicular IGF ligand regulating the growth of bovine antral follicles. In preantral follicles the expression of mRNA encoding type 1 IGF receptor but absence of endogenous IGF-I or -II mRNA expression, highlights a probable endocrine mechanism for the IGF regulation of preantral follicle growth.

Specific non-genomic, membrane-localized binding sites for progesterone in the bovine corpus luteum.

Fractionation of bovine corpus luteum (CL) homogenates on continuous sucrose density gradients with and without preincubation with 3H-progesterone demonstrated high levels of tracer binding and high content of endogenous progesterone associated with particulate membrane fractions. Analysis of gradient fractions for a range of luteal plasma membrane and intracellular organelle marker enzyme activities indicated that endogenous progesterone content and 3H-progesterone-binding activity were associated with fractions enriched in luteal plasma membrane markers. This was confirmed by pretreatment of homogenates with the saponin, digitonin, prior to fractionation. Digitonin perturbed the buoyant density of luteal surface membrane markers and 3H-progesterone binding to a similar extent, but did not perturb the buoyant densities of other intracellular markers to the same degree. Interestingly, digitonin pretreatment also increased the proportion of progesterone tracer that entered the gradients. We consistently failed to demonstrate significant binding of 3H-progesterone to membrane fractions incubated with progesterone tracer in vitro. However, when digitonin was included in the in vitro binding assay, we observed a dramatic, dose-dependent stimulation of 3H-progesterone binding by digitonin. Other radiolabeled steroids tested (3H-cortisol, 3H-testosterone) bound poorly in the presence or absence of digitonin. 3H-Progesterone binding in the presence of optimal digitonin concentrations increased linearly with increasing luteal membrane concentration; was dependent on the pH, duration, and temperature of incubation; and low levels of progesterone (68 nM) competed for tracer binding. A range of other steroids tested (androgens, estrogens, corticosteroids, steroid precursors) competed at higher concentrations (10- to 100-fold) or did not compete at all for 3H-progesterone binding. There was no correlation between the hydrophobicity of various steroids and their ability to compete for binding. Moreover, a number of agonists and antagonists specific for the genomic progesterone receptor, agonists of peripheral benzodiazepine receptors, and inhibitors of a range of steroidogenic enzymes did not compete for 3H-progesterone binding. Western blots confirmed that detergent-solubilized progesterone-binding sites could be resolved from cytochrome P450 side-chain cleavage and 3beta-hydroxysteroid dehydrogenase. Moreover, extraction of bound steroid from the binding site and HPLC demonstrated identity to progesterone, suggesting that no metabolism of the progesterone tracer had occurred during incubation. Progesterone binding to membranes of large luteal cells was higher compared with binding to small luteal cells, and levels were similar in membranes prepared from CL at all stages of the luteal phase. We suggest that bovine luteal progesterone-binding sites may play a role either in sequestration of newly synthesized progesterone or in the mediation of autocrine and/or paracrine actions of progesterone in the CL.