Growth and cellular proliferation of pig corpora lutea throughout the oestrous cycle

Corpora lutea were obtained from gilts on days 2, 4, 8, 12, 15 or 18 after oestrus. Luteal fresh masses and DNA contents increased linearly (P < 0.01) from day 2 to day 12 and day 2 to day 15, respectively. Changes in the ratio of protein:DNA were greatest between days 2 and 4 and days 15 and 18, whereas changes in DNA content were relatively small during the same intervals. Thus, a major component of changes in the size of the corpus luteum during the early and late periods of the luteal phase was cellular hypertrophy. Proliferation of luteal cells in vivo (nuclear incorporation of 5-bromo-2-deoxyuridine, a thymidine analogue) was greatest on day 2 and decreased exponentially (P < 0.01) throughout the oestrous cycle. Results from co-localization of 5-bromo-2-deoxyuridine and factor VIII (von Willebrand factor), a marker of endothelial cells, or 5-bromo-2-deoxyuridine and 3 beta-hydroxysteroid dehydrogenase, a marker of steroidogenic cells, indicated that some of the luteal steroidogenic cells proliferated early in luteal development. However, during early and mid-cycle, most of the luteal cell proliferation occurred in the endothelial cells. Thus, during growth of the pig corpus luteum, which is extremely rapid, most of the proliferating luteal cells are vascular endothelial cells. This observation is consistent with the high vascularity and blood flow of the mature corpus luteum and implies a critical role for angiogenesis in luteal development in the pig, as has been proposed for several other mammalian species.     

Angiogenesis in the corpus luteum

The corpus luteum (CL) is a site of intense angiogenesis. Within a short period, this is followed either by controlled regression of the microvascular tree in the non-fertile cycle, or maintenance and stabilisation of the new vasculature a conceptual cycle. The molecular regulation of these diverse aspects is examined. The CL provides a unique model system in which to study the cellular and molecular regulation of angiogenesis. Vascular endothelial growth factor (VEGF) has been found to have a major role in the CL. By targeting its action at specific stages of the luteal phase in vivo by antagonists, profound inhibitory effects on luteal angiogenesis and function are observed.     

The expression of prolactin and its cathepsin D-mediated cleavage in the bovine corpus luteum vary with the estrous cycle

In the corpus luteum (CL), blood vessels develop, stabilize, and regress. This process depends on the ratio of pro- and antiangiogenic factors, which change during the ovarian cycle. The present study focuses on the possible roles of 23,000 (23K) prolactin (PRL) in the bovine CL and its antiangiogenic NH(2)-terminal fragments after extracellular cleavage by cathepsin D (Cath D). PRL RNA and protein were demonstrated in the CL tissue, in luteal endothelial cells, and in steroidogenic cells. Cath D was detected in CL tissue, cell extracts, and corresponding cell supernatants. In the intact CL, 23K PRL levels decreased gradually, whereas Cath D levels concomitantly increased between early and late luteal stages. In vitro, PRL cleavage occurred in the presence of acidified homogenates of CL tissue, cells, and corresponding cell supernatants. Similar fragments were obtained with purified Cath D, and their appearance was inhibited by pepstatin A. The aspartic protease specific substrate MOCAc-GKPILF~FRLK(Dnp)-D-R-NH(2) was cleaved by CL cell supernatants, providing further evidence for Cath D activity. The 16,000 PRL inhibited proliferation of luteal endothelial cells accompanied by an increase in cleaved caspase-3. In conclusion, 1) the bovine CL is able to produce PRL and to process it into antiangiogenic fragments by Cath D activity and 2) PRL cleavage might mediate angioregression during luteolysis.     

Gelatinases, endonuclease and Vascular Endothelial Growth Factor during development and regression of swine luteal tissue

Background  

The development and regression of corpus luteum (CL) is characterized by an intense angiogenesis and angioregression accompanied by luteal tissue and extracellular matrix (ECM) remodelling. Vascular Endothelial Growth Factor (VEGF) is the main regulator of angiogenesis, promoting endothelial cell mitosis and differentiation. After the formation of neovascular tubes, the remodelling of ECM is essential for the correct development of CL, particularly by the action of specific class of proteolytic enzymes known as matrix metalloproteinases (MMPs). During luteal regression, characterized by an apoptotic process and successively by an intense ECM and luteal degradation, the activation of Ca⁺⁺/Mg⁺⁺-dependent endonucleases and MMPs activity are required. The levels of expression and activity of VEGF, MMP-2 and -9, and Ca⁺⁺/Mg⁺⁺-dependent endonucleases throughout the oestrous cycle and at pregnancy were analyzed.     

Ever-changing cell interactions during the life span of the corpus luteum: Relevance to luteal regression

The corpus luteum (CL) undergoes dramatic morphological and functional changes throughout its lifespan. It initially develops from cells that remain in the follicle following ovulation. Eventually the mature CL is composed of multiple, distinctive cell types including steroidogenic cells (small and large luteal cells) and other cell types (endothelial cells, pericytes, fibroblasts, and immune cells). Robust angiogenesis accompanies CL formation, establishing an elaborate blood vessel network at mid cycle. In the absence of embryonic signals, the CL will regress in a process triggered by prostaglandin F2α (PG). Luteal demise in the responsive gland is characterized by cessation of steroid production, angio-regression, and apoptotic cell death, brought about by leukocyte infiltration, inflammatory responses, and diminished angiogenic support. However, the young immature CL is resistant or refractory to the luteolytic actions of PG. Evidence based on functional genomics and other studies highlight the roles played by endothelial, immune, and steroidogenic luteal cells and their interactions in the PG-responsive vs. PG-refractory CL.     

Apoptosis does not affect the vasculature of the corpus luteum of pregnancy in the rat

There has been much research into the mechanics of angiogenesis and many studies have demonstrated that newly formed vessels regress during angiogenesis. This vascular involution has been shown to involve basement membrane dissolution and endothelial cell apoptosis. The corpus luteum provides an ideal in vivo model to study physiologic angiogenesis and studies have shown that involution of newly formed vessels occurs during corpus luteum regression. However, few studies to date have investigated the role of apoptosis on the vasculature which develops during pregnancy. By the use of the TUNEL technique to detect apoptotic cells and immunohistochemistry to distinguish between endothelial cells and pericytes, this present study demonstrated that the vasculature of the corpus luteum of pregnancy in the rat does not undergo apoptosis.     

Cholesterol transport and steroidogenesis by the corpus luteum

The synthesis of progesterone by the corpus luteum is essential for the establishment and maintenance of early pregnancy. Regulation of luteal steroidogenesis can be broken down into three major events; luteinization (i.e., conversion of an ovulatory follicle), luteal regression, and pregnancy induced luteal maintenance/rescue. While the factors that control these events and dictate the final steroid end products are widely varied among different species, the composition of the corpus luteum (luteinized thecal and granulosa cells) and the enzymes and proteins involved in the steroidogenic pathway are relatively similar among all species. The key factors involved in luteal steroidogenesis and several new exciting observations regarding regulation of luteal steroidogenic function are discussed in this review.     

Role of vascular endothelial growth factor in ovarian physiology - an overview

In the female reproductive system, as in a few adult tissues, angiogenesis occurs as a normal process and is essential for normal tissue growth and development. In the ovary, new blood vessel formation facilitates oxygen, nutrients, and hormone substrate delivery, and also secures transfer of different hormones to targeted cells. Ovarian follicle and the corpus luteum (CL) have been shown to produce several angiogenic factors, however, vascular endothelial growth factor (VEGF) is thought to play a paramount role in the regulation of normal and abnormal angiogenesis in the ovary. Expression of VEGF in ovarian follicles depends on follicular size. Inhibition of VEGF expression results in decreased follicle angiogenesis and the lack of the development of mature antral follicles. The permeabilizing activity of VEGF is thought to be involved in follicle antrum formation and in the ovulatory process. In the CL, VEGF expression corresponds to different patterns of angiogenesis during its lifespan. In most the species, higher VEGF expression in the early luteal phase is essential for the development of a high-density capillary network in the CL. However, high VEGF expression may be still maintained in the mid-luteal phase to increase vascular permeability that results in enhancement of luteal function. During gestation, VEGF is thought to be important for the persistence of the CL function for a longer than in the nonfertile cycle period of time. Further elucidation of specific roles of VEGF in ovarian physiology may help to understand the phenomenon of luteal insufficiency and reveal novel strategies of ovarian angiogenesis manipulation to alleviate infertility or to control fertility.     

Isolation and characterization of microvascular endothelial cells from developing corpus luteum.

We have isolated and characterized microvascular endothelial cells from the developing rabbit corpus luteum. The isolated cells express Factor VIII-related antigen and angiotensin-converting enzyme, internalize acetylated low-density lipoprotein, and form capillary-like tubules in collagen gel cultures. Of the mitogens tested, only basic fibroblast growth factor stimulated the proliferation of these cells. Transforming growth factor-beta 1 and tumor necrosis factor-alpha strongly inhibited the proliferation of these endothelial cells. Platelet-derived growth factor, epidermal growth factor, insulin-like growth factor-1, histamine, prostaglandins, sex steroids, and interleukin-6 (interferon-beta 2) had no effect on the proliferation of these microvascular endothelial cells from the corpus luteum, whereas interleukin-1 alpha and 1 beta were mildly inhibitory. Endothelial cells are an essential component of corpus luteum physiology. Therefore, the availability of these cells will allow us to investigate the potential interactions between endothelial cells and luteal cells in vitro.     

Superoxide dismutase activity, lipid peroxide production and corpus luteum steroidogenesis during natural luteolysis and regression induced by oestradiol deprivation of the ovary in pseudopregnant rabbits.

The relationship of oxygen free radicals to corpus luteum function in rabbits was explored during various stages of pseudopregnancy, including natural and induced luteal regression. Induced luteolysis was achieved during mid-pseudopregnancy by removal of an oestradiol capsule placed at the onset of pseudopregnancy, which suppressed ovarian oestradiol production. Activity of manganese superoxide dismutase (Mn SOD) was significantly and positively correlated with ovarian progesterone production (P < 0.01) throughout pseudopregnancy and during natural regression. Oestradiol deprivation for 12, 24 or 72 h resulted in declines in Mn SOD activity and progesterone secretion, although Mn SOD rose and corpus luteum steroidogenesis was restored to normal when the capsule was replaced for 48 h before assessment, having been removed for 24 h. Lipid peroxide and progesterone concentrations were not correlated, although a significant rise in lipid peroxides in the luteal tissue was detected after deprivation of oestradiol for 72 h. Changes in progesterone production and Mn SOD activity were not associated with alterations in concentration of prostaglandin F metabolite. These data suggest that Mn SOD may be involved in regulating function of the corpus luteum during pseudopregnancy in rabbits and that oxygen free radicals may play a role in regression of corpus luteum in this species.     

Progesterone is a cell death suppressor that downregulates Fas expression in rat corpus luteum

In female rats, apoptotic cell death in the corpus luteum is induced by the prolactin (PRL) surge occurring in the proestrous afternoon during the estrous cycle. We have previously shown that this luteolytic action of PRL is mediated by the Fas/Fas ligand (FasL) system. During pregnancy or pseudopregnancy, apoptosis does not occur in the corpus luteum. Progesterone (P4), a steroid hormone secreted from luteal steroidogenic cells, attenuated PRL-induced apoptosis in cultured luteal cells in a dose-dependent manner. P4 significantly decreased the expression of mRNA of Fas, but not FasL, in cultured luteal cells prepared from both proestrous and mid-pseudopregnant rats. These data indicate that P4 suppresses PRL-induced luteal cell apoptosis via reduction of the expression level of Fas mRNA in the corpus luteum, suggesting that P4 acts as an important factor that can change the sensitivity of corpus luteum to PRL.     

Intraluteal release of angiotensin II and progesterone in vivo during corpora lutea development in the cow: effect of vasoactive peptides.

The newly formed corpus luteum (CL) develops rapidly and has the features of active vascularization and mitosis of steroidogenic cells. Such local mechanisms must be strictly regulated by the complex relationship between angiogenic growth factors and vasoactive peptides such as angiotensin (Ang) II, atrial natriuretic peptide (ANP), and endothelin (ET)-1. Thus, the objective of the present study was to determine 1) the changes in vasoactive peptides and progesterone (P) concentrations within the developing CL, along with the changes in concentration in ovarian venous plasma (OVP) and jugular venous plasma (JVP) in the cow, 2) the effects of CL exposure to vasoactive peptides on Ang II and P secretion, and 3) the expression of mRNA for ANP type C receptor in the bovine CL and endothelial cells (ETC) from bovine developing CL. A microdialysis system (MDS) was surgically implanted into multiple CL of six cows on Day 3 after a GnRH injection that induced superovulation, and a catheter was simultaneously inserted into the ovarian vein. The Ang II concentration in OVP was higher than that in JVP throughout the experiment, while the intraluteal release of Ang II was stable. During the experimental period, the concentrations of other vasoactive peptides (ANP and ET-1) showed no clear changes in plasma and were below detectable levels in the MDS perfusate. Exposure of CL to Ang II using the MDS stimulated P release, while exposure to ANP enhanced Ang II release within the developing CL. However, ET-1 had no effect on either P or Ang II release. The expression of mRNA for ANP type C receptor was mainly observed in early CL and ETC. The results suggest that the ET-Ang-ANP system in the preovulatory follicle switches to an Ang-ANP system to enhance both the angiogenesis and steroidogenesis that are actively occurring in developing CL.     

Expression of mRNA encoding insulin-like growth factors I and II and the type 1 IGF receptor in the bovine corpus luteum at defined stages of the oestrous cycle

Previous studies have implicated insulin-like growth factors I and II (IGF-I and -II), in the regulation of ovarian function. The present study investigated the localization of mRNA encoding IGF-I and -II and the type 1 IGF receptor using in situ hybridization to determine further the roles of the IGFs within the bovine corpus luteum at precise stages of the oestrous cycle. Luteal expression of mRNA encoding IGF-I and -II and the type 1 IGF receptor was detected throughout the oestrous cycle. The expression of IGF-I mRNAvaried significantly during the oestrous cycle. IGF-I mRNA concentrations were significantly higher on day 15 than on day 10, and IGF-I mRNA in the regressing corpus luteum at 48 h after administration of exogenous prostaglandin was significantly greater than in the early or mid-luteal phase (days 5 and 10). In contrast, there was no significant effect of day of the oestrous cycle on expression of mRNA for IGF-II and the type 1 IGF receptor in the corpus luteum. Expression of IGF-II mRNA was localized to a subset of steroidogenic luteal cells and was also associated with cells of the luteal vasculature. mRNA encoding the type 1 IGF receptor was widely expressed in a pattern indicative of expression in large and small luteal cells. These data demonstrate that the bovine corpus luteum is a site of IGF production and reception throughout the luteal phase. Furthermore, this study highlights the potential of IGF-II in addition to IGF-I in the autocrine and paracrine regulation of luteal function.     

GnRH action on luteal steroidogenesis during pregnancy.

The results of our study presented here establishes that gonadotropin-releasing hormone (GnRH) acts directly on the corpus luteum, leading to suppressed production and release of progesterone and thus disrupting pregnancy. A GnRH-agonist (GnRH-Ag) treatment suppressed the luteal and serum progesterone levels. This suppression is neither mediated by a fall in ovarian testosterone production nor its conversion to estradiol. Although the treatment suppressed the nuclear estradiol-receptor content and binding sites for LH in the corpus luteum, it had no effect on the luteal binding sites for GnRH and prolactin within 24 h. GnRH-Ag augmented the plasma levels of luteinizing hormone, decreased the magnitude of nocturnal surges of prolactin, and had no effect on luteal cyclic adenosine 5'-monotriphosphate levels. Yet, the treatment had no effect on the luteal content of free cholesterol. We have also demonstrated, for the first time, the presence of steroidogenic acute regulatory protein and peripheral benzodiazepine receptor in the rat corpus luteum, and the suppression of these proteins by GnRH-Ag leads to reduced steroidogenesis by the corpus luteum. Concomitantly, P450 side-chain cleavage enzyme, its activity, and its mRNA content and 3beta-hydroxy-steroid dehydrogenase content in the corpus luteum decreased. The treatment suppressed the plasma levels of pregnenolone and 20alpha-dihydroprogesterone. These data suggest that the suppression of luteal steroidogenesis by GnRH-Ag may be due to its inhibitory effect on the cholesterol transport and/or on the enzymes involved in the steroidogenic pathway. Furthermore, based on other observations made in our laboratory, we propose a hypothesis that an endogenous GnRH is present in the corpus luteum/ovary during pregnancy in the rat and that this GnRH may play a physiological role in the regulation, maintenance, and/or termination of pregnancy.     

Platelet-derived growth factors and receptors in the rat corpus luteum: localization and identification of an effect on luteogenesis

Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) play a vital role in regulating cell growth and angiogenesis. In this study, the expression of the family of PDGFs and PDGFRs in the ovarian corpus luteum were identified and characterized, and an effect of their activity on development of the corpus luteum revealed. Gonadotropin-stimulated immature rats were utilized as a model of induced ovulation, luteogenesis, and pseudopregnancy. Levels of ovarian mRNA for Pdgfb and Pdgfd, and their receptor, Pdgfrb, increased significantly as early as 4 h after human chorionic gonadotropin (hCG) injection in immature rats primed with equine chorionic gonadotropin (eCG). Gonadotropin regulation of Pdgfb expression was confirmed by in vitro promoter-reporter assays, which showed a 2- to 3-fold increase in Pdgfb promoter activity in response to luteinizing hormone (LH). Inhibition studies implicated protein kinase A, phosphatidylinositol 3-kinase and mitogen activated protein kinase signaling pathways in the LH-induced upregulation. In the corpus luteum, PDGFA, PDGFB, PDGFC, and PDGFRA were localized to a population of luteal parenchymal/steroidogenic cells. PDGFRB was expressed primarily in what appeared to be cells of the luteal microvasculature. Intraovarian injection of an inhibitor of PDGF receptor activity, the tyrphostin AG1295, prior to injection of hCG in eCG-primed immature rats resulted in a significant 21.86%+/-11.15% decrease in corpora lutea per treated ovary in comparison to the contralateral vehicle-injected control ovary. In addition, the treated ovary of 3 of 16 rats showed widespread hemorrhage throughout the entire ovary, indicating a possible role for PDGF receptor activity in maintenance of the ovarian vasculature.     

Differences of microvascular endothelium in the bovine corpus luterum of pregnancy and the corpus luteum of the estrous cycle

Summary— The purpose of the present study was to investigate potential modulations of endothelial cells of the bovine corpus luteum (CL) during pregnancy. Luteal endothelia of pregnant and non-pregnant cows were isolated and purity of cultures was verified by flow cytometric quantification of three independent endothelial markers (von Willebrand factor, angiotensin converting enzyme, Bandeiraea simplicifolia agglutinin I ligands). Different cellular parameters including light and electron microscopical investigation of morphology and growth characteristics as well as quantification of cellular lectin binding sites were compared. Extensive heterogeneity between luteal endothelial cells in pregnant and non-pregnant animals could be demonstrated, reflected in functional attributes like angiogenic activity, ultrastructural characteristics and the quantitative expression of cellular carbohydrates. Two different morphological types of cells (‘cobblestone growth pattern’ and ‘arcuate growth pattern’) were isolated from the CL of pregnancy as well as from the cyclic CL. Spontaneous angiogenic activities, including cellular migration in band-like structures and formation of ring-like structures, were observed in endothelial cells isolated from the CL of pregnant cows exclusively. This strongly suggests that microvascular luteal endothelium of pregnant animals, in contrast to the one of non-pregnant animals, is able to produce quantitatively and/or qualitatively specific angiogenesis factor(s). Heterogeneity between luteal endothelial cells in the pregnant and non-pregnant animal could also be demonstrated by quantification of lectin (Bandeiraea simplicifolia agglutinin I, concanavalin A, Dolichos biflorus agglutinin, Ulex europaeus agglutinin I, wheat germ agglutinin) binding sites: quantitative expression of specific endothelial cell surface carbohydrates could be correlated to be status of pregnancy, thus emphasizing the actual need of quantification of lectin binding.