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.     

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.     

The Corpus Luteum of the Dog: Source and Target of Steroid Hormones?

Aim of this paper is to review our present understanding on the endocrine control of luteal function in the bitch and to add some new data generated in our laboratories in support of the hypothesis of a paracrine/autocrine role of corpus luteum (CL) derived steroid hormones. Luteal lifespan in non-pregnant dogs often exceeds that of pregnant dogs, where luteal regression terminates in a rapid luteolysis, immediately prior to parturition. In non-pregnant dogs, luteal regression occurs independently of a uterine luteolysin and in spite of increased gonadotropic support during the last third of dioestrus. The CL is the only source of progesterone (P(4)) maintaining pregnancy, and they have the capacity to synthesize oestrogens as substantiated by expression of the CYP19 (aromatase) gene observed in this study. Our data demonstrated that lutein and non-lutein cells of the canine CL express in a rather constant manner the progesterone receptor (PR) and the oestrogen receptor, classifying them as targets for an autocrine/paracrine activity of CL-derived steroids. Therefore, a functional role of P(4) within a positive loop feedback system, including StAR and 3β-hydroxysteroid dehydrogenase, has been postulated.     

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.     

Regulation of Corpus Luteum Function in Cattle – an Overview

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

Effect of exogenous ovine placental lactogen on basal and prostaglandin-stimulated progesterone production by porcine luteal cells

The ability of ovine placental lactogen (oPL) to stimulate progesterone secretion of porcine luteal cells isolated from ovaries in different stages of the oestrous cycle and to support the luteotropic action of PGE2 or to protect the corpus luteum (CL) against the luteolytic action of PGF2 alpha was investigated. oPL in all doses used had no effect on progesterone production of cells isolated from early developing corpora lutea while in doses of 1 and 10 ng/ml it increased oestradiol secretion by this type of cells. In doses of 1, 10 and 100 ng/ml it also increased progesterone secretion of cells isolated from mature corpora lutea in a dose-dependent manner. No influence on progesterone production of cells isolated from regressing corpora lutea was observed. oPL added to the culture media had no effect on PGE2-stimulated progesterone production by cells isolated from mature corpora lutea. However, it exerted a protective effect against the luteolytic action of PGF2 alpha observed in cultures treated with PGF2 alpha alone or in combination with PGE2 in a ratio of 4:1. These studies provide evidence that oPL is luteotropic and supports progesterone production in swine. The fact that oPL acted directly on ovarian steroidogenesis suggests that it may also play some role under non-pregnant physiological conditions. Future studies of structural and functional proteins secreted by the porcine conceptus will help resolve this uncertainty.     

Steroids as local regulators of ovarian activity in domestic animals

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

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.     

Ovarian function in ruminants

The purpose of this overview is to highlight important steps of ovarian regulation during follicle development, ovulation and the life span of corpus luteum (CL) in ruminants. The ovarian cycle is central to reproductive function. It is characterized by repeating patterns of cellular proliferation, differentiation and transformation that encompass follicular development and ovulation as well as the formation, function and regression of the CL. In the first part, the importance and regulation of final follicle growth and especially of angiogenesis and blood flow during folliculogenesis, dominant follicle development and CL formation are described. Our results underline the importance of growth factors especially of insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) for development and completion of a dense network of capillaries (angiogenesis) during follicle growth and CL formation. In the second part, the regulation of CL function by endocrine/paracrine and autocrine acting regulators is discussed. There is evidence that besides the main endocrine hormones luteinizing hormone (LH) and growth hormone (GH) local regulators as growth factors, peptides, steroids and prostaglandins are important modulators of luteal function. During early CL development until midluteal stage oxytocin (OT), prostaglandins and progesterone (P) itself stimulate luteal cell proliferation and function supported by the luteotropic action of a number of growth factors. The still high mRNA expression, protein concentration and localization of VEGF, FGF and IGF family members in the cytoplasm of luteal cells during midluteal stage suggest that they play pivotal role in the maintenance (survival) of this endocrine tissue. The major function of the CL is to secrete P. Progesterone itself regulates the length of the estrous cycle via influencing the timing of the luteolytic PGF2alpha signal from the endometrium. At the end of a nonfertile cycle, the regression of CL commences, steroidogenic capacity is lost (functional luteolysis), cell death is initiated, and tissue involution as well as resorption occurs within a few days (structural luteolysis). The cascade of mediators during luteolysis is very complex and still awaits elucidation. Evidence is given for participation of blood flow, inflammatory cytokines, vasoactive peptides (angiotensin II and endothelin-1), and decrease of the classical luteotropic mediators.     

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.     

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.     

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.     

Hysterectomy during the luteal period in dogs chronically treated with bromocriptine

To determine whether prolactin has luteolytic properties during the first part of the luteal period, hysterectomy was performed in four dogs, in which prolactin had been chronically suppressed by bromocriptine administration. The concentration of progesterone in the peripheral blood decreased upon hysterectomy during the first part of the luteal phase and regained normal values after about seven days. The progesterone patterns during the perisurgical period in these dogs were similar to those patterns observed in dogs hysterectomised without bromocriptine treatment. It is concluded therefore that, in the dog, luteolytic properties can not be attributed to prolactin.     

Cell‐specific Distribution of Progesterone Receptors in the Bovine Ovary

This study describes the localization of progesterone receptors (PR) in the bovine ovary. Ovaries were obtained from 11 non‐pregnant and two pregnant cows. Progesterone receptors were visualized by immunohistochemistry on paraffin sections. Nuclear staining for PR was observed in cells of the follicles, corpora lutea, theca layers, surface epithelium, tunica albuginea, and in superficial and deep stroma cells. No staining was noticed in apoptotic bodies of atretic follicles. Expression of PR in follicle cells indicates an intrafollicular role of progesterone. The higher expression in thecal cells compared with follicle cells indicates that thecal cells mediate some effects of progesterone on the follicular development. Superficial stroma cells showing high expression might have a similar influence on primordial and primary follicles. In general, luteal cells had a lower expression than follicle cells, which may be explained by the down‐regulatory effect of locally produced progesterone. The lower expression in luteal cells during pregnancy can be due to the longer life span of this corpus luteum and concomitant degeneration of its PR. The high and rather constant expression of PR in cells of the surface epithelium remains to be elucidated.     

Chemoattractant properties of conditioned medium from equine corpora lutea collected at various stages of the oestrous cycle

This study investigated the chemotactic activity of equine CL at different stages of the oestrous cycle. The purpose of this was to ascertain whether luteal tissue itself contributes to the massive influx of leucocytes around the time of natural and induced luteal regression. Corpora lutea were collected at different stages of dioestrus and after treatment with PGF2alpha. Culture medium harvested after incubation of luteal tissue for 20 h was chemotactic for both polymorphonuclear and mononuclear cells in late dioestrus (before functional regression) as well as after natural and induced luteal regression. By contrast, midluteal tissue showed no chemotactic activity. This is the first report of the ability of equine luteal tissue actively to recruit inflammatory cells in vitro and supports our earlier findings that this infiltration starts prior to functional luteolysis. We hypothesise that this early influx of inflammatory cells may play an active role in luteal regression. Further research is needed to identify the specific chemotactic factor(s).     

Hysterectomy during the luteal period in dogs chronically treated with bromocriptine

Summary

To determine whether prolactin has luteolytic properties during the first part of the luteal period, hysterectomy was performed in four dogs, in which prolactin had been chronically suppressed by bromocriptine administration. The concentration of progesterone in the peripheral blood decreased upon hysterectomy during the first part of the luteal phase and regained normal values after about seven days. The progesterone patterns during the perisurgical period in these dogs were similar to those patterns observed in dogs hysterectomised without bromocriptine treatment. It is concluded therefore that, in the dog, luteolytic properties can not be attributed to prolactin.     

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.     

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.     

Distribution of estrogen receptor alpha in the dominant follicles and corpus luteum at the three stages of estrous cycle in Japanese black cows

Distribution of estrogen receptor alpha (ERalpha) in the dominant follicle (DF) and corpus luteum (CL) at the three stages of estrous cycle in Japanese Black cows was evaluated by means of immunohistochemistry. Ovarian dynamics were observed twice daily using ultrasonography until the ovariectomy performed on Day 7 (First group, n=3), Day 10 (Second group, n=3) and Day 18 (Third group, n=3) (Day 0=estrus). Expression of ERalpha represented by immunohistological staining intensity in cells was determined using a light microscope equipped with a digital camera. A tendency toward higher expression were observed in theca interna (TI) of DF when compared with those in mural granulosa cells (mGC), antral granulosa cells (aGC) and theca externa (TE). ERalpha expression in the Third group was lower than that in the First Group in mGC, and it was also lower than that in the second group in TE (P<0.05). ERalpha expression in luteal cells was higher than those in the stromal cells in CL. No significant difference of ERalpha expression was observed within luteal or stromal cells, except in the Second group in the luteal cells, in which significantly higher expressions than that in the Third group (P<0.05) were observed. The results showed that, 1) ERalpha was present in developing DF on Day 7, early regressing DF on Day 10 and preovulatory DF on Day 18, especially in the TI, and a few were localized in the mGC, and 2) ERalpha was highly expressed in the luteal cells and the expression decreased in combination with regression of CL.     

Progesterone stimulation by LH involves the phospholipase-C pathway in bovine luteal cells

Luteinizing hormone (LH)-stimulated steroidogenesis in luteal cells is known to be mediated through the activation of cyclic AMP (cAMP)-dependent protein kinase, and to be also modulated by calcium-dependent mechanisms. In the present study, we tested the hypothesis that LH stimulates progesterone (P4) production in bovine luteal cells through activation of phospholipase (PL) C by using a cell culture system. Bovine mid-luteal cells (Days 8-12 of the estrous cycle) were cultured for 24 h and then exposed to a PLC inhibitor (U-73122; 10 microM) with or without LH (10 ng/ml) for 4 h. U-73122 blocked LH-stimulated P4 production without affecting cAMP accumulation. Moreover, exposure of luteal cells to PLC increased P4 production in a dose-dependent manner. These results support the hypothesis that the luteotropic action of LH in bovine luteal cells is mediated not only by activation of adenylate cyclase but also by activation of PLC.