Possible role of growth hormone, IGFs, and IGF-binding proteins in the regulation of ovarian function in large farm animals.

The aim of the study and short review was to present evidence that growth hormone (GH), locally produced insulin-like growth factors (IGFs), and IGF-binding proteins (IGFBPs) may have an important role in the control of ovarian function. There is clear evidence for a distinct GH-receptor mRNA expression and protein production in follicles (oocytes and granulosa-cumulus cells) and corpus luteum (CL). In hypophysectomized ewes, GH and LH are necessary for normal CL development. IGF-1 mRNA in the follicles is expressed in theca interstitial cells (TIC) and granulosa cells (GC) with already higher levels in the TIC before follicle selection. In contrast, IGF-2 is mainly expressed in the TIC. The IGFR-1 mRNA is expressed in both the TIC and GC, with increasing levels in GC during the final development of dominant follicles. IGF-1 is a very potent stimulator of progesterone and oxytocin release in GC. IGFBP-1, -2, -3, -4, -5, and -6 have been isolated from follicular fluid or ovarian tissue. Studies indicate that IGFBP expression and production in the developing follicle is dependent on both cell type and follicle size and is regulated by IGF-1 and gonadotropins. The highest expression of IGF-1 and IGFR-1 mRNA was demonstrated during the early luteal phase. Distinct receptors for IGF-1 and IGF-2 were present in CL membrane preparations at all stages investigated. Intense immunostaining for IGF-1 was observed mainly in bovine large and small luteal cells and in a limited number of endothelial cells. In contrast, IGF-2 protein was localized in perivascular fibroblast and pericytes of the capillaries. With the use of a microdialysis system, we found that in vitro and in vivo IGF-1, IGF-2, and GH stimulated the release of progesterone in cultures of luteal cells or intact tissues. In conclusion, there is clear evidence for a central role of the IGFs, IGFBPs, and GH in follicular development and CL function.     

The expression of thrombopoietin and its receptor during different physiological stages in the bovine ovary

Thrombopoietin (TPO) is known to be involved in megakaryocytopoiesis, but its role in the control of ovarian function is unknown in cattle. The aims of this study were to demonstrate the expression of TPO and its receptor (c-MPL) in detail in bovine corpus luteum (CL) obtained from different stages of the oestrous cycle and during pregnancy--and to demonstrate that TPO/c-MPL system is expressed clearly in bovine follicles. Real-time RT-PCR (qPCR) and ELISA were applied to investigate mRNA expression of examined factors and TPO protein, respectively. In this investigation, increases in the concentrations of TPO protein and the mRNA expression of TPO and c-MPL were noticed during both early luteal stage and late luteal stage of the oestrous cycle. Furthermore, the expression of TPO/c-MPL system does not show any significant regulation in the CL throughout pregnancy. Highest co-expression of TPO/c-MPL system in both theca interna (TI) and granulosa cells (GC) in small follicles (<10 mm in diameter) was observed in this study that may suggest the possible role of TPO/c-MPL system in proliferation of TI and GC cells. To conclude, the results demonstrate the possible involvement of locally produced TPO/c-MPL system as a 'physiological filter' in bovine ovary where they may promote cell selection by inducing proliferation of viable cells and scavenging non-viable cells and thereby may play an important role in modulation of ovarian function.     

Expression and Localization of Gap Junctional Connexins 26 and 43 in Bovine Periovulatory Follicles and in Corpus Luteum During Different Functional Stages of Oestrous Cycle and Pregnancy

The aim of this study was to characterize the regulation of connexins (Cx26 and Cx43) in the bovine ovary (experiment 1–3). Experiment 1: ovaries containing preovulatory follicles or corpora lutea (CL) were collected at 0, 4, 10, 20, 25 (follicles) and 60 h (CL) relative to injection of GnRH. Experiment 2: CL were assigned to the following stages: days 1–2, 3–4, 5–7, 8–12, 13–16, >18 (after regression) of oestrous cycle and of early and late pregnancy (<4 and >4 months). Experiment 3: induced luteolysis, cows on days 8–12 were injected with PGF2α analogue (Cloprostenol), and CL were collected by transvaginal ovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after PGF2α injection. Real‐time RT‐PCR was applied to investigate mRNA expression and immunofluorescence was utilized for protein localization. Cx26 mRNA increased rapidly 4 h after GnRH injection (during LH surge) and decreased afterwards during the whole experimental period. Cx43 mRNA expression decreased continuously after GnRH application. Cx26 mRNA in CL increased significantly in the second part of oestrous cycle and after regression. In contrast, the highest mRNA expression for Cx43 in CL was detected during the early luteal phase. After induced luteolysis the mRNA expression of Cx26 increased significantly at 24 h. As shown by immunofluorescence, Cx26 was predominantly localized in the connective tissue and blood vessels of bovine CL, whereas Cx43 was present in the luteal cells and blood vessels. This resulted in a strong increase of Cx26 expression during the late luteal phase and after luteal regression. Subsequently, Cx43 expression was distinctly decreased after luteal regression. These data suggest that Cx26 and Cx43 are involved in the local cellular mechanisms participating in tissue remodelling during the critical time around periovulation as well as during CL formation (angiogenesis), function and regression in the bovine ovary.     

Expression of Lymphangiogenic Vascular Endothelial Growth Factor Family Members in Bovine Corpus Luteum

The aim of this study was to evaluate mRNA expression, protein concentration and localization of the assumedly important lymphangiogenic factors VEGFC and VEGFD and the receptor FLT4 in bovine corpora lutea (CL) during different physiological stages. In experiment 1, CL were collected in a slaughterhouse and stages (days 1–2, 3–4, 5–7, 8–12, 13–16, >18) of oestrous cycle and month <3, 3–5, 6–7 and >8 of pregnancy. In experiment 2, prostaglandin F2α (PGF)‐induced luteolysis was performed in 30 cows, which were injected with PGF analogue on day 8–12 (mid‐luteal phase), and CL were collected before and 0.5, 2, 4, 12, 24, 48 and 64 h after PGF injection. The mRNA expression was characterized by RT‐qPCR. All three factors were clearly expressed and showed significant changes during different groups and periods examined in both experiments. Protein concentrations of VEGFD and FLT4 measured by ELISA were not detectable in early cyclic CL but increased to higher plateau levels during pregnancy. After PGF‐induced luteolysis FLT4 protein showed an increase within 2–24 h after the injection. FLT4 localization by immunohistochemistry in the cytoplasm of luteal cells was relatively weak in early CL. It increased in late CL and especially in CL during pregnancy. During pregnancy, a positive FLT4 staining in both the nucleus and cytoplasm of lymphatic endothelial cells in peripheral tissue was observed. In conclusion, our results lead to the assumption that lymphangiogenic factors are produced and regulated in CL and may be involved in mechanisms regulating CL function, especially during pregnancy.     

The mRNA expression of the members of the IGF-system in bovine corpus luteum during induced luteolysis

The components of the IGF-system were shown to be differentially regulated in bovine antral follicles and corpora lutea (CL) during different stages of the estrous cycle, and to have important functions for specific stages. The aim of this study was to investigate the detailed pattern of mRNA expression of most constituents of the IGF-system and their possible involvement in prostaglandin (PG)F2-induced luteolysis in the bovine CL. Therefore, cows in the mid-luteal phase (days 8–12) were injected with the PGF2-analogue Cloprostenol, and CL were collected by transvaginal ovariectomy at 2, 4, 12, 48 and 64 h after PGF2-injection. Real-time RT-PCR using SYBR Green I detection was employed to determine mRNA expressions of the following factors: ubiquitin (UBQ), insulin-like growth factor I (IGF I), IGF II, IGF-receptor type 1 (IGFR-1), growth hormone receptor (GH-R) and IGF-binding proteins-1–6 (IGFBP-1–6). Total extractable RNA decreased with ongoing luteolysis. IGFBP-1 mRNA was significantly up-regulated at 2 h after PGF2 and maximal at 4 h with a 34-fold increase. IGFBP-5 mRNA was significantly up-regulated after 12 h with a maximum of an 11-fold increase at 64 h. For GH-R, IGFR-1, IGF II, IGFBP-3 and -4 mRNA expression, we found a significant down-regulation in certain stages. There was a significant up-regulation for IGFBP-2 and -6 mRNA at 64 h after induced luteolysis. There were no significant changes in IGF I mRNA expression. In conclusion, the IGF-system with all its components seems to play an important role in the very complex process of PGF2-induced luteolysis in bovine CL.     

Toll‐like receptor and related cytokine mRNA expression in bovine corpora lutea during the oestrous cycle and pregnancy

Improving our understanding of the mechanisms controlling the corpus luteum (CL) and its role in regulating the reproductive cycle should lead to improvements in the sustainability of today's global animal industry. The corpus luteum (CL) is a transient endocrine organ composed of a heterogeneous mixture steroidogenic, endothelial and immune cells, and it is becoming clear that immune mechanisms play a key role in CL regulation especially in luteolysis. Toll‐like receptors (TLR) mediate innate immune mechanisms via the production of pro‐inflammatory cytokines, especially within various tissues, although the role of TLR within CL remains unknown. Thus, the objectives of this study were to characterize TLR mRNA expression in the CL during the oestrous cycle and in pregnancy (day 30–50), and to examine the role of TLR signalling in luteal cells. Corpora lutea were collected at various stages of the cycle and pregnancy and analysed for TLR and cytokine mRNA expression. In addition, luteal cells were cultured with the TLR4 ligand (lipopolysaccharide, LPS) for 24 h to evaluate the role of TLR4 in regulating luteal function. Toll‐like receptors 1, 2, 4, 6, tumour necrosis factor alpha (TNF), interferon gamma (IFN‐G), and interleukin (IL)‐12, mRNA expressions were greatest in regressing CL compared with earlier stages (p < .05), whereas no change was observed for IL‐6 mRNA expression. Cytokine mRNA expression in cultured luteal cells was not altered by LPS. Based on these data, one or more of the TLRs found within the CL may play a role in luteolysis, perhaps via pro‐inflammatory cytokine mRNA expression.     

Regulation of Corpus Luteum Function in Cattle – an Overview

The corpus luteum (CL) is a transient reproductive gland that produces progesterone (P), required for the establishment and maintenance of pregnancy. Although the regulation of bovine luteal function has been studied for several decades, many of the regulatory mechanisms involved are incompletely understood. We are far from understanding how these complex mechanisms function in unison. The purpose of this overview is to stress important steps of regulation during the lifetime of CL. In the first part, the importance and regulation of angiogenesis and blood flow during CL formation is described. The results underline the importance of growth factors especially of vascular endothelial growth factor A (VEGF A) and basic fibroblast growth factor (FGF-2) for development and completion of a dense network of capillaries. In the second part, the regulation of function by endocrine/paracrine- and autocrine-acting regulators is discussed. There is now more evidence that besides the main endocrine hormones LH and GH local regulators as growth factors, peptides, steroids and prostaglandins are important modulators of luteal function. During early CL development until mid-luteal stage oxytocin, prostaglandins and P itself stimulate luteal cell proliferation and function supported by the luteotropic action of a number of growth factors. The still high mRNA expression, protein concentration and localization of growth factors [VEGF, FGF-1, FGF-2, insulin-like growth factors (IGFs)] in the cytoplasm of luteal cells during mid-luteal stage suggest maintenance (survival) functions for growth factors. In the absence of pregnancy regression (luteolysis) of CL occurs. Progesterone itself regulates the length of the oestrous cycle by influencing the timing of the luteolytic signal prostaglandin F2alpha (PGF2alpha) from the endometrium. The cascade of mediators afterwards is very complex and still not well-elucidated. Evidence is given for participation of blood flow, inflammatory cytokines, vasoactive peptides (angiotensin II and endothelin-1), reactive oxygen species, angiogenic growth factors (VEGFs, FGFs, IGFs) and decrease of the classical luteotropic components as LH-R, GH-R, P450(scc) and 3beta-HSD. Despite of differences in methodology and interpretations, progress has been made and will continue to be made.     

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.     

Bovine Endothelial Cells Interact with Fully-luteinized, but Not Luteinizing, Granulosa Cells in the mRNA Expression of Endothelin-1 System in Response to Prostaglandin F2α

The corpus luteum (CL) undergoes regression by prostaglandin (PG)F(2alpha) from uterus and endothelin-1 (ET-1) plays an important role during luteolysis as a local mediator of PGF(2alpha) in the cow. Endothelial cells (EC) and luteal cells are main cell types making up the CL and their interactions are vital for CL function. We aimed to examine the relevance of interactions between EC and luteal cells on stimulation of genes which involved ET-1 synthesis by PGF(2alpha). We further focused the impact of maturity of luteal cells on the stimulation of the genes. To make a microenvironment which resembles the CL, we used bovine aortic endothelial cells (BAEC) and luteinizing or fully-luteinized granulosa cells (GC) and evaluated the effect of PGF(2alpha) on the expression for mRNA of ET-1 system by using real-time RT-PCR. PGF(2alpha) stimulated the expression of preproET-1 and endothelin converting enzyme-1 mRNA only in the co-cultures of BAEC with fully-luteinized GC, but not with luteinizing GC. The data suggest that interactions between BAEC and fully-luteinized GC enhance the capability of BAEC to produce ET-1 in response to PGF(2alpha). This mechanism may contribute to the local induction of luteolytic action of PGF(2alpha) which is dependent on the age/maturation of the CL.     

The effect of IGF-1 and FSH on the in vitro development of caprine secondary follicles and on the IGF-1, IGFR-I and FSHR mRNA levels

This study verified the in vitro effects of IGF-1, FSH or both on caprine preantral follicle development and mRNA levels encoding IGF-1, IGFR-1 and FSHR. Secondary follicles were cultured for six days with FSH, IGF-1 or IGF-1+FSH. The results showed that IGF-1 and/or FSH addition did not influence follicular development for six days. The interaction between IGF-1 and FSH increased the mRNA levels of IGF-1 and FSHR, and FSH increased the expression of the IGFR-1 mRNA. Thus, IGF-1 and/or FSH increased IGF-1, IGFR-1 and FSHR mRNA levels in in vitro cultured caprine secondary follicles, but they did not influence their development after six days of in vitro culture.     

Expression pattern of HIF1alpha and vasohibins during follicle maturation and corpus luteum function in the bovine ovary

The aim of this study was to characterize expression patterns of hypoxia‐inducible factor‐1alpha (HIF1A) and vasohibin family members (VASH1 and VASH2) during different stages of ovarian function in cow. Experiment 1: Antral follicle classification occurred by follicle size and estradiol‐17beta (E2) concentration in the follicular fluid into 5 groups (<0.5, 0.5–5, 5–40, 40–180 and >180 E2 ng/ml). Experiment 2: Corpora lutea (CL) were assigned to the following stages: days 1–2, 3–4, 5–7, 8–12, 13–16 and >18 (after regression) of oestrous cycle and of pregnancy (months 1–2, 3–4, 6–7, >8). Experiment 3: Cows on days 8–12 were injected with a prostaglandin F2alpha (PGF) analogue and CL were collected before and 0.5, 2, 4, 12, 24, 48 and 64 hr after PGF injection. Expression of mRNA was measured by qPCR, steroid hormone concentration by EIA and localization by immunohistochemistry. HIF1A mRNA expression in our study increases significantly in follicles during final maturation. The highest HIF1A mRNA expression was detected during the early luteal phase, followed by a significant decrease afterwards. In contrast, the mRNA of vasohibins in small follicle was high, followed by a continuous and significant downregulation in preovulatory follicles. The obtained results show a remarkable inverse expression and localization pattern of HIF1A and vasohibins during different stages of ovarian function in cow. These results lead to the assumption that the examined factors are involved in the local mechanisms regulating angiogenesis and that the interactions between proangiogenic (HIF1A) and antiangiogenic (vasohibins) factors impact all stages of bovine ovary function.     

Steroids as local regulators of ovarian activity in domestic animals

The presented overview gives clear evidence for steroids as local regulators of follicular and luteal activity. In the follicle, estrogen receptor-alpha (ERalpha) and ERbeta expression are demonstrated in cow, ewe and pig. Besides species specific effects in general, there is evidence that estradiol-17beta (E(2)) exerts a dose-dependent inhibition on the secretion of progesterone (P(4)) by both theca interna cells (TI) and granulosa cells (GC). GC enhance the ability of the TI to produce androstendione by supplying them with progestin precursor. Androgen produced by TI enhances the ability of the GC to make E(2), and high concentrations of E(2) in the preovulatory follicle inhibit 3beta-HSD in both TI and GC and thus, may promote the use of the pathway Delta(5) for TI androgen production. The authors suggest that E(2) acts within the follicle to exert positive feedback on androgen and E(2) production, and exerts mitotic and anti-atretic or anti-apoptotic effects on follicular cells. Parts of the E(2)-mediated local action are regulated by stimulating effects on hormone receptors (LH, FSH, oxytocin). Gap junctions permit transfer of nutrients and cytokines to and from the avascular GC and oocyte, and formation is stimulated by estrogens. In bovine corpus luteum (CL) there is evidence that P(4) may directly regulate the production of P(4), oxytocin and prostaglandins (PGs) in a cycle dependent fashion. In most of domestic animal species, there is clear evidence for CL production of E(2) with clear stimulatory and luteotropic effects on P(4), and an intraluteal circuit that involves paracrine effects of E(2), oxytocin and PGF(2alpha) (especially in pigs). In contrast, there are species (ruminants, mares) in which the evidence for important local effects of E(2) is less clear, although expression of ERalpha, ERbeta and progesterone receptor (PR) is documented. Progesterone is very important for the regulation of CL lifetime by effects on the endometrium and release of the luteolytic signal PGF(2alpha). In conclusion, steroids as local regulators of ovarian activity are now documented and may stimulate further research in this field.     

Expression and Localization of Vascular Endothelial Growth Factor and its Receptors in the Corpus Luteum During Oestrous Cycle in Water Buffaloes (Bubalus bubalis)

The aim of this study was to document the expression and localization of VEGF system comprising of VEGF isoforms (VEGF 120, VEGF 164 and VEGF 188) and their receptors (VEGFR1 and VEGFR2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle. Real‐time RT‐PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors. In general, all the components of VEGF system (the VEGF isoforms and their receptors) were found in the water buffalo CL during the oestrous cycle. The mRNA as well as protein expression of VEGF system was highest during the early and mid‐luteal phase, which later steadily decreased (p < 0.05) after day 10 to reach the lowest level in regressed CL. As demonstrated by immunohistochemistry, VEGF protein was localized predominantly in luteal cells; however, VEGFR1 and VEGFR2 were localized in luteal cells as well as in endothelial cells. In conclusion, the dynamics of expression and localization of VEGF system in buffalo corpora lutea during the luteal phase were demonstrated in this study, indicating the possible role of VEGF system in the regulation of luteal angiogenesis and proliferation of luteal as well as endothelial cells through their non‐angiogenic function.     

Effect of the preovulatory LH surge on bovine follicular progesterone receptor mRNA expression

A growing body of evidence indicates that intrafollicular progesterone receptor signaling pathways are obligatory for follicle rupture. However, the intrafollicular localization and regulation of progesterone receptor expression during the periovulatory period in cattle are not known. In this study, we determined the effect of the preovulatory gonadotropin surge on localization and expression of progesterone receptor mRNA in bovine periovulatory follicular and luteal tissue. Ovaries containing preovulatory follicles or new corpora lutea (CL) were collected at approximately 0, 6, 12, 18, 24 (preovulatory follicles) and 48 h (CL) after a GnRH-induced LH surge (n=5-8 per timepoint). Expression of progesterone receptor mRNA was detected in periovulatory follicular and luteal tissue at all timepoints examined. Relative levels of progesterone receptor mRNA were dramatically upregulated within 6h after the LH surge compared to all other time points (P<0.0001). In situ hybridization analysis revealed that the significant increase in progesterone receptor mRNA expression was localized to the granulosal layer of preovulatory follicles. Our results indicate that progesterone receptor mRNA expression is upregulated specifically in the granulosal layer of bovine preovulatory follicles following the LH surge. Progesterone receptor signaling pathways may help mediate the effects of the preovulatory LH surge on follicle rupture in cattle.     

Age‐related changes in the bovine corpus luteum function and progesterone secretion

Decreased fertility associated with maternal ageing is a well‐known critical problem, and progesterone (P4) concentration decreases during the menopause transition in women. The corpus luteum (CL) secretes P4, thereby supporting the implantation and maintenance of pregnancy. It is proposed that a bovine model is suitable for studying age‐associated decline of fertility in women because the physiology of cows is similar to that of women and cows have a greater longevity compared with other animal models. Thus, we investigated the age‐dependent qualitative changes and inflammatory responses in the bovine CL. In vivo experiment: Cows were divided into three groups, namely, young (mean age: 34.8 months), middle (80.1 months) and aged (188.9 months). Blood samples were collected on days 7 and 12 during the estrous cycle. In vitro experiments: Cows were divided into young (mean age: 27.6 months) and aged (183.1 months). The CL tissues of these groups were collected from a local slaughterhouse and used for tissue culture experiments. An in vivo experiment, plasma P4 concentration in aged cows was significantly lower than that in young cows, whereas no difference was found regarding the area of CL. An in vitro examination in the bovine CL tissues showed that the luteal P4 concentration, P4 secretion, and mRNA expression of StAR and 3β‐HSD were lower in aged cows compared with young cows, especially in the early luteal phase. However, no differences were detected in the mRNA expression of inflammation‐ and senescence‐related factors and inflammatory responses to lipopolysaccharides between the CL tissues from young and aged cows, indicating that an age‐dependent increase in inflammation is not involved in the luteal function. P4 production and secretion from the bovine CL diminish in old cows, especially during the early luteal phase, suggesting that senescence may affect the luteal function in cows.     

Prostaglandin F2α Stimulates Endothelial Nitric Oxide Synthase Depending on the Existence of Bovine Granulosa Cells: Analysis by Co‐culture System of Endothelial Cells,Smooth Muscle Cells and Granulosa Cells

Prostaglandin F_2α (PGF_2α) induces luteolysis in the mid but not in the early luteal phase; despite this, both the early and the mid corpus luteum (CL) have PGF_2α receptor (FPr). We previously indicated that the luteal blood flow surrounding the CL drastically increases prior to a decrease of progesterone (P) in the cows, suggesting that an acute increase of luteal blood flow may be an early sign of luteolysis in response to PGF_2α and that this may be induced by a vasorelaxant nitric oxide (NO). The aim of this study was to investigate the luteal stage‐dependent and the site‐restricted effect of PGF_2α and NO on the mRNA expressions and P secretion. To mimic the local luteal region both of peripheral and central areas of the CL, we utilized co‐cultures using bovine aorta endothelial cells (EC), smooth muscle cells (SMC) and luteinizing granulosa cells (GC) or fully‐luteinized GC. PGF_2α stimulated the expression of endothelial NO synthase (eNOS) mRNA at 0.5 h in mix‐cultures of EC and SMC with fully‐luteinized GC but not with luteinizing GC. The expression of eNOS mRNA in EC was increased by PGF_2α at 1 h only when EC was cultured together with fully‐luteinized GC but not with luteinizing GC. In all co‐cultures, PGF_2α did not affect the mRNA expression of FPr. Treatment of NO donor inhibited P secretion at 0.5 h. In conclusion, the present study suggests that the coexistence of the mature luteal cells (fully‐luteinized GC) with EC/SMC may be crucial for acquiring functional NO synthesis induced by PGF_2α.     

Angiogenesis in The Ovary – The Most Important Regulatory Event for Follicle and Corpus Luteum Development and Function in Cow – An Overview

In the ovary, the development of new capillaries from pre‐existing ones (angiogenesis) is a complex event regulated by numerous local factors. The dominant regulators of angiogenesis in ovarian follicles and corpora lutea are the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin‐like growth factor (IGF), angiopoietin (ANPT) and hypoxia‐inducible factor (HIF) family members. Antral follicles in our study were classified according to the oestradiol‐17‐beta (E2) content in follicular fluid (FF) and were divided into five classes (E2 < 0.5, 0.5–5, 5–20, 20–180 and >180 ng/ml FF). The corresponding sizes of follicles were 5–7, 8–10, 10–13, 12–14 and >14 mm, respectively. Follicle tissue was separated in theca interna (TI) and granulosa cells (GC). The corpora lutea (CL) in our study were assigned to the following stages: days 1–2, 3–4, 5–7, 8–12 13–16 and >18 of the oestrous cycle and months 1–2, 3–4, 6–7 and >8 of pregnancy. The dominant regulators were measured at mRNA and protein expression levels; mRNA was quantified by RT‐qPCR, hormone concentrations by RIA or EIA and their localization by immunohistochemistry. The highest expression for VEGF‐A, FGF‐2, IGF‐1 and IGF‐2, ANPT‐2/ANPT‐1 and HIF‐1‐alpha was found during final follicle maturation and in CL during the early luteal phase (days 1–4) followed by a lower plateau afterwards. The results suggest the importance of these factors for angiogenesis and maintenance of capillary structures for final follicle maturation, CL development and function.