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

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

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.     

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.     

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.     

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.     

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.     

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).     

Nitric oxide induces apoptosis in bovine luteal cells

We previously showed in in vivo and in vitro studies that nitric oxide (NO) is engaged in luteolysis in cattle. Nitric oxide produced locally in the bovine corpus luteum (CL) inhibits progesterone (P4) synthesis and is suggested to be a component of the luteolytic cascade induced by uterine prostaglandin (PG) F2alpha. In the present study, the molecular mechanisms of NO action during structural luteolysis were studied in cultured bovine luteal cells (Days 15-17 of the estrous cycle). The effects of the NO donor (NONOate; 10(-4)M) on DNA fragmentation, cell viability, P4 production and caspase-3 activity were compared with those of PGF2alpha (10(-6)M). Moreover, mobilization of intracellular calcium [Ca2+]i and gene expressions of Fas-L, Fas, bcl-2, bax, and caspase-3 in the cells were determined by semi-quantitative RT-PCR after NONOate treatment. Caspase-3 activity was examined calorimetrically. Contrary to PGF2alpha NONOate decreased cell viability. DNA fragmentation after NONOate treatment increased by more than with PGF22alpha. NONOate increased mobilization of [Ca2+]i in the cells. Although the NO donor did not affect Fas-L and bcl-2 gene expression, it stimulated Fas and bax mRNA and caspase-3 expression. The ratio of bcl-2 to bax mRNA level decreased in the cells treated with NONOate. Moreover, NONOate stimulated caspase-3 activity more effectively than PGF2alpha. The overall results suggest that NO is a luteolytic factor that plays a crucial role in regulation of the estrous cycle in structural luteolysis by inducing apoptosis of luteal cells in cattle.     

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.     

IFN‐τ Acts in a Dose‐Dependent Manner on Prostaglandin Production by Buffalo Endometrial Stromal Cells Cultured in vitro

Interferon‐τ (IFN‐τ) has been recognized as the primary embryonic signal responsible for maternal recognition of pregnancy. Uterine endometrium produces both prostaglandin F_2α (PGF_2α) and prostaglandin E₂ (PGE₂). PGF_2α is responsible for the luteolysis; however, PGE₂ favours establishment of pregnancy by its luteoprotective action. In this study, the dose‐response effect of recombinant bovine IFN‐τ (rbIFN‐τ) on prostaglandin (PG) production by buffalo endometrial stromal cells cultured in vitro was studied. Buffalo endometrial stromal cells were isolated by double enzymatic digestion, initially with trypsin III followed by a cocktail of trypsin III, collagenase type II and DNase I and subsequently cultured till confluence. Further, cells were treated with different doses of rbIFN‐τ (0.001, 0.01, 0.1, 1.0 and 10 μg/ml) and keeping a separate set of control. Culture supernatant was collected after 6, 12 and 24 h of treatment. PG levels in the culture supernatant were measured by enzyme immune assay (EIA) and total cellular protein estimated by Bradford method. Results indicated that buffalo endometrial stromal cells following rbIFN‐τ treatment enhanced the secretion of both PGE₂ and PGF_2α, and also its ratio in a strict dose‐dependent manner with a significant increase (p < 0.01) in PGE₂ production at 1 μg/ml dose of rbIFN‐τ and maximal stimulation for both PG was observed at 10 μg/ml. Further, both PG production and its ratio were increased significantly (p < 0.01) in a time‐dependent fashion in all the groups at 6, 12 and 24 h post‐treatment with highest level achieved at 24 h as compared with control. Absolute levels of PGE₂ remained higher than PGF_2α indicating PGE₂ as the major PG produced by endometrial stromal cells. The dose‐dependent response of rbIFN‐τ signifies the importance of optimum concentration of IFN‐τ for the embryonic development especially during the critical period to establish successful pregnancy.     

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.     

Current knowledge about the processes of luteolysis and maternal recognition of pregnancy in camelids

Camelids have many unique reproductive features that considerably differ from those of other domestic species. Females are induced ovulators with subsequent development of a corpus luteum (CL) with a short lifespan. Plasma progesterone concentration starts to increase on day 4, peaks on day 8–9 and, in non-pregnant animals, basal concentration is reached around day 10–11 post-induction of ovulation. Luteolytic pulses of prostaglandin F_2α (PGF_2α) are firstly detected on day 7 or 8 (approximately on day 5–6 after ovulation), with maximal luteolytic peaks observed between days 9 and 11 post-mating, in coincidence with a high endometrial expression of cyclooxygenase 2, a limiting enzyme in prostaglandins synthesis. Unlike other species, oxytocin seems not to be involved in the luteolytic process in these species. The CL is the main source of progesterone secretion, and its function is required to support pregnancy. Despite constant research efforts, aspects of reproduction and maternal recognition of pregnancy in camelids remain not fully understood. A transient decrease and subsequent recovery in plasma progesterone concentration are observed after day 9 post-mating in pregnant animals in association with a pulsatile release of PGF_2α and a transitory decrease in CL vascularization. Thus, embryo recognition should occur between days 8 and 12 post-mating. In camels, conceptus tissues exhibit aromatizing activity with the capacity to synthesize large amounts of oestradiol. Similarly, llama blastocysts secrete oestradiol-17β during the preimplantation stage, with a higher production during the elongation period. An increase in the endometrial expression of oestrogen receptor α is also observed on day 12 post-mating. All these evidences suggest that oestrogen could be the signal released by the embryo at the time of its recognition in camelids. Besides, nearly 98% of pregnancies are carried out in the left horn. A decrease in the endometrial expression of mucin 1 and 16 genes has been reported, suggesting that these changes are crucial for successful embryo implantation; however, no differences have been observed between horns. Thus, maternal recognition of pregnancy in camelids is a particularly complex process that must occur in a concise time to allow the rescue of the CL and embryo survival.     

Effects of Flunixin Meglumine and Prostaglandin F2α Treatments on the Development and Quality of Bovine Embryos In Vitro

Assisted reproduction procedures, such as embryo transfer (ET) and artificial insemination (AI), in cattle could induce the secretion of prostaglandin F₂‐alpha (PGF₂α) from uterine horns which may in turn interrupt embryo development and implantation. This study investigated the effect of flunixin meglumine (FM), prostaglandin F2 alpha (PGF2α) and FM combined with PGF2α supplementation in culture medium (IVC‐II) on the development and quality of in vitro produced bovine embryos. The development rate of embryos was significantly higher in the FM group (33.3%) than in control (24.3%), PGF₂α (23.9%) and FM + PGF₂α groups (24.5%). The percentage of hatched blastocysts was also higher (p < 0.05) in the FM group (41.2%) than in the control (27.8%) and PGF₂α groups (19.8%). While, there was no significant difference in total cell number in all experimental groups, the number of apoptotic cells was significantly higher in the PGF₂α group (8.2 ± 6.6) than in the control (4.7 ± 3.2), FM (4.7 ± 2.5) and FM + PGF₂α (4.9 ± 3.4) groups. Detected by real‐time PCR, secreted vesicle seminal protein 1 (SSLP1) and prostaglandin G/H synthase 2 (PTGS2) gene expression decreased (p < 0.05) in the PGF₂α group. However, SSLP1 and PTGS2 gene expression in the FM + PGF₂α group returned to their baseline levels, similar to the control and FM groups. Caspase 3 (CAPS3) gene expression increased in the PGF₂α group compared with other groups (p < 0.05). In conclusion, addition of FM in vitro culture significantly improved embryo development as well as alleviated the negative impact of PGF₂α.     

Factors Affecting Synchronization and Conception Rate after the Ovsynch Protocol in Lactating Holstein Cows

Objectives were to evaluate risk factors affecting ovulatory responses and conception rate to the Ovsynch protocol. Holstein cows, 466, were submitted to the Ovsynch protocol [day 0, GnRH‐1; day 7, prostaglandin (PG) F_2α; day 9, GnRH‐2] and 103 cows were inseminated 12 h after GnRH‐2. Information on parity, days in milk at GnRH‐1, body condition, milk yield, exposure to heat stress, pre‐synchronization with PGF_2α and the use of progesterone insert from GnRH‐1 to PGF_2α was collected. Ovaries were scanned to determine responses to treatments. Overall, 54.7%, 10.6%, 2.2%, 81.1%, 9.0%, 91.5% and 36.9% of the cows ovulated to GnRH‐1, multiple ovulated to GnRH‐1, ovulated before GnRH‐2, ovulated to GnRH‐2, multiple ovulated to GnRH‐2, experienced corpus luteum (CL) regression and conceived, respectively. Ovulation to GnRH‐1 was greater in cows without a CL at GnRH‐1, cows with follicles >19 mm and cows not pre‐synchronized with PGF_2α 14 days before GnRH‐1. Multiple ovulations to GnRH‐1 increased in cows without CL at GnRH‐1 and cows with follicles ≤19 mm at GnRH‐1. Ovulation before GnRH‐2 was greater in cows without CL at PGF_2α. Ovulation to GnRH‐2 increased in cows that received a progesterone insert, cows with a CL at GnRH‐1, cows with follicles not regressing from the PGF_2α to GnRH‐2, cows with larger follicles at GnRH‐2, cows that ovulated to GnRH‐1 and cows not pre‐synchronized. Multiple ovulations after GnRH‐2 increased in cows with no CL at GnRH‐1, multiparous cows and cows that multiple ovulated to GnRH‐1. Conception rate at 42 days after AI increased in cows with body condition score > 2.75 and cows that ovulated to GnRH‐2. Strategies that optimize ovulation to GnRH‐2, such as increased ovulation to GnRH‐1, should improve response to the Ovsynch protocol.     

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.     

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.     

Presence of multiple corpora lutea affects the luteolytic response to prostaglandin F2α in lactating dairy cows

Since the 1970s, luteolytic doses used for synchronizing estrus in dairy cattle have remained unchanged. This study aimed to evaluate the dose-response effect of prostaglandin F_2α (PGF_2α), which is used for synchronizing estrus, and subsequent fertility in cows with two or more corpora lutea (CL). The study population consisted of 1,683 cows with a single CL (1CL), 501 cows with multiple CL receiving a single dose of PGF_2α (2CL1), and 252 cows with multiple CL receiving a 1.5 × PGF_2α dose (2CL1.5). Cows with a single CL (n = 1,245) showed estrus significantly (P < 0.01) earlier (3.01 ± 1.23 days; mean ± SD) than cows with multiple CL (n = 287; 3.33 ± 1.69 days). Using 1CL cows as reference, the odds ratio (OR) for the estrus response in 2CL1 cows was 0.13 (P < 0.0001), whereas the ORs for estrus response and pregnancy of 2CL1.5 cows were 1.8 (P = 0.0001) and 1.7 (P = 0.001), respectively. Based on the results for only the 2CL1 cows, the OR for the estrus response was 0.7 (P = 0.01) for cows producing ≥ 45 kg of milk at treatment, compared to the remaining cows producing < 45 kg of milk. Our results showed that the presence of multiple CL reduced the estrus response to that induced by a single PGF_2α dose and milk production was inversely associated with this response, whereas an increased PGF_2α dose improved the estrus response. Therefore, an increase in the standard PGF_2α dose is recommended.