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.     

Nitric oxide in bovine corpus luteum: Possible mechanisms of action in luteolysis

Although prostaglandin (PG) F_2α is considered as the principal luteolytic factor, its action on the bovine corpus luteum (CL) is mediated by other intraovarian factors. Among them, nitric oxide (NO) seems to play a mandatory role in luteolysis. In this article we review the background and current status of work on possible roles of NO in the CL function, based on available information and our own experimental data. NO is produced in all three main types of bovine CL cells: steroidogenic, endothelial and immune cells. PGF_2α and some luteolytic cytokines (tumor necrosis factor, interferon) increase NO production and stimulate NO synthase expression in the bovine CL. NO inhibits progesterone production, stimulates the secretion of PGF_2α and leukotriene C4, reduces the number of viable luteal cells and, finally, participates in functional luteolysis. NO induces the apoptotic death of CL cells by the modulation of bcl‐2 family gene expression and the stimulation of caspase‐3 gene expression and activity. However, this simple molecule shows both luteolytic and luteotropic actions during the estrous cycle in ruminants. The aim of this overview is to present and discuss the recent findings crucial for understanding NO role in the process of CL regression in cattle.     

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.     

Regulation of uterine function by cytokines in cows: Possible actions of tumor necrosis factor-α, interleukin-1α and interferon-τ

When animals do not become pregnant, regression of the corpus luteum (CL) is essential for normal cyclicity because it allows the development of a new ovulatory follicle. Luteal regression is caused by a pulsatile release of prostaglandin (PG) F_2α from the uterus in the late luteal phase in most mammals including cattle. Although it has been proposed in ruminants that pulsatile PGF_2α secretion is generated by a positive feedback loop between luteal and/or hypophyseal oxytocin and uterine PGF_2α, the bovine endometrium may possess other mechanisms for initiation of luteolytic PGF_2α secretion. There is increasing evidence that several cytokines mainly produced by immune cells modulate CL and uterine function in many species. Tumor necrosis factor‐α (TNF‐α) stimulates PGF_2α output from bovine endometrium not only at the follicular phase but also at the late luteal phase. Administration of TNF‐α at a high concentration prolongs luteal lifespan, whereas administration of a low concentration of TNF‐α accelerates luteal regression in cows. The data obtained from the authors’ previous in vitro and in vivo studies strongly suggest that TNF‐α is a crucial factor in regulating luteolysis in cows. The authors’ recent study has shown that interleukin‐1α mediates PG secretion from bovine endometrium as a local regulator. Furthermore, interferon‐τ (IFN‐τ) suppresses the action of TNF‐α on PGF_2α synthesis by the bovine endometrium in vitro, suggesting that IFN‐τ plays a luteoprotective role by inhibiting TNF‐α‐induced PGF_2α production in early pregnancy. The purpose of the present review is to summarize current understanding of the endocrine mechanisms that regulate uterine function by cytokines during the estrous cycle and early pregnancy in cows.     

Luteolytic Effect of Prostaglandin F2α on Bovine Corpus Luteum Depends on Cell Composition and Contact

Prostaglandin F_2α (PGF_2α) is a main luteolytic factor in vivo; however, its direct luteolytic influence on steroidogenic cells of bovine corpus luteum (CL) is controversial and not fully understood. The aim of the study was to clarify PGF_2α action on bovine CL in different in vivo and in vitro conditions and to examine whether the contact among all main types of CL cells is necessary for luteolytic PGF_2α action. In experiment 1, the bovine CL (day 15 of the oestrous cycle) was perfused using in vivo microdialysis system with dinoprost (an analogue of PGF_2α) for 0.5 h. Dinoprost caused a short‐time increase in progesterone (P4), whose concentration decreased thereafter (at 6‐, 10‐, 12‐ and 24‐h after treatment). In experiment 2, the direct effect of PGF_2α on P4 accumulation in CL steroidogenic cells cultured in monolayer (day 15 of the cycle) was determined. PGF_2α after 24 h of incubation increased P4 accumulation in steroidogenic CL cells. In experiment 3 steroidogenic, endothelial CL and immune cells (day 15 of the cycle) were incubated with PGF_2α in cocultures for 24 h in glass tubes and the levels of P4, stable metabolites of nitric oxide (NO) and leukotriene (LT) C₄ were determined. Although PGF_2α treatment increased P4 secretion in homogeneous steroidogenic CL cell culture, the decrease in P4 secretion in cocultures of all types of CL cells was observed. The secretion of NO and LTC₄ increased after the treatment of PGF_2α both in pure cultures of CL cells and in cocultures. The interactions between endothelial and immune cells with steroidogenic CL cells are needed for luteolytic PGF_2α action within the bovine CL. Our results indicate that the cell coculture model, including the main types of CL cells, is the most approximate to study PGF_2α role in vitro.     

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.     

Leukotrienes Affect Secretory Function of Ovarian Cells In Vitro*

The aim of this study was to determine which cells are the source of production and target for leukotriene (LTs) action within the bovine ovary. Luteal (CL, days 14–16 of the oestrous cycle), steroidogenic cells (LSC) and endothelial cells (LEC) of the bovine corpus luteum (CL), and granulosa cells (GC) were isolated enzymatically, cultured in a monolayer and incubated with LTC₄, LTB₄, Azelastine (an antagonist of LTC₄) or Dapsone (an antagonist of LTB₄). Then cells were collected for determination of mRNA expression for LT receptors (LTRs) and 5‐lipoxygenase (5‐LO) by real time RT‐PCR, and media were collected for determination of prostaglandin (PG)E₂, F_2α, progesterone (P4; LSC only), endothelin‐1 (ET‐1; LEC only) and 17‐β oestradiol (E2; GC only). The greatest mRNA expression for LTR‐II and 5‐LO were found in LEC, whereas LTR‐I mRNA expression did not differ among cell types. The level of PGE₂ increased after LTs treatment in each type of ovarian cell, excluding LTC₄ treatment in LEC. The secretion of PGF_2α was also increased by LTs, but decreased after LTB₄ treatment of LSC. In GC cultures, both LTs stimulated E2 secretion; in LEC cultures, LTB₄ stimulated whereas LTC₄ inhibited P4 secretion; in LEC cultures, LTC₄ stimulated but LTB₄ inhibited ET‐1 secretion. The results show that LTs are produced locally and are involved in PGs production/secretion in all examined cells (LSC, LEC and GC) of bovine ovary. Leukotriene treatment modulate secretion of E2, by GC, P4 by LSC and ET‐1 by LEC, which indicates that LTs are involved in regulation of ovarian secretory functions.     

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.     

Leukotrienes are Auto‐/Paracrine Factors in the Bovine Corpus Luteum: An In Vitro Study

The aim of this study was to determine leukotrienes (LTs) functions in the bovine corpus luteum (BCL) during the oestrous cycle. In steroidogenic CL cells we examined the effect of luteotropic [LH, prostaglandin E₂ (PGE₂)] and luteolytic (PGF_2α, cytokines) factors on: the levels of LTB₄ and C₄, the expression of 5‐lipoxygenase (LO), LT receptors type I (LTR‐I) and LTR‐II, and the effects of LTB₄ and C₄ stimulations on the levels of progesterone (P4), PGE₂, F_2α and nitric oxide (NO) metabolites. Both luteolytic and luteotropic factors stimulated 5‐LO expression on days 2–4 and 17–19 of the cycle. Leukotriene receptors type I expression increased after PGE₂ and tumour necrosis factor α with interferon γ (TNF/IFN) stimulation on days 2–4 of the cycle. Leukotriene receptor type II expression increased after PGE_2α and TNF/IFN stimulation on days 2–4 and 17–19 of the cycle, and LTR‐II expression on days 8–10 of the cycle was unchanged after cell stimulation with any factor. Leukotriene B₄ level increased after BSC incubation with luteotropic factors during all examined days of the cycle and after cytokine stimulation at early‐ and mid‐luteal stages, whereas luteolytic factors stimulated LTC₄ secretion over the entire cycle. Leukotriene B₄ stimulated P4 secretion at the mid‐luteal stage and stimulated NO secretion during all examined phases. Leukotriene B₄ stimulated PGE₂ secretion at the early‐ and mid‐luteal stage. Leukotriene C₄ inhibited P4 secretion at the mid‐ and regressing‐luteal stage, stimulated NO (entire cycle) and PGF_2α at mid‐ and regressing‐luteal phases. Leukotrienes modulate steroidogenic cells functions, depending on the stage of the cycle. Leukotriene B₄ plays a luteotropic role stimulating P4 and PGE₂ secretions; LTC₄ stimulates the secretion of luteolytic factors and enhances the luteolytic cascade within BCL.     

Expression of Aldo-keto Reductase 1C23 in the Equine Corpus Luteum in Different Luteal Phases

Regression of the corpus luteum (CL) is characterized by a decay in progesterone (P₄) production (functional luteolysis) and disappearance of luteal tissues (structural luteolysis). In mares, structural luteolysis is thought to be caused by apoptosis of luteal cells, but functional luteolysis is poorly understood. 20α-hydroxysteroid dehydrogenase (20α-HSD) catabolizes P₄ into its biologically inactive form, 20α-hydroxyprogesterone (20α-OHP). In mares, aldo-keto reductase (AKR) 1C23, which is a member of the AKR superfamily, has 20α-HSD activity. To clarify whether AKR1C23 is associated with functional luteolysis in mares, we investigated the expression of AKR1C23 in the CL in different luteal phases. The luteal P₄ concentration and levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA were higher in the mid luteal phase than in the late and regressed luteal phases (P<0.05), but the level of 3β-HSD protein was higher in the late luteal phase than in the regressed luteal phase (P<0.05). The luteal 20α-OHP concentration and the level of AKR1C23 mRNA were higher in the late luteal phase than in the early and mid luteal phases (P<0.05), and the level of AKR1C23 protein was also highest in the late luteal phase. Taken together, these findings suggest that metabolism of P₄ by AKR1C23 is one of the processes contributing to functional luteolysis in mares.     

The Role of the Endometrium in Endocrine Regulation of the Animal Oestrous Cycle

A critical analysis of the results of research in the function of the endometrium was carried out and a view point presented. The role of the endometrium in endocrine regulation of the oestrus cycle can be summarized as follows: 1. The transfer of prostaglandin F_2α (PGF_2α) from the uterus to an ovary, which causes luteolysis, occurs mainly via the lymphatic pathways. 2. The system of retrograde transfer of PGs enables PGF_2α and PGE₂ to reach the myometrium and endometrium with arterial blood at high concentration. In the luteal phase, PGF_2α, together with the increasing concentration of progesterone, constricts the arterial vessels of the uterus; in the follicular phase and in early pregnancy, PGE₂ together with oestrogen and embryonic signals, relaxes the arterial vessels. In addition, this system protects the corpus luteum from premature luteolysis during the cycle and luteolysis during early pregnancy. 3. In days 10–12 of the cycle, the blood flow in the uterus decreases by 60–70% in pigs and around 90% in sheep. This causes ischaemia and local hypoxia confirmed by the presence of hypoxia inducible factor and thus remodelling of the endometrium commences. 4. The pulsatile elevations in PGF_2α concentration occurring in the blood flowing out of the uterus during the period of luteolysis and the next few days, do not result from increased PGF_2α synthesis as suggested in numerous studies. They are the effect of excretion of PGF_2α and its metabolites together with lymph and venous blood and tissue fluids in which prostaglandin accumulates.     

Local regulation of corpus luteum development and regression in the cow: Impact of angiogenic and vasoactive factors

The corpus luteum (CL) of the estrous cycle in the cow is a dynamic organ which has a life time of approximately 17-18 days. The main function of the CL is to secrete a large amount of progesterone (P) thereby supporting the achievement of pregnancy. As the CL matures, the steroidogenic cells establish contact with many capillaries and the matured CL is composed of many vascular endothelial cells that account for up to 50% of all CL cells. The bovine CL produces several major angiogenic and vasoactive foctors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietin-1 and -2 (ANPT-1 and -2), prostaglandin F_2α (PGF_2α), endothelin-1 (EDN1), angiotensin II (Ang II) and nitric oxide (NO). These factors regulate P secretion directly and/or indirectly within the CL. Moreover, different actions of PGF_2α in the early cycle CL (non-luteolytic) and the mid cycle CL (luteolytic) may provide insight into the luteolysis cascade in the cow. The aim of the present review is to describe the current concepts of the local mechanisms for the cascade of development and regression of the bovine CL as regulated by luteal angiogenic and vasoactive factors.     

Hormonal control of oestrous cycle in the ewe (a review)Contrôle hormonal de l'oestrus chez la brebis: revueHormonelle Kontrolle des Brunstzyklus beim Mutterschaf

During the cycle, the secretion of progesterone by the corpus luteum inhibits the positive feedback of oestrogens and thus prevents the LH discharge, and also primes the central nervous system for oestrous behaviour. Prostaglandin F_2α has been identified as the hormone produced by the uterus which causes luteal regression. The LH discharge leading to ovulation follows the demise of the CL. None of the characteristics of the LH surge (duration, maximum level, total release) can be related to ovulation rate. However, the interval from onset of oestrus to the beginning of the LH discharge is greater in highly prolific breeds than in less prolific ones.The knowledge of these physiological processes leading to oestrus and ovulation makes possible the control of ovarian activity in the ewe. In cyclic females, the control of the timing of the LH discharge and ovulation can be obtained either by inducing luteolysis with PGF_2α or its synthetic analogues after day 4–5 of the cycle, or by artificially lengthening the luteal phase with exogenous progesterone or progestagens.During the seasonal and post-partum anoestrus, PGF_2α is ineffective and progesterone or progestagens alone are generally unable to induce oestrus and ovulation. Addition at the end of progestagen treatment of inducers of follicular growth and LH release is necessary. Both PMSG and synthetic GnRH are used for this purpose.     

The Effect of Dose and Type of Cloprostenol on the Luteolytic Response of Dairy Cattle during the Ovsynch Protocol under Different Oestrous Cycle and Physiological Characteristics

The objective of this study was to characterize the effect of dose and type of cloprostenol (CLO) on the luteolytic response of dairy cattle during the Ovsynch protocol under different oestrus cycle and physiological characteristics. Twelve non‐lactating dairy cows and 111 lactating dairy cows were used in three experiments. In Experiment I, cows were synchronized so that they had only a 5.5‐ to 6‐day‐old corpus luteum (CL) at the time of the prostaglandin F_2α (PGF_2α) treatment of Ovsynch. In Experiment II, cows were synchronized so that they had at least a CL of approximately 14 days old at the time of PGF_2α treatment and an accessory CL if they had responded to the first GnRH of Ovsynch. Furthermore, in each experiment, cows received either a standard or a double dose of d‐CLO as the luteolytic treatment. In Experiment III, lactating cows were blocked by parity and assigned to one of three luteolytic treatments during Ovsynch: 500 μg d,l‐CLO, 150 or 300 μg of d‐CLO. In Experiment I, the dose of d‐CLO had an effect (p = 0.08) on the percentage of cows with full luteolysis, but not in Experiment II (p > 0.1). More cows in Experiment II had full luteolysis than did cows of Experiment I (87% vs 58%, respectively; p = 0.007). In Experiment III, 87.1%, 84.4% and 86.2% lactating dairy cows had full luteolysis and 37.8%, 36.8% and 36.1% of cows became pregnant after treatment with 500 μg d,l‐CLO, 150 or 300 μg of d‐CLO, respectively (p > 0.05).     

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.     

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.     

Influence of prostaglandin F2α analogues on the secretory function of bovine luteal cells and ovarian arterial contractility in vitro

Although prostaglandin (PG) F_2α analogues are routinely used for oestrus synchronisation in cattle, their effects on the function of the bovine corpus luteum (CL), and on ovarian arterial contractility, may not reflect the physiological effects of endogenous PGF_2α. In the first of two related experiments, the effects of different analogues of PGF_2α (aPGF_2α) on the secretory function and apoptosis of cultured bovine cells of the CL were assessed. Enzymatically-isolated bovine luteal cells (from between days 8 and 12 of the oestrous cycle), were stimulated for 24 h with naturally-occurring PGF_2α or aPGF_2α (dinoprost, cloprostenol or luprostiol). Secretion of progesterone (P4) was determined and cellular [Ca²⁺]_i mobilisation, as well as cell viability and apoptosis were measured.Naturally-occurring PGF_2α and dinoprost stimulated P4 secretion (P < 0.05), whereas cloprostenol and luprostiol did not influence P4 synthesis. The greatest cytotoxic and pro-apoptotic effects were observed in the luprostiol-treated cells, at 37.3% and 202%, respectively (P < 0.001). The greatest effect on [Ca²⁺]_i mobilisation in luteal cells was observed post-luprostiol treatment (200%; P < 0.001).In a second experiment, the influence of naturally-occurring PGF_2α and aPGF_2α on ovarian arterial contraction in vitro, were examined. No differences in the effects of dinoprost or naturally-occurring PGF_2α were found across the studied parameters. The effects of cloprostenol and luprostiol on luteal cell death, in addition to their effects on ovarian arterial contractility, were much greater than those produced by treatment with naturally-occurring PGF_2α.     

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.     

Follicular dynamics and changes in oestradiol‐17β, progesterone and LH profiles following PGF2α induced oestrus in early and late ovulating Beetal goats

This study compares the factors associated with variable interval to oestrus and ovulation between early versus late ovulating goats following PGF_2α administration. The time of ovulation in Beetal goats (n = 38) was monitored through transrectal ultrasound at every 6 hr following a single dose of PGF_2α (experiment 1). Variations in oestrus and ovulation times were further explored through the changes in follicular dynamics, endocrine profiles and behaviour in another set of goats (n = 13) following single PGF_2α given randomly during the luteal phase (experiment 2). The ovulation time varied between 60 and 96 hr, and 57% of ovulations occurred by 72 hr following PGF_2α (experiment 1). Accordingly, the goats (n = 13) in the second experiment were retrospectively divided either into early and/or late ovulating, that is, ≤72 and/or ≥84 hr following PGF_2α. The onset of oestrus, peak estradiol‐17β concentration and LH surge after PGF_2α was first observed in early than late ovulating goats (p < 0.05). The goats ovulating early had larger follicle and smaller CL in diameter at the time of PGF_2α administration than those ovulating late (5.4 ± 0.2 vs. 4.3 ± 0.2 mm and 10 ± 0.6 vs. 11.8 ± 0.3 mm, respectively; p < 0.05). Likewise, plasma progesterone concentration tended to be lower (p = 0.087) in early than late ovulating goats. In conclusion, the size of dominant follicle and CL at the time of PGF_2a determines the interval to ovulation following a single dose of PGF_2a during the luteal phase.