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.     

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.     

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.     

Prostaglandin F2alpha (PGF2alpha) independent and dependent regulation of the bovine luteal endothelin system

We have examined the genes of the endothelin system that are targets for regulation by prostaglandin F2alpha (PGF2alpha). The effects of a luteolytic dose of PGF2alpha ) on the mRNA encoding endothelin converting enzyme-1 (ECE-1), pre-pro endothelin-1 (pp ET-1) and the ET receptors ETA, ETB, in bovine corpus luteum (CL) during the early (days 1 and 4), mid (day 10) or late (day 17) luteal phases were examined. The effect of the PGF(2alpha) treatment on ECE-1 protein, Big ET-1 and the biologically active mature ET-1 peptide were also examined. Most importantly, the direct ECE-1 activity was determined. Before day 10 of the cycle, in a PGF2alpha-independent manner, the amounts of mRNA encoding ET-1, ECE-1, ETA, and ETB were increased steadily from day 1. After day 10 of the cycle, expression of mRNA encoding pp ET-1 and ETA acquired responsiveness to exogenous PGF2alpha and both genes were up-regulated by the PGF2alpha treatment. This effect of PGF2alpha was also detected for the proteins corresponding to the mature ET-1. The enzymatic activity of ECE-1 remained unchanged throughout the lifespan of the CL in spite of the detected changes in mRNA and protein. The results suggest that the luteal endothelin system is regulated in a PGF2alpha-independent and -dependent manner. Importantly, an alteration in luteal ET-1 availability is most likely achieved by modulating the expression of mRNA encoding pp ET-1 and not by the amount or activity of ECE-1. This interpretation is supported by the observation that the activity of ECE-1 remained unchanged throughout the ovarian cycle. The combined effects of greater ET-1 availability and gene expression encoding the ETA receptor in the late luteal phase could render the CL, at this developmental stage, more sensitive or responsive to ET-1. If the luteal tissue is responsive to the available ET-1 during the early phase of the ovarian cycle, an additional role for ET-1 should be considered beyond mediating the luteolytic actions of PGF2alpha. Agents blocking the actions of ET-1 might be the best approach to interfere with the luteal ET system and test its physiological role(s) in vivo.     

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 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.     

Changes in the messenger RNA expressions of the endothelin-1 and angiotensin systems in mature follicles of the superovulated bovine ovary

The aim of the present study was to examine the messenger RNA expressions of the endothelin and angiotensin systems during the periovulatory phase in gonadotrophin releasing hormone (GnRH)-treated cows. Ovaries were collected by transvaginal ovariectomy (n=5 cows/group), and the follicles (n=5, one follicle/cow) were classified into the following groups: before GnRH administration (control, before LH surge), 3-5 h after GnRH (during LH surge), 10 h after GnRH; 20 h after GnRH, 25 h after GnRH (peri-ovulation), and early corpus luteum (CL) (Days 2-3). Expression of mRNA was investigated using quantitative real-time PCR. The expression of angiotensin converting enzyme (ACE) mRNA significantly decreased immediately after onset of the LH surge and remained at low levels. The levels of angiotensin II receptor type 1 (AT1R) and type 2 (AT2R) expression during the periovulatory period significantly decreased compared with other periods. The concentration of angiotensin II in follicular fluid began to increase 10 h after GnRH treatment and further increased as ovulation approached. The level of ET-1 mRNA significantly decreased 10 h after GnRH treatment compared with the levels before GnRH treatment and those of the early CL period. The expression of ETR-A and ETR-B mRNA during the periovulatory period were lower than in other periods. The expression of ECE-1 mRNA began to decrease in the LH surge period and significantly decrease in the periovulatory period compared with other periods. These results suggest that the vasoactive peptides angiotensin and endothelin may be associated with final maturation of follicles.     

A cooperative action of endothelin-1 with prostaglandin F(2alpha) on luteal function in the cow

Prostaglandin F(2alpha) (PGF(2alpha)) is the primary luteolysin in the cow, and luteal endothelin-1 (ET-1) interacts with PGF(2alpha) during the process of luteolysis. In contrast, a developing corpus luteum (CL) is refractory to exogenous administration of PGF(2alpha). Thus, the present study was aimed to investigate the functional relationship between ET-1 and PGF(2alpha) in the mid-CL (PGF(2alpha)-sensitive) and early-CL (PGF(2alpha)-refractory). In the mid-CL model, cows (n = 6/treatment) were assigned to receive one of five types of treatments on day 10 of the estrous cycle: (1) an injection of saline; control, (2) a 500 microg of PGF(2alpha) analogue (sufficient dose to induce luteolytis); full-PG, (3) an intraluteal injection of 0.25 mg ET-1; ET-1, (4) a 125 micro g of PGF(2alpha) (insufficient dose to induce luteolytis); 1/4PG or (5) an intraluteal injection of 0.25 mg ET-1 after administration of a insufficient dose of PGF(2alpha) analogue; 1/4PG/ET. In the early-CL model, cows were assigned to receive one of two types of treatments on day 5 of the estrous cycle: (1) a sufficient dose of PGF(2alpha) analogue; PG (n = 5) or (2) an intraluteal injection ET-1 after a sufficient dose of PGF(2alpha); PG/ET (n = 7). In the mid-CL model, 1/4PG/ET resulted in a rapid reduction of progesterone (P) concentrations similar to that in full-PG from the next day. However, the levels of P in 1/4PG/ET (1.5-2.5 ng/ml) kept significantly higher than that in full-PG (< 0.5 ng/ml). ET-1 or 1/4PG did not decrease plasma P concentrations (4-6 ng/ml). The plasma ET-1 levels increased with the full-PG administration. In the early-CL model, both treatments had no effect on plasma P increase and ET-1 levels. The overall results indicate that the intraluteal ET-1 injection after administration of insufficient dose of PGF(2alpha) induces the depression of P secretion in vivo during the mid luteal phase in the cow, supporting the concept that ET-1 is one of a local mediator of functional luteolysis in the cow. The result further indicates that the early-CL is not only PG-refractory but also ET-1-refractory.     

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 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 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.     

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.     

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.     

The yin and yang of corpus luteum-derived endothelial cells: balancing life and death

A dense network of capillaries irrigates the corpus luteum (CL) allowing an intricate cross talk between luteal steroiodgenic and endothelial cell (EC) types. Indeed, luteal endothelial cells (LEC) play pivotal roles throughout the entire CL life-span. Microvascular endothelial cells are locally specialized to accommodate the needs of individual tissues, therefore unraveling the characteristics of LEC is imperative in CL physiology. Numerous studies demonstrated that endothelium-derived endothelin-1 (ET-1) is upregulated by the luteolytic hormone-prostaglandin F2alpha (PGF2alpha) and functions as an important element of the luteolytic cascade. To have a better insight on its synthesis and action, members of ET system (ET-1, ET converting enzyme -ECE-1 and ET(A) and ET(B) receptors) were quantified in LEC. The characteristic phenotype of these cells, identified by high ET-1 receptor expression (both ET(A), ET(B)) and low ET-1 and ECE-1 levels, was gradually lost during culture suggesting that luteal microenvironment sustains the selective phenotype of its resident endothelial cells. Proper vascularization and endothelial cell activity per se are essential for normal CL function. Therefore, factors affecting vascular growth are expected to play major role in the regulation of luteal function. Concomitantly with the angiogenic process, luteal PGF2alpha and its receptors (PGFR) are induced and maintained during most of the CL life-span, suggesting a possible role of PGF2alpha in LEC proliferation and function. Dispersed LEC expressed PGFR and incubation with the prostaglandin stimulated mitogen-activated protein kinase (MAPK) signaling cascade. PGF2alpha activated p42/44 MAPK phosphorylation also in long-term cultured LEC. In this cell type, PGF2alpha increased cell number, 3H-Thymidine incorporation and cell survival. Additionally, PGF2alpha rapidly and transiently stimulated the expression of immediate-early response genes, i.e. c-fos and c-jun mRNA, further suggesting a mitogenic effect for this prostaglandin in LEC. These data imply that PGF2alpha may assume different and perhaps opposing roles depending on luteal microenvironment.     

Local mechanisms for luteolysis in the cow: Novel roles of vasoactive substances in the luteolytic cascade within the corpus luteum

The corpus luteum (CL) in the estrous cycle in the cow is a dynamic organ which has a lifespan of approximately 17–18 days. As the CL matures, the steroidogenic cells establish contact with many capillary vessels and the CL is composed of a large number of vascular endothelial cells that can account for up to 50% of the bovine CL. Furthermore, luteal cells and endothelial cells secrete several vasoactive substances such as prostaglandin F_2α (PGF_2α), endothelin‐1 and angiotensin II. These vasoactive substances also function in regulating progesterone secretion in an autocrine/paracrine manner in the CL. The blood vessels and endothelial cells in the CL therefore have an essential role in the luteal function in the cow. Endometrial PGF_2α, the primary luteolysin in the cow, stimulates luteal vasoactive substances during luteolysis. Moreover, luteal vasoactive substances may have key roles in the regulation of luteolysis to induce vasodilatation, vasoconstriction and angiolysis. This review describes the current concept for possible roles of vasoactive substances in the luteolytic cascade within the bovine CL.     

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.     

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.     

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.     

Intrauterine infusion of BQ-610, an endothelin type A receptor antagonist, delays luteolysis in dairy heifers

Three separate in vivo experiments were conducted to evaluate the putative role of endothelin-1 (ET-1) during luteal regression in heifers. In Experiment 1, a single intraluteal injection of 500 μg BQ-610 [(N,N-hexamethylene) carbamoyl-Leu-d-Trp (CHO)-d-Trp], a highly specific endothelin A (ET_A) receptor antagonist, did not diminish the decline in plasma progesterone following a single exogenous injection of 25 mg prostaglandin F2 alpha (PGF₂) administered at midcycle of the estrous cycle. In Experiment 2, six intrauterine infusions of 500 μg BQ-610 given every 12 h on days 16–18 delayed spontaneous luteolysis, as evidenced by an extended elevation (P = 0.054) of plasma progesterone concentration. In Experiment 3, heifers were administered six intrauterine infusions of BQ-610 or saline on days 16–19, and peripheral blood samples were collected from day 11 to 16 (before infusion), hourly on days 16–19 (during infusion), and on days 20–25 (after infusion). BQ-610 treated heifers had markedly higher (P < 0.0001) levels of plasma progesterone compared with saline controls, and this effect was most notable during the infusion period (treatment by period interaction; P ≤ 0.05). Heifers infused with BQ-610 also had higher progesterone levels on day 21 (treatment by time interaction; P ≤ 0.05). Mean plasma concentrations of 13,14-dihydro-15-keto-PGF₂ (PGFM), the primary metabolite of PGF₂, were measured in the samples collected hourly and were not different (P ≥ 0.05) between treatments. These results indicate that the in vivo antagonism of the ET_A receptor can delay functional luteolysis, and supports the theory that ET-1 regulates luteal function in ruminants.