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.     

Expression of prostaglandin F(2α) (PGF(2α)) receptor and its isoforms in the bovine corpus luteum during the estrous cycle and PGF(2α)-induced luteolysis

Prostaglandin F(2α) (PGF(2α)) induces luteolysis via a specific receptor, PTGFR. Although PTGFR mRNA expression in the bovine corpus luteum (CL) has been studied previously, changes in PTGFR protein and its localization are not fully understood during the life span of the CL. In addition to full-length PTGFR, several types of PTGFR isoforms, such as PTGFRα (type I) and PTGFRζ (type II), were reported in the bovine CL, suggesting isoform-specific luteal action. Full-length PTGFR mRNA in the bovine CL increased from the early to the mid-luteal phase and decreased during luteolysis, whereas PTGFR protein remained stable. PTGFR protein was localized to both luteal and endothelial cells and was expressed similarly during the life span of the CL. Like full-length PTGFR mRNA, PTGFRα and PTGFRζ mRNA also increased from the early to mid-luteal phases, and mRNA of PTGFRζ, but not PTGFRα, decreased in the regressing CL. During PGF(2α)-induced luteolysis, the mRNAs of full-length PTGFR, PTGFR,α and PTGFRζ decreased rapidly (from 5 or 15 min after PGF(2α) injection), but PTGFR protein decreased only 12 h later. Silencing full-length PTGFR using small interfering RNA prevented PGF(2α)-stimulated cyclooxygenase-2 (PTGS2) mRNA induction. By contrast, PGF(2α) could stimulate vascular endothelial growth factor A (VEGFA) mRNA even when full-length PTGFR was knocked down, thus suggesting that PGF(2α) may stimulate PTGS2 via full-length PTGFR, whereas VEGFA is stimulated via other PTGFR isoforms. Collectively, PTGFR protein was expressed continually in the bovine CL during the estrous cycle, implying that PGF(2α) could function throughout this period. Additionally, the bovine CL expresses different PTGFR isoforms, and thus PGF(2α) may have different effects when acting via full-length PTGFR or via PTGFR isoforms.     

Expression and localization of IGF family members in bovine antral follicles during final growth and in luteal tissue during different stages of estrous cycle and pregnancy

The objectives of the study were to monitor the detailed pattern for mRNA expression (RT-PCR and RPA) of IGFs, IGFR-1, IGFBPs, GHR and localization of protein (immunohistochemistry) for IGF-1 and IGFR-1 in bovine follicle classes during final maturation and different corpus luteum (CL) stages during estrous cycle and during pregnancy. A relative high expression of IGF-1 in theca interna (TI) was observed before selection (E<0.5ng/mL). In GC, mRNA expression increased after selection. In contrast, IGF-2 was mainly expressed in the TI. The IGFR-1 mRNA was present in the TI and GC with increasing levels during final development. The expression results were confirmed by localization of IGF-1 and IGFR-1 proteins in GC and TI. There is clear evidence for the local expression of IGFBPs in TI and GC compartment with clear regulatory differences. In CL, the highest mRNA expression of IGF-1, IGF-2 and IGFR-1 was observed during early luteal phase, followed by a decrease, and then by a tendency of an increase during the mid and late luteal phases of the cyclic CL. This level remained low during pregnancy. Intense immunostaining for IGFR-1 in CL was observed mainly in large luteal cells. Evidence for a mRNA for all six IGFBPs were obtained with distinct differences for BP-3, -4 and -5. In conclusion, this comprehensive study gives clear evidence for an important role of the IGFs and IGFBPs in bovine follicular development and CL function. The relative amounts of IGFBPs may ultimately determine ovarian IGF action.     

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.     

Acquisition of luteolytic capacity involves differential regulation by prostaglandin F2alpha of genes involved in progesterone biosynthesis in the porcine corpus luteum

Luteolytic capacity is defined as the ability of corpora lutea (CL) to undergo luteolysis after prostaglandin (PG) F2alpha treatment. The mechanisms causing acquisition of luteolytic capacity are not yet identified but CL without luteolytic capacity have PGF2alpha receptors and respond to PGF2alpha with some changes in gene expression. Inhibition of progesterone biosynthesis is a key feature of luteolysis and therefore we postulated that genes involved in progesterone biosynthesis would be regulated by PGF2alpha differently in CL with or without luteolytic capacity. Gilts on day 9 after estrus (lack luteolytic capacity) or day 17 of pseudopregnancy (with luteolytic capacity) were treated with saline or a PGF2alpha analog (cloprostenol) and CL were collected 0.5 (Experiment I) or 10 h (Experiment II) later. In Experiment III, large luteal cells from CL on day 9 or 17 were cultured for 1, 12 and 24h with or without PGF2alpha. PGF2alpha decreased LDL receptor mRNA (27%), steroidogenic acute regulatory protein (StAR) mRNA (41%), StAR protein (75%), LH receptor mRNA (55%), and LH receptor protein (45%) at 10 h after treatment in day 17 but not day 9 CL. PGF2alpha increased DAX-1 mRNA at 0.5 h (43%) and 10 h (46%) after PGF2alpha in day 17 but not day 9 CL but decreased 3betaHSD mRNA ( approximately 20% at 10 h) in both days 9 and 17 CL. In vitro, PGF2alpha decreased StAR mRNA at 12 h only in day 17 luteal cells; however, continuous treatment with PGF2alpha for 24 h decreased StAR mRNA in both days 9 and 17 luteal cells. Thus, luteolytic capacity involves a critical change in responsiveness of DAX-1, StAR, and LH receptor to PGF2alpha that results in inhibition of luteal progesterone biosynthesis.     

Blood flow: a key regulatory component of corpus luteum function in the cow

Prostaglandin F2alpha (PGF2alpha) is the primary luteolysin in the cow. During the early luteal phase, the corpus luteum (CL) is resistant to the luteolytic effect of PGF2alpha. Once mature, the CL becomes responsive to PGF2alpha and undergoes luteal regression. These actions of PGF2alpha coincide with changes in luteal blood flow (BF): PGF2alpha has no effect on BF in the early CL, but acutely increases BF in the peripheral vasculature of the mature CL within 30 min of PGF2alpha injection. During spontaneous luteolysis, luteal BF increases on Days 17-18 of the estrous cycle, prior to any decrease in plasma progesterone (P). The increase in luteal BF is synchronous with an increase in plasma PGFM levels, suggesting that pulsatile release of PGF2alpha from uterus stimulates the increase in luteal BF. Serial biopsies of these CL showed that mRNA expression for endothelial nitric oxide synthase (eNOS) together with endothelin-1 (ET-1) and angiotensin converting enzyme (ACE) increases on Days 17-18 when the luteal BF is elevated. On Day 19 when plasma P level firstly decreases, eNOS mRNA returns to the basal level whereas ET-1 and ACE mRNA remains elevated. Cyclooxygenase-2 (COX-2) mRNA expression increases on Day 19. In support of these data, an in vivo microdialysis study revealed that luteal ET-1 and angiotensin II (Ang II) secretion increases and precedes PGF2alpha secretion during spontaneous luteolysis. In conclusion, we show for the first time that an acute increase of BF occurs in the peripheral vasculature of the mature CL together with increases in eNOS expression and ET-1 and Ang II secretion in the CL during the early stages of luteolysis in the cow. We propose that the increase in luteal BF may be induced by NO from large arterioles surrounding the CL, and simultaneously uterine or exogenous PGF2alpha directly increases ET-1 and Ang II secretion from endothelial cells of microcapillary vessels within the CL, thereby suppressing P secretion by luteal cells. Taken together, our results indicate that an acute increase in luteal BF occurs as a first step of luteolysis in response to PGF2alpha. Therefore, local BF plays a key role to initiate luteal regression in the cow.     

Expressions of estrogen receptors in the bovine corpus luteum: cyclic changes and effects of prostaglandin F2alpha and cytokines

Estrogen (E) exerts its function by binding to two intracellular estrogen receptors, ERalpha and ERbeta. Although ERs have been reported to be expressed in the bovine corpus luteum (CL), the mechanisms that control ER expression in the bovine CL are not fully understood. To determine the possible regulatory mechanisms of ERalpha and ERbeta that meditate distinct E functions, we examined 1) the changes in the protein expressions of ERs in the CL throughout the luteal phase and 2) the effects of prostaglandin (PG) F2alpha, tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) on the expressions of ERs in cultured bovine luteal cells. Western blot analyses revealed that ERalpha and ERbeta proteins were expressed throughout the luteal phase. The ERalpha protein level was high at the early luteal (Days 2-3 after ovulation) and mid-luteal stages (Days 8-12) and was extremely low at the regressed luteal stage (Days 19-21). The ERbeta protein level increased from the early to developing luteal stage, remained at the same level at the mid-luteal stage and decreased thereafter. The ratio of ERbeta to ERalpha was higher in the regressed stage than in the other stages. Luteal cells obtained from mid-stage CLs (Days 8-12) were incubated with PGF2alpha (0.01-1 microM), TNFalpha (0.0145-1.45 nM) or IFNgamma (0.0125-1.25 nM) for 24 h. PGF2alpha and TNFalpha inhibited ERa and ERbeta mRNA expressions. IFNgamma suppressed ERbeta mRNA expression but did not affect the expression of ERalpha mRNA. However, the ERalpha and ERbeta protein levels were not affected by any of the above treatments. These data indicate that PGF2alpha, TNFalpha and IFNgamma regulate ERalpha and ERbeta mRNA expressions in bovine luteal cells. Moreover, the changes in the ERbeta/ERalpha ratio throughout the luteal phase suggest that ERalpha is associated with luteal maintenance. Therefore, a dramatic decrease in ERalpha at the regressed luteal stage could result in progression of structural luteolysis in the bovine CL.     

Effect of intraluteal injection of endothelin type A receptor antagonist on PGF2alpha-induced luteolysis in the cow

Endothelin-1 (ET-1) is a luteolytic mediator in the bovine corpus luteum (CL), and its action appears to be via endothelin type A receptor (ETR-A). Thus, the aim of the present study was to determine the effect of ETR-A antagonist on PGF2alpha-induced luteolysis in the cow. Cows on days 10-12 of the estrous cycle were subjected to five intraluteal injections of the ETR-A antagonist LU 135252 in saline or only saline at -0.5, 2, 4, 6, and 8 h after PGF2alpha administration (=0 h). Serial luteal biopsies were conducted to determine the expression of mRNA in the luteal tissue. There were no significant differences in the decrease in plasma progesterone (P) concentrations and the mRNA expressions of steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase/Delta5, Delta4-isomerase between the ETR-A antagonist-treated group and the control group. However, the start of the decline in CL volume and blood flow area surrounding the CL was delayed for almost two days in the ETR-A antagonist-treated group compared to the control group. The mRNA expression of preproET-1 and endothelin type B receptor increased in both groups, while the ETR-A mRNA remained unchanged. In addition, caspase-3 mRNA expression increased significantly at 24 h in the control group only and its level was higher than that of the ETR-A antagonist-treated group. Thus, the present study suggests that ET-1 regulates structural luteolysis via ETR-A by controlling blood vessel contraction in the CL of the cow.     

Expression of insulin-like growth factor binding protein (IGFBP)-3, and the effects of IGFBP-2 and -3 in the bovine corpus luteum.

The present study was conducted to gain insight into the insulin-like growth factor (IGF) system in the bovine corpus luteum (CL). Specific aims were to measure the levels of IGF binding protein-3 (IGFBP-3) and RNA encoding IGFBP-3 in the CL throughout diestrus, and to investigate the effects of IGFBP-2 and -3 on IGF-I-stimulated progesterone (P4) production and IGF-I-receptor binding. Bovine CL were collected from a local abattoir and classified according to stage of diestrus based on anatomical characteristics. Corpora lutea from early, mid and late diestrus were each analyzed for the presence of IGFBP-3 by ligand blot analysis, and for RNA encoding IGFBP-3 by Northern blot analysis. Dissociated cells from mid-cycle CL were treated with IGF-I, IGFBP-2 or -3, or a combination of IGF-I and IGFBP-2 or -3. The effect of IGFBP-2 and IGFBP-3 on [(125)I] IGF-I binding to its receptor on CL plasma membranes also was investigated. IGFBP-3 protein and RNA expression were higher in early CL, compared to mid or late CL (p < 0.05). IGF-I stimulated P4 production in a dose-dependant manner (p < 0.05). IGFBP-2 and -3 blocked the stimulatory effect of IGF-I on P4 production (p < 0.05). Both IGFBP-2 and -3 inhibited [(125)I]-IGF-I binding to its receptor in a dose-dependant manner. These results demonstrate that IGFBP-3 protein and RNA are expressed predominantly during early diestrus in the bovine CL. Moreover, both IGFBP-2 and -3 can modulate IGF-I actions in the CL by interfering with binding of IGF-I to its receptor.     

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.     

Expression of mRNA for cell adhesion molecules in the bovine corpus luteum during the estrous cycle and PGF2alpha-induced luteolysis

Cell-to-cell interaction via cell contact-dependent pathway is essentially important for maintenance and regulation of corpus luteum (CL) integrity and its physiological actions. The objective of the present study was to evaluate the mRNA expression of the cell adhesion molecules (CAMs) that are constituent factors of gap junctions [connexin (Cx) 43] and adherence junctions (VE-, E-, N-cadherin) in two types of endothelial cells from the mid CL and in CL tissue during the estrous cycle and PGF(2alpha)-induced luteolysis in the cow. Specific mRNA expression for Cx43 and N-cadherin was detected in cytokeratin-positive (CK+) and cytokeratin-negative (CK-) luteal endothelial cells (EC) and fully luteinized granulosa cells (LGC). E-cadherin mRNA was expressed in CK+EC and LGC, but not in CK-EC. VE-cadherin mRNA was expressed in both CK+ and CK-EC. During the estrous cycle, Cx43 mRNA expression was significantly lower in the regressing CL. VE-cadherin expression also tended to increase in the mid CL and increased significantly in the regressing CL. E-cadherin mRNA expression was higher in the early and late CL than in the mid- and regressing CL. N-cadherin mRNA expression gradually increased from the early to late CL followed by a decrease in the regressing CL. During PGF(2alpha)-induced luteolysis, Cx43 mRNA expression appeared to increase, and VE-cadherin and E-cadherin mRNA significantly increased at 24 h. N-cadherin mRNA expression decreased 2 and 4 h after PGF(2alpha) administration. Collectively, expression of the mRNAs for CAMs was different in the two types of luteal endothelial cells and fully luteinized granulosa cells and changed independently in the CL during the estrous cycle and PGF(2alpha)-induced luteolysis in the cow. The results suggest that CAMs play physiological roles in cell-to-cell communication to regulate both gap and adherence junctions during CL development and regression in the cow.     

Role of tumor necrosis factor-alpha and nitric oxide in luteolysis in cattle

Although prostaglandin (PG) F2alpha is known to be a principal luteolytic factor, its action on the bovine corpus luteum (CL) is mediated by other intra-ovarian factors. Tumor necrosis factor-alpha (TNFalpha) and its specific receptors are present in the bovine CL with the highest expressions at luteolysis. TNFalpha in combination with interferon-gamma reduced progesterone (P4) secretion, increased PGF2alpha and leukotriene C4 (LTC4) production, and induced apoptosis of the luteal cells in vitro. Low concentrations of TNFalpha caused luteolysis, which resulted in a decreased level of P4, and increased levels of PGF2alpha, LTC4 and nitrite/nitrate (stable metabolites of nitric oxide-NO) in the blood. Inhibition of local NO production counteracts spontaneous and PGF2alpha-induced luteolysis. Therefore, NO is a likely candidate for the molecule that mediates PGF2alpha and TNFalpha actions during luteolysis. Both PGF2alpha and TNFalpha increase NO concentrations in blood, and stimulate NO synthase expression on protein level in the bovine CL cells. NO stimulates PGF2alpha and LTC4 secretion, inhibits P4 production and reduces the number of viable luteal cells. TNFalpha and NO induce apoptotic death of the CL by modulating expression of bcl-2 family genes and by stimulating expression and activity of caspase-3. The above findings indicate that TNFalpha and NO play crucial roles in functional and structural luteolysis in cattle.     

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.     

Expression of matrix metalloproteinases in bovine luteal cells induced by prostaglandin F2α, interferon γ and tumor necrosis factor α

We recently demonstrated that luteal cells flow out from the ovary via lymphatic vessels during luteolysis. However, the regulatory mechanisms of the outflow of luteal cells are not known. Matrix metalloproteinases (MMPs) can degrade the extracellular matrix and basal membrane, and tissue inhibitors of matrix metalloproteinases (TIMPs) inhibit the activity of MMPs. To test the hypothesis that MMP expression in luteal cells is regulated by luteolytic factors, we investigated the effects of prostaglandin F2α (PGF), interferon γ (IFNG) and tumor necrosis factor α (TNF) on the mRNA expression of MMPs and TIMPs in cultured luteal cells. Luteal cells obtained from the CL at the mid-luteal stage (days 8–12 after ovulation) were cultured with PGF (0.01, 0.1, 1 μM), IFNG (0.05, 0.5, 5 nM) and TNF (0.05, 0.5, 0.5 nM) alone or in combination for 24 h. PGF and IFNG significantly increased the expression of MMP-1 mRNA. In addition, 1 μM PGF in combination with 5 nM IFNG stimulated MMP-1 and MMP-9 mRNA expression significantly more than either treatment alone. In contrast, IFNG significantly decreased the level of MMP-14 mRNA. The mRNA expression of TIMP-1, which preferentially inhibits MMP-1, was suppressed by 5 nM INFG. One μM PGF and 5 nM IFNG suppressed TIMP-2 mRNA expression. These results suggest a new role of MMPs: luteal MMPs stimulated by PGF and IFNG break down the extracellular matrix surrounding luteal cells, which accelerates detachment from the CL during luteolysis, providing an essential prerequisite for outflow of luteal cells from the CL to lymphatic vessels.     

Endometrial expression of the insulin-like growth factor system during uterine involution in the postpartum dairy cow

Rapid uterine involution in the postpartum period of dairy cows is important to achieve a short interval to conception. Expression patterns for members of the insulin-like growth factor (IGF) family were determined by in situ hybridisation at day 14 ± 0.4 postpartum (n = 12 cows) to investigate a potential role for IGFs in modulating uterine involution. Expression in each uterine tissue region was measured as optical density units and data were analysed according to region and horn. IGF-I mRNA was localized to the sub-epithelial stroma (SES) of inter-caruncular and caruncular endometrium. Both IGF-II and IGF-1R expression was detected in the deep endometrial stroma (DES), the caruncular stroma and myometrium. IGFBP-2, IGFBP-4 and IGFBP-6 mRNAs were all localised to the SES of inter-caruncular and caruncular uterine tissue, and in the DES and caruncular stroma, with IGFBP-4 mRNA additionally expressed in myometrium. IGFBP-3 mRNA was only detectable in luminal epithelium. IGFBP-5 mRNA was found in myometrium, inter-caruncular and caruncular SES and caruncular stroma. These data support a role for IGF-I and IGF-II in the extensive tissue remodelling and repair which the postpartum uterus undergoes to return to its non-pregnant state. The differential expression of binding proteins between tissues (IGFBP-3 in epithelium, IGFBP-2, -4, -5 and -6 in stroma and IGFBP-4 and -5 in myometrium) suggest tight control of IGF activity within each compartment. Differential expression of many members of the IGF family between the significantly larger previously gravid horn and the previously non-gravid horn may relate to differences in their rate of tissue remodelling.     

Endothelin-1 receptors and biosynthesis in the corpus luteum: molecular and physiological implications

Endothelin-1 (ET-1), a 21-amino acid peptide was initially identified as a potent vasoconstrictor, ET-1 plays an important role in the female reproductive cycle: its quick ascent during luteal regression, ability to inhibit steroidogenesis in vitro and in vivo, combined with the observation that the luteolytic effects of prostaglandin F2alpha (PGF2alpha) were delayed by pretreatment with ET-1 receptors type A (ETA) antagonists suggest that this peptide functions as an important element of the luteolytic cascade. The observation that ETA receptor expression was inversely correlated with steroidogenesis in luteal cells; namely factors which stimulated steroidogenesis inhibited ETA receptor levels is also in accord with the inhibitory role of ET-1 in corpus luteum (CL) function. Contrary to the mature mid cycle CL, the CL of early cycle is refractory to PGF2alpha-induced luteolysis. PGF2alpha administered at early luteal phase (day 4 of the cycle) failed to increase luteal ET-1 gene expression or its ETA receptors. In contrast, both genes were markedly induced in mid cycle CL exposed to PGF2alpha. ET-1 gene is transcribed as prepro ET-1 (ppET-1) and the active form of peptide is derived from the inactive intermediate big ET-1, by endothelin-converting enzyme-1 (ECE-1), therefore alterations in mature ET-1 levels can be achieved by modulating the expression of ppET-1 and/or ECE-1. Analysis using in situ hybridization and enriched luteal cell subpopulations showed that both steroidogenic and endothelial cells of the CL expressed high levels of ECE-1 mRNA. The ppET-1 mRNA, on the other hand, was only expressed by resident endothelial cells, suggesting that luteal parenchymal and endothelial cells cooperate in the biosynthesis of mature bioactive ET-1. A significant, four-fold elevation in ECE-1 expression (mRNA and protein levels) occurred during the transition of the CL from early to mid luteal phase. This increase was accompanied by a significant rise in ET-1 peptide. Surprisingly however, ppET-1 mRNA levels remained similar during early and mid luteal phase. Collectively, these studies demonstrate that: (a) the various components of ET-1 system (ET-1/ECE-1/ETA) are dynamically and independently regulated during bovine luteal life span. (b) The CL becomes PGF2alpha-responsive only when both ppET-1 and ECE-1 genes are expressed at a level which enable an uninterrupted ET-1 biosynthesis.     

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.