Kathryn J. Woad

Effect of Dietary Energy and Protein on Bovine Follicular Dynamics and Embryo Production In Vitro: Associations with the Ovarian Insulin-Like Growth Factor System

Abstract 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.

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.

The genetic basis of premature ovarian failure

Premature ovarian failure (POF) is a common condition, affecting approximately 1:100 women. It is characterised by amenorrhea, hypoestrogenism, and elevated gonadotrophin levels in women under the age of 40. It is often an unexpected and distressing diagnosis, which coincides with infertility and menopausal symptoms. There is a well recognised genetic basis to the development of POF. Our laboratory has identified several candidate genes associated with POF.

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.

INVESTIGATING THE ASSOCIATION BETWEEN INHIBIN ALPHA GENE PROMOTER POLYMORPHISMS AND PREMATURE OVARIAN FAILURE

OBJECTIVE To determine whether variants in the promoter region of the inhibin alpha gene (INHA) are associated with premature ovarian failure (POF). DESIGN Mutational analysis of the INHA gene promoter in women with POF. SETTING Academic institution. PATIENT(S) Patients with POF (n = 194) and controls (n = 162) from New Zealand and Slovenia. INTERVENTION(S) Peripheral blood samples were screened for known polymorphisms in the INHA promoter (c.-16C-->T, c.-124A-->G, and an imperfect TG repeat at approximately -300 base pairs). Genotyping was performed by restriction fragment length polymorphism, forced restriction fragment length polymorphism, and nondenaturing high-performance liquid chromatography analysis. MAIN OUTCOME MEASURE(S) Genotypic status of INHA promoter polymorphisms. RESULT(S) Significant differences in INHA promoter allele frequencies were observed between POF patient populations and controls. Significant reductions in allele frequency were observed for the -16T allele (New Zealand POF) and -124G allele (total POF) and for INHA promoter haplotypes C (New Zealand POF) and D (Slovenian POF). CONCLUSION(S) We conclude that INHA promoter variants are associated with the development of POF.

FGF2 is crucial for the development of bovine luteal endothelial networks in vitro.

The development of the corpus luteum requires angiogenesis, and involves the complex interplay between factors such as vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). However, the relative role of these factors remains to be elucidated. This study used a new physiologically relevant mixed luteal cell culture system to test the hypotheses that: a) FGF2 and VEGFA are critical for bovine luteal angiogenesis; and b) local luteal PDGF signalling stimulates the formation of endothelial networks. Cells were treated with receptor tyrosine kinase inhibitors against VEGFA (SU1498), FGF2 (SU5402) or PDGF (AG1295) activity. After 9 days in culture, endothelial cells were immunostained for von Willebrand factor (VWF) and quantified by image analysis. Highly organised intricate endothelial networks were formed in the presence of exogenous VEGFA and FGF2. The inhibition of FGF2 activity reduced the total area of VWF staining versus controls (>95%; P<0.001). Inhibition of VEGF and PDGF activity reduced the endothelial network formation by more than 60 and 75% respectively (P<0.05). Progesterone production increased in all treatments from day 1 to 7 (P<0.001), and was unaffected by FGF2 or PDGF receptor kinase inhibition (P>0.05), but was reduced by the VEGF receptor inhibitor on days 5 and 7 (P<0.001). In conclusion, this study confirmed that VEGF signalling regulates both bovine luteal angiogenesis and progesterone production. However, FGF2 was crucial for luteal endothelial network formation. Also, for the first time, this study showed that local luteal PDGF activity regulates bovine luteal endothelial network formation in vitro.

Fibroblast growth factor 2 is a key determinant of vascular sprouting during bovine luteal angiogenesis

Fibroblast growth factor (FGF) 2 and vascular endothelial growth factor (VEGF) A are thought to be key controllers of luteal angiogenesis; however, their precise roles in the regulation and coordination of this complex process remain unknown. Thus, the temporal and spatial patterns of endothelial network formation were determined by culturing mixed cell types from early bovine corpora lutea on fibronectin in the presence of FGF2 and VEGFA (6 h to 9 days). Endothelial cells, as determined by von Willebrand factor immunohistochemistry, initially grew in cell islands (days 0-3), before undergoing a period of vascular sprouting to display a more tubule-like appearance (days 3-6), and after 9 days in culture had formed extensive intricate networks. Mixed populations of luteal cells were treated with SU1498 (VEGF receptor 2 inhibitor) or SU5402 (FGF receptor 1 inhibitor) or control on days 0-3, 3-6 or 6-9 to determine the role of FGF2 and VEGFA during these specific windows. The total area of endothelial cells was unaffected by SU1498 treatment during any window. In contrast, SU5402 treatment caused maximal reduction in the total area of endothelial cell networks on days 3-6 vs controls (mean reduction 81%; P<0.001) during the period of tubule initiation. Moreover, SU5402 treatment on days 3-6 dramatically reduced the total number of branch points (P<0.001) and degree of branching per endothelial cell island (P<0.05) in the absence of changes in mean island area. This suggests that FGF2 is a key determinant of vascular sprouting and hence critical to luteal development.

FSH receptor gene variants are rarely associated with premature ovarian failure

FSH receptor (FSHR) gene variants have been associated with premature ovarian failure (POF). Genomic DNA from New Zealand women with POF (n=80) and control women (n=80) was screened for variants in FSHR exons 7 and 10. FSHR exon 7 variants, including the c.566C>T Finnish founder mutation (p.Ala189Val), were not detected. Previously reported FSHR exon 10 polymorphisms were identified in both groups with similar allelic distributions. A novel heterozygous FSHR exon 10 variant c.1411A>T, p.Ile471Phe was observed in one woman with a family history of POF, but not her affected siblings. It is concluded that variants in exons 7 and 10 of FSHR are not frequently associated with the development of POF in the New Zealand population.

A high incidence of polymorphic CYP2C19 variants in archival blood samples from Papua New Guinea

There is considerable inter-ethnic variability in the incidence of CYP2C19 genetic poor metabolisers (var/var). About 3 per cent of Caucasians are CYP2C19 var/var. By contrast, an extremely high incidence (70 per cent) is observed in the Melanesian island of Vanuatu. The colonisation of the Pacific Islands is believed to have involved migration through Papua New Guinea (PNG), and hence a high incidence may also be expected in this population. The reported incidence in PNG was only 36 per cent, however. PNG is a country of extensive ethnic diversity, and the incidence of the CYP2C19 var/var in other regional populations of PNG is currently not established. In this study, restriction fragment length polymorphism-polymerase chain reaction analysis of archival blood serum samples was used to determine the prevalence of the CYP2C19*2 and *3 variant alleles in three different ethnic and geographically isolated populations of PNG. In the largest population studied (Iruna), the frequency of both variant CYP2C19 alleles was high (0.37 and 0.34, respectively). Specifically, the frequency of the CYP2C19*3 allele was significantly higher than in the PNG (East Sepik) population reported previously (0.34 vs 0.16; p <0.0001). In the Iruna population, 48.9 per cent of the samples were homozygous variants for CYP2C19*2 or *3, which although higher was not statistically different from the East Sepik population (36 per cent). The results of this study indicated that other regional populations of PNG also have a relatively high incidence of the CYP2C19 genetic polymorphism compared with Caucasian populations. The high incidence reported in Vanuatu, however, may be due to genetic drift rather than a PNG founder population, as the Vanuatu population is dominated by the CYP2C19*2 allele, with a lower contribution from the *3 allelic variant.

Fibroblast growth factor 2 induces the precocious development of endothelial cell networks in bovine luteinising follicular cells

The transition from follicle to corpus luteum represents a period of intense angiogenesis; however, the exact roles of angiogenic factors during this time remain to be elucidated. Thus, the roles of vascular endothelial growth factor (VEGF) A, fibroblast growth factor (FGF) 2 and LH in controlling angiogenesis were examined in the present study. A novel serum-free luteinising follicular angiogenesis culture system was developed in which progesterone production increased during the first 5 days and was increased by LH (P<0.01). Blockade of signalling from FGF receptors (SU5402; P<0.001) and, to a lesser extent, VEGF receptors (SU1498; P<0.001) decreased the development of endothelial cell (EC) networks. Conversely, FGF2 dose-dependently (P<0.001) induced the precocious transition of undeveloped EC islands into branched networks associated with a twofold increase in the number of branch points (P<0.001). In contrast, VEGFA had no effect on the area of EC networks or the number of branch points. LH had no effect on the area of EC networks, but it marginally increased the number of branch points (P<0.05) and FGF2 production (P<0.001). Surprisingly, progesterone production was decreased by FGF2 (P<0.01) but only on Day 5 of culture. Progesterone production was increased by SU5402 (P<0.001) and decreased by SU1498 (P<0.001). These results demonstrate that FGF and VEGF receptors play a fundamental role in the formation of luteal EC networks in vitro, which includes a novel role for FGF2 in induction of EC sprouting.