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.

The expression, regulation and function of secreted protein, acidic, cysteine-rich in the follicle-luteal transition.

The role of the tissue remodelling protein, secreted protein, acidic, cysteine-rich (SPARC), in key processes (e.g. cell reorganisation and angiogenesis) that occur during the follicle-luteal transition is unknown. Hence, we investigated the regulation of SPARC in luteinsing follicular cells and potential roles of SPARC peptide 2.3 in a physiologically relevant luteal angiogenesis culture system. SPARC protein was detected mainly in the theca layer of bovine pre-ovulatory follicles, but its expression was considerably greater in the corpus haemorrhagicum. Similarly, SPARC protein (western blotting) was up-regulated in luteinising granulosa but not in theca cells during a 6-day culture period. Potential regulatory candidates were investigated in luteinising granulosa cells: LH did not affect SPARC (P>0.05); transforming growth factor (TGF) B1 (P<0.001) dose dependently induced the precocious expression of SPARC and increased final levels: this effect was blocked (P<0.001) by SB505124 (TGFB receptor 1 inhibitor). Additionally, fibronectin, which is deposited during luteal development, increased SPARC (P<0.01). In luteal cells, fibroblast growth factor 2 decreased SPARC (P<0.001) during the first 5 days of culture, while vascular endothelial growth factor A increased its expression (P<0.001). Functionally, KGHK peptide, a SPARC proteolytic fragment, stimulated the formation of endothelial cell networks in a luteal cell culture system (P<0.05) and increased progesterone production (P<0.05). Collectively, these findings indicate that SPARC is intricately regulated by pro-angiogenic and other growth factors together with components of the extracellular matrix during the follicle-luteal transition. Thus, it is possible that SPARC plays an important modulatory role in regulating angiogenesis and progesterone production during luteal development.

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.

Expression of renin-angiotensin system components in the early bovine embryo.

The renin-angiotensin system (RAS), mainly associated with the regulation of blood pressure, has been recently investigated in female reproductive organs and the developing foetus. Angiotensin II (Ang II) influences oviductal gamete movements and foetal development, but there is no information about RAS in the early embryo. The aim of this study was to determine whether RAS components are present in the pre-implantation embryo, to determine how early they are expressed and to investigate their putative role at this stage of development. Bovine embryos produced in vitro were used for analysis of RAS transcripts (RT-PCR) and localisation of the receptors AGTR1 and AGTR2 (immunofluorescent labelling). We also investigated the effects of Ang II, Olmesartan (AGTR1 antagonist) and PD123319 (AGTR2 antagonist) on oocyte cleavage, embryo expansion and hatching. Pre-implanted embryos possessed AGTR1 and AGTR2 but not the other RAS components. Both receptors were present in the trophectoderm and in the inner cell mass of the blastocyst. AGTR1 was mainly localised in granular-like structures in the cytoplasm, suggesting its internalisation into clathrin-coated vesicles, and AGTR2 was found mainly in the nuclear membrane and in the mitotic spindle of dividing trophoblastic cells. Treating embryos with PD123319 increased the proportion of hatched embryos compared with the control. These results, the first on RAS in the early embryo, suggest that the pre-implanted embryo responds to Ang II from the mother rather than from the embryo itself. This may be a route by which the maternal RAS influences blastocyst hatching and early embryonic development.

Nutritional effects on oocyte and embryo development in mammals: implications for reproductive efficiency and environmental sustainability.

The environment in which a breeding female lives prior to conception and during the early stages of her pregnancy has striking effects on oocytes developing in the ovarian follicle and on early embryos in the reproductive tract. Of the various environmental factors known to affect oocyte and embryo development, altered nutrition during this critical period has been particularly well studied. Alterations in the quantity of food consumed or the composition of the diet imposed solely during the pre-mating period affect oocyte maturity, blastocyst yield, prenatal survival and the number of offspring born alive. Importantly, nutrition at this time also affects the quality of embryos and resultant offspring, with increasing evidence from a variety of species showing that peri-conception nutrition can alter behaviour, cardiovascular function and reproductive function throughout post-natal life. In livestock species, it is important to devise nutritional strategies that improve reproductive efficiency and the quality of offspring but that do not add to the environmental footprint of the production system and which recognize likely changes in feedstuff availability arising from predicted changes in climate.

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.

Supra-basal progesterone concentrations during the follicular phase are associated with development of cystic follicles in dairy cows.

The objective of this study was to test the hypothesis that supra-basal concentrations of progesterone during the follicular phase are associated with the development of follicular cysts. Twenty-five non-lactating dairy cows were used in the study, which was performed over five identical replicate trials. Luteolysis was induced during the mid-luteal phase. Transrectal ultrasonography was performed daily to determine the occurrence/timing of ovulation. Plasma samples were collected for progesterone, oestradiol and luteinizing hormone (LH) analysis. Three cows failed to ovulate (cystic anovulatory) but did ovulate in a subsequent replicate (cystic ovulatory). Eight cows from the appropriate replicates were used as control cows (normal group). Follicular growth patterns and plasma oestradiol concentrations were similar between the three groups. However, the plasma progesterone concentrations during the follicular phase were twofold higher in the cystic anovulatory group (P < 0.01). Furthermore, no LH surge was detected in these animals. While LH pulse amplitude was similar between groups, LH pulse frequency in the cystic anovulatory group was attenuated (P < 0.05). In conclusion, the formation of follicular cysts were preceded by elevated plasma progesterone concentrations and the suppression of the LH surge.

BMP-2 and -6 modulate porcine theca cell function alone and co-cultured with granulosa cells.

Bone morphogenetic proteins (BMPs) are emerging as a family of proteins crucial in the regulation of fertility and ovulation rate. We have shown that porcine theca cells express BMP receptors, however, there is a paucity of information regarding the effect(s) of BMPs on theca cell function. The purpose of this study was to investigate the effects of BMP-2 and -6 on theca cells cultured under serum-free conditions in terms of steroidogenesis, cAMP release and proliferation. The study was further extended to determine whether BMP responses in theca cells are affected by the addition of granulosa cells to the culture system. Both BMPs suppressed progesterone and androstenedione synthesis by theca cells (P < 0.05) after 144 h in culture. Oestradiol synthesis was suppressed (P < 0.05) by BMP-2, but not BMP-6, and theca cell proliferation was stimulated (P < 0.05) by BMP-6, but not BMP-2, after 144 h in culture. Both BMP-2 and -6 inhibited cAMP release (P < 0.05) by theca cells. Furthermore, progesterone and androstenedione synthesis by co-cultured theca and granulosa cells were suppressed (P < 0.05) whereas cell proliferation was stimulated (P < 0.05). These results provide strong evidence for a functional BMP system in the porcine ovary and that theca cells are responsive to BMPs in terms of steroidogenesis and proliferation. BMP-2 and -6 may have a role as luteinisation inhibitors in this polyovular species.

Oocyte-somatic cell-endocrine interactions in pigs.

Oocyte-somatic cell communication is bi-directional and essential for both oocyte and follicular granulosa and theca cell function and development. We have shown that the oocyte secretes factors that stimulate porcine granulosa cell proliferation in serum-free culture, and suppress progesterone production, thereby preventing premature luteinisation. Possible candidates for mediating some of these effects are the bone morphogenetic proteins (BMPs) that belong to the transforming growth factor beta family. They are emerging as a family of proteins critical for fertility and ovulation rate in several mammals, and they are expressed in various cell types in the ovary. We have evidence for a functional BMP system in the porcine ovary and BMP receptors are present in the egg nests in the fetal ovary and in the granulosa cells, oocytes and occasional theca cells throughout subsequent development. In addition to paracrine interactions in the ovary, the porcine oocyte and its developmental potential can also be influenced by nutritional manipulation in vivo. We have demonstrated that feeding a high plane of nutrition to gilts for 19 days prior to ovulation increased oocyte quality compared to control animals fed a maintenance diet, as determined by oocyte maturation in vitro. This was associated with a number of changes in circulating reproductive and metabolic hormones and also in the follicular fluid in which the oocyte is nurtured. Further studies showed a similar increase in prenatal survival on Day 30 of gestation, demonstrating a direct link between oocyte quality/maturation and embryo survival. Collectively, these studies emphasise the importance of the interactions that occur between the oocyte and somatic cells and also with endocrine hormones for ovarian development, and ultimately for the production of oocytes with optimal developmental potential.

Evidence for a functional bone morphogenetic protein (BMP) system in the porcine ovary.

Bone morphogenetic proteins (BMPs) play important roles in controlling fertility and ovulation rate. There is however, little information on the BMP system in the ovary of a large polyovular species. The aims of the present study were to investigate BMP-2 and -6 protein expression in the porcine ovary, their effects on granulosa cells in culture and their mechanism of action. Cells and oocytes were recovered from healthy antral follicles 2-6mm in diameter. When assessed by Western blotting, oocytes and follicular fluid contained BMP-2 and -6. In addition, BMP-2 and -6 were observed in granulosa cells and BMP-2 was also found in theca cells. Granulosa cells were cultured in a serum-free system for 144 h in the presence of increasing doses (0, 3, 30 and 100 ng/ml) of BMP-2 or BMP-6. Both BMPs suppressed progesterone production in a dose-dependent manner after 48 h (P<0.001) and 144 h (P<0.05). Only BMP-6 stimulated cell proliferation at 100 ng/ml (P<0.05). Investigation into the mechanism of action found that BMP-2 and -6 decreased cyclic adenosine monophosphate (cAMP) production (P<0.01), expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) protein (P<0.001) and steroidogenic acute regulatory protein (StAR) (BMP-6 only; P<0.05). This supports the hypothesis that BMP-2 and -6 act as luteinization inhibitors. In conclusion, these findings provide evidence for the presence of a complex signalling mechanism in the porcine ovary and suggest that both BMP-2 and -6 may act in a paracrine manner to control granulosa cell function in this large polyovulatory species.

Effects of 17beta-estradiol on in vitro maturation of pig oocytes in protein-free medium.

The present study examined the effects of 17 beta-estradiol (E(2)) on in vitro maturation and subsequent in vitro fertilization of pig oocytes matured with or without cumulus cells. When E(2) (10 ng/ml) was added to the protein-free maturation medium, the proportions of cumulus-enclosed oocytes that underwent germinal vesicle breakdown and reached metaphase II were significantly reduced (P<0.05), and cumulus expansion was also significantly inhibited (P<0.05) compared with the control (no E(2) added). Although oocytes matured in the presence of E(2) were penetrated by sperm in vitro at the same level as the control, the incidences of male pronuclear (MPN) formation and activated oocytes were significantly lower (P<0.05) than the control. These inhibitory effects of E(2) were prevented when the medium was supplemented with E(2) together with its antagonist, ICI 182,780 (1 microg/ml), although the presence of the antagonist alone in the medium had no effect on the maturation and fertilization in vitro of oocytes. In cumulus-free oocytes, E(2) had no effect on nuclear maturation and penetration in vitro, but low MPN formation was observed in oocytes matured in the presence and absence of E(2). When cumulus-enclosed oocytes were cultured in the presence of progesterone (P(4); 600 ng/ml) alone or together with E(2), no significant differences in nuclear maturation, cumulus expansion or penetration in vitro were observed compared with control oocytes. The concentration of P(4) in maturation medium was significantly (P<0.01) lower when cumulus-enclosed oocytes were cultured for 44 h in the medium with E(2) than in medium without E(2). These results indicate that E(2) inhibits both nuclear and cytoplasmic maturation of cumulus-enclosed pig oocytes, and that this inhibition can be prevented by an E(2) antagonist or P(4). This E(2) inhibition may occur indirectly via the cumulus cells and inhibition of P(4) synthesis.

Immunohistochemical localization of the bone morphogenetic protein receptors in the porcine ovary.

The bone morphogenetic protein (BMP) family is emerging as playing a crucial role in regulating normal follicle growth and determining ovulation rate. BMPs exert their effects via BMP receptors (BMPR-IA, -IB and -II). However, there is a paucity of information relating to the expression of the BMPRs within the ovary of large polyovular species such as the pig. Furthermore, there is a lack of information on the expression of BMPRs by fetal ovaries of any species. The purpose of this study was to investigate temporal and spatial expression of the BMPRs in the porcine ovary, at different developmental stages. Immunohistochemistry for BMPR-IA, BMPR-IB and BMPR-II was performed using sections from paraffin wax-embedded ovaries, obtained from fetal (n = 15), prepubertal (n = 3) and cycling postpubertal (n = 4) pigs. Results confirmed the presence of all three receptors in the fetal egg nests and in the granulosa cell layer of follicles ranging from primordial to late antral stages. Immunostaining was also observed in oocytes, theca layer, corpus luteum and ovarian surface epithelium. The expression of BMPRs by fetal ovaries may be related to follicle formation, whereas expression in pre- and post-pubertal animals indicates BMPs are involved in regulating porcine ovarian follicle growth.

Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells in vitro.

Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2-6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0-10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids. Oocyte secreted factors were shown to stimulate both granulosa cell proliferation (P < 0.001) and oestradiol production (P < 0.001) by granulosa cells throughout culture. In contrast, oocyte secreted factors suppressed granulosa cell progesterone production after both 48 and 144 hours (P < 0.001). Thecal cell numbers were increased by oocyte secreted factors (P = 0.02), together with a suppression in progesterone and androstenedione synthesis after 48 hours (P < 0.001) and after 144 hours (P = 0.02), respectively. Oocyte secreted factors also increased viable cell numbers (P < 0.001) in co-cultures together with suppression of progesterone (P < 0.001) and oestradiol (P < 0.001). In granulosa cell only cultures, SCF increased progesterone production in a dose dependent manner (P < 0.001), whereas progesterone synthesis by theca cells was reduced in a dose dependent manner (P = 0.002). Co-cultured cells demonstrated an increase in progesterone production with increasing SCF dose (P < 0.001) and an increase in oestradiol synthesis at the highest dose of SCF (100 ng/ml). In summary, these findings demonstrate the presence of a co-ordinated paracrine interaction between somatic cells and germ cells, whereby oocyte derived signals interact locally to mediate granulosa and theca cell function. SCF has a role in modulating this local interaction. In conclusion, the oocyte is an effective modulator of granulosa-theca interactions, one role being the inhibition of luteinization.

Immunocytochemical localization of angiotensin II receptor subtypes 1 and 2 in the porcine fetal, prepubertal and postpubertal ovary.

There is considerable evidence for a mammalian ovarian renin-angiotensin system, which may influence ovulation, angiogenesis and steroidogenesis via the autocrine and/or paracrine actions of the biologically active product of the cascade, angiotensin II (AngII). There are two characterized AngII receptors--type 1, AT1 and type 2, AT2. We report the localization of these receptor subtypes within porcine fetal, prepubertal and postpubertal ovaries. Positive staining for AT1 and AT2 receptors was observed in egg nests in all fetal ovaries studied, as well as in a defined two-cell layer at the ovarian periphery. In prepubertal tissue, positive AT1 and AT2 staining was localized to granulosa cells adjacent to the basement membrane of pre-antral and antral follicles, with no staining in the thecal layer. There was immunostaining for both receptors in prepubertal oocytes and zona pellucida. In postpubertal tissue, positive AT1 and AT2 immunostaining was localized to areas of putative neovascularization, the zona pellucida and the oocyte. Further AT1 staining was located to the postpubertal antral follicle granulosa cells. The results indicate that there are higher densities of AT1 receptors than AT2 receptors in the porcine fetal, prepubertal and postpubertal ovary, and this has profound implications for the role of AngII in ovarian development.

Follicular factors regulating oocyte maturation and quality.

Maturation of the oocyte can be divided into two different aspects: nuclear maturation and cytoplasmic maturation. The spontaneous nature of nuclear maturation in oocytes removed from the follicle and cultured in vitro was observed in mammals as early as 1935. However, oocytes cultured in basic conditions are deficient in some cytoplasmic factors and are, therefore, developmentally incompetent. Data from large domestic species indicate that although oocytes matured in vitro in supplemented media can develop after fertilization, they require the presence of follicular factors during culture to ensure their developmental competence. The importance of follicular maturation on the capacity of oocytes to achieve fertilization and early embryonic development can be studied by reproducing some important events in vitro. Follicular supplementation may be in the form of follicular fluid, granulosa cells or follicle-conditioned media, and there is evidence that the maturational status of the follicle used for co-culture influences subsequent male pronuclear formation. Studies in this laboratory on the prolific Chinese Meishan pig, which has significantly higher early embryonic survival than conventional European breeds, indicate crucial differences in the pattern of follicle development and, therefore, the intrafollicular environment in which the oocytes are nurtured. It is suggested that this produces oocytes of improved 'quality' and this hypothesis is supported by experiments both in vivo and in vitro. Ultimately, it is hoped that these studies on large domestic animals will lead to identification of the follicular factors that influence oocyte quality.