Identification and physical mapping of genes expressed in the corpus luteum in cattle

A representational difference analysis was performed to identify genes expressed in the corpus luteum of cattle. The corpus luteum is an ovarian structure that is essential for the establishment and maintenance of pregnancy. Knowledge of gene expression and function of corpus luteum will be important to improve fertility in humans and domestic animals. Housekeeping genes were removed from the corpus luteum representation (tester) using skeletal muscle as the subtracting agent (driver). A total of 80 clones of the final subtraction product were analysed by sequencing and 11 new bovine gene sequences were identified (pBTCL1-11). The sequences were mapped to segments of 10 different chromosomes using a somatic cell hybrid panel and a radiation hybrid panel. With one exception the locations are in agreement with published comparative maps of cattle and man. Expression in corpus luteum was verified by RT-PCR for all the 11 clones.     

Microvascular endothelial cells of the corpus luteum

The cyclic nature of the capillary bed in the corpus luteum offers a unique experimental model to examine the life cycle of endothelial cells, involving discrete physiologically regulated steps of angiogenesis, blood vessel maturation and blood vessel regression. The granulosa cells and theca cells of the developing antral follicle and the steroidogenic cells of the corpus luteum produce and respond to angiogenic factors and vasoactive peptides. Following ovulation the neovascularization during the early stages of corpus luteum development has been compared to the rapid angiogenesis observed during tumor formation. On the other end of the spectrum, the microvascular endothelial cells are the first cells to undergo apoptosis at the onset of corpus luteum regression. Important insights on the morphology and function of luteal endothelial cells have been gained from a combination of in vitro and in vivo studies on endothelial cells. Endothelial cells communicate with cells comprising the functional unit of the corpus luteum, i.e., other vascular cells, steroidogenic cells, and immune cells. This review is designed to provide an overview of the types of endothelial cells present in the corpus luteum and their involvement in corpus luteum development and regression. Available evidence indicates that microvascular endothelial cells of the corpus luteum are not alike, and may differ during the process of angiogenesis and angioregression. The contributions of vasoactive peptides generated by the luteal endothelin-1 and the renin-angiotensin systems are discussed in context with the function of endothelial cells during corpus luteum formation and regression. The ability of two cytokines, tumor necrosis factor alpha and interferon gamma, are evaluated as paracrine mediators of endothelial cell function during angioregression. Finally, chemokines are discussed as a vital endothelial cell secretory products that contribute to the recruitment of eosinophils and macrophages. The review highlights areas for future investigation of ovarian microvascular endothelial cells. The potential clinical applications of research directed on corpus luteum endothelial cells are intriguing considering reproductive processes in which vascular dysfunctions may play a role such as ovarian failure, polycystic ovary syndrome (PCOS), and ovarian hyperstimulation syndrome (OHSS).     

Endocrine and local control of the primate corpus luteum

The primate corpus luteum is a transient endocrine gland that differentiates from the ovulatory follicle midway through the ovarian (menstrual) cycle. Its formation and limited lifespan is critical for fertility, as luteal-derived progesterone is the essential steroid hormone required for embryo implantation and maintenance of intra-uterine pregnancy until the placenta develops. It is well-established that LH and the LH-like hormone, CG, are the vital luteotropic hormones during the menstrual cycle and early pregnancy, respectively. Recent advances, particularly through genome analyses and cellular studies, increased our understanding of various local factors and cellular processes associated with the development, maintenance and repression of the corpus luteum. These include paracrine or autocrine factors associated with angiogenesis (e.g., VEGF), and that mediate LH/CG actions (e.g., progesterone), or counteract luteotropic effects (i.e., local luteolysis; e.g., PGF_2α). However, areas of mystery and controversy remain, particularly regarding the signals and events that initiate luteal regression in the non-fecund cycle. Novel approaches capable of gene “knockdown” or amplification”, in vivo as well as in vitro, should identify novel or underappreciated gene products that are regulated by or modulate LH/CG actions to control the functional lifespan of the primate corpus luteum. Further advances in our understanding of luteal physiology will help to improve or control fertility for purposes ranging from preservation of endangered primate species to designing novel ovary-based contraceptives and treating ovarian disorders in women.     

Production of prostacyclin by different cell types of the goat ovary

A sensitive platelet aggregation-inhibition assay was used to quantitate the production of prostacyclin by different cell types of the goat ovary. The assay could detect as low as 0.16 ng in the test sample. Different cell types i.e. granulosa, theca and corpus luteum or the total ovarian homogenate were incubated at 37° C for 10 minutes with or without 0.2mM arachidonic acid. Rat aortic strips were incubated under similar conditions as a positive control. Under basal conditions the amount of prostacyclin produced by corpus luteum cells was higher compared to that by granulosa cells. When the precursor of prostaglandins (arachidonic acid) was provided the production markedly increased in corpus luteum, granulosa, and ovarian homogenate as well as in aortic strips. Theca cells did not produce detectable levels of prostacyclin even when the precursor was provided. Trapidil did not alter the basal but enhanced the archidonic acid-stimulated prostacyclin production in homogenate and granulosa cells with no further increase in corpus luteum cells. U-51605 decreased basal as well as arachidonic acid-stimulated prostacyclin production in all the cell types. The prostacyclin production in ovaries is compartmentalized suggesting a possible role in ovarian physiology.     

Detailed analytical subcellular fractionation of non-pregnant porcine corpus luteum reveals peroxisomes of normal size and significant UDP-glucuronosyltransferase activity in the high-speed supernatant

A detailed subfractionation of the non-pregnant porcine corpus luteum (CL) was performed employing differential centrifugation. Marker enzyme assays (i.e., lactate dehydrogenase for the cytosol, NADPH-cytochrome P450 reductase for the endoplasmatic reticulum, catalase (CAT) for peroxisomes, glutamate dehydrogenase for the mitochondrial matrix and acid phosphatase for lysosomes) in all subfractions obtained exhibited a pattern of distribution similar to that observed with rat liver. These subfractions should be useful in connection with many types of future studies. In disagreement with previous biochemical and morphological studies, peroxisomes (identified on the basis of catalase activity and by Western blotting of catalase and of the major peroxisomal membrane protein (PMP-70)) sedimented together with mitochondria (i.e., at 5000 x g(av) for 10 min) and not in the post-mitochondrial fraction prepared at 30,000 x g(av) for 20 min by Peterson and Stevensson. No other classical peroxisomal enzymes were detectable in the porcine ovary, raising questions concerning the function of peroxisomes in this organ. Furthermore, UDP-glucuronosyltransferase (UGT), generally considered to be an integral membrane protein anchored in the endoplasmatic reticulum, was recovered in both the cytosolic (i.e., the supernatant after centrifugation at 50,000 x g(av) for 1h) and the microsomal fraction of the porcine corpus luteum, even upon further centrifugation of the former. In contrast, UGT sediments exclusively in the microsomal fraction upon subfractionation of the liver and ovary from rat.     

The Mare Model to Study the Effects of Ovarian Dynamics on Preantral Follicle Features

Ovarian tissue collected by biopsy procedures allows the performance of many studies with clinical applications in the field of female fertility preservation. The aim of the present study was to investigate the influence of reproductive phase (anestrous vs. diestrous) and ovarian structures (antral follicles and corpus luteum) on the quality, class distribution, number, and density of preantral follicles, and stromal cell density. Ovarian fragments were harvested by biopsy pick-up procedures from mares and submitted to histological analysis. The mean preantral follicle and ovarian stromal cell densities were greater in the diestrous phase and a positive correlation of stromal cell density with the number and density of preantral follicles was observed. The mean area (mm²) of ovarian structures increased in the diestrous phase and had positive correlations with number of preantral follicles, follicle density, and stromal cell density. Biopsy fragments collected from ovaries containing an active corpus luteum had a higher follicle density, stromal cell density, and proportion of normal preantral follicles. In conclusion, our results showed: (1) the diestrous phase influenced positively the preantral follicle quality, class distribution, and follicle and stromal cell densities; (2) the area of ovarian structures was positively correlated with the follicle and stromal cell densities; and (3) the presence of an active corpus luteum had a positive effect on the quality of preantral follicles, and follicle and stromal densities. Therefore, herein we demonstrate that the presence of key ovarian structures favors the harvest of ovarian fragments containing an appropriate number of healthy preantral follicles.     

Ghrelin ovarian cell expression in patients with polycystic ovary syndrome: an immunohistochemical evaluation.

INTRODUCTION: The influence of ghrelin on different organs has been studied recently, e.g. in the regulation of pituitary hormone release, regulation of energy homeostasis, glucose metabolism and insulin secretion, cell proliferation, and reproductive function. However, the etiology of polycystic ovary syndrome has not been fully explained. The aim of our study was to estimate the presence of ghrelin in polycystic ovaries cells and evaluation of the relationship between ghrelin occurrence and cells proliferation. METHODS: In the present work we have compared ten polycystic ovaries with ovaries without pathology as the control group. We used immunohistochemical method to detect ghrelin. The cells proliferation was evaluated by Ki 67 proliferation index. RESULTS: Ghrelin immunostaining was demonstrated in cytoplasm of ovarian secondary interstitial cells and in atretic corpus luteum. The cell nuclei were ghrelin positive in granulosa, theca layers of follicular cyst in both groups as well as in luteal cells of young corpus luteum in healthy ovaries. Ki 67 immunostaining was observed in granulosa and theca layers of follicular cyst in polycystic and healthy ovaries. CONCLUSIONS: It is possible that local ghrelin expression plays an important role in the direct control of ovarian development and function and ghrelin may participate in patomechanism of PCOS.     

Physiological and immunocytochemical evidence for a new concept of blood flow regulation in the corpus luteum

To explain the high rate of blood flow in the corpus luteum, we hypothesize that luteal blood vessels offer minimal resistance to flow and are incapable of vasomotion. This hypothesis was tested in rabbits at mid-pseudopregnancy by measuring blood flow in the corpus luteum and ovarian stroma with tracer-labeled microspheres at three levels of arterial blood pressure, which was manipulated by constricting the aorta above the ovarian artery. In addition, the distribution of vascular smooth muscle in the ovary was evaluated with morphological and immunocytochemical techniques. Decreases in arterial pressure were paralleled by reductions in blood flow in the corpus luteum, whereas ovarian stromal blood flow was unchanged. Consistent with our hypothesis, there was no change in the low level of vascular resistance offered by blood vessels in the corpus luteum, supporting the view that they are maximally dilated and incapable of autoregulation. Morphologically, the vessels within the corpus luteum appeared as large sinusoidal capillaries without smooth muscle, providing an anatomical explanation for the lack of vasomotor control demonstrated physiologically. The absence of vascular smooth muscle was confirmed with immunocytochemistry using an antibody against the muscle-specific intermediate filament, desmin. The fluorescein-labeled antibody decorated arteries and arterioles within the ovarian stroma and near the capsule of the corpus luteum, but did not decorate vessels in the corpus luteum of pseudopregnancy, providing additional evidence that the vessels of the corpus luteum lack the smooth muscle investment necessary to change vascular caliber. From these findings, we have proposed a novel scheme to explain intraovarian blood flow regulation. Vascular resistance in the ovarian stroma, as in most tissues, is acutely regulated by dilation or constriction of intratissue arterioles. In contrast, vascular resistance within the corpus luteum is modeled as a relatively invariable parameter, fixed at a low level by the morphological characteristics of the luteal vasculature. Therefore, the corpus luteum operates on a linear (maximally "vasodilated") pressure-flow curve, does not actively regulate intratissue blood flow, and is subject to acute regulation of perfusion only through changes in extra-luteal vessels.     

Effect of prostaglandins on the steroidogenesis in human ovarian tissues

Although prostaglandins are luteolytic in some species, in conditions they stimulate progesterone production in the corpus luteum (1). Apart from this effect prostaglandins may also stimulate other steps in the steroidogenic sequence e.g. corticosteroidogenesis in superfused rat adrenal glands (2) and aromatization of testosterone by perfused human placenta (3). With this possibility in view and also because of paucity of data on the effect of prostaglandins on steroidogenesis in human ovarian tissues we have been studying under conditions the effect of prostaglandins on progesterone formation in human corpora lutea and on the utilization of C₂₁ steroids by the luteal and follicular compartments of the ovary. These studies are still in progress. However, the data obtained so far indicates that in addition to stimulating progesterone synthesis in the corpus luteum prostaglandins may also affect other steps in steroidogenesis in human ovarian tissues. We wish to report here in brief these preliminary results.     

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.     

Endocrine profiles and embryo quality in superovulated Japanese Black cattle

The relationships between plasma concentrations of progesterone (P(4)), estradiol-17beta (E(2)) and lutenizing hormone (LH) and embryo yield and quality were examined in 40 superovulated Japanese Black cattle. The results indicated that the ovarian function, especially the function of corpus luteum on the first treatment day, is an important factor for reliable superovulation in cattle. The levels of plasma E(2) and LH at estrus were related to embryo yield and quality.     

HIF-1在卵巢黄体发育过程中对血管新生的调控作用

在哺乳动物中,卵巢黄体（corpus luteum,CL）是由破裂排卵后的卵泡所形成的,也是血管增生比较激烈的地方。尤其是在卵巢黄体早期发育阶段,这种快速形成的致密毛细血管网可以确保产生激素的细胞获得氧气、营养和合成激素等所必要的前体,同时释放大量的激素用于早期妊娠的建立和维持。目前的研究已经表明,血管内皮生长因子（vascular endothel ial growth factor,VEGF）作为重要的促血管生成因子,在卵巢黄体发育过程中对血管增生具有至关重要的调节作用,而VEGF作为转录因子HIF－1的下游靶基因,受缺氧诱导因子HIF－1信号通路的调控。该文一方面对卵巢黄体发育过程中VEGF依赖性血管增生的调控机制进行概述,另一方面就转录因子H1F－1对VEGF的转录激活调控机制进行系统阐述,从而揭示HIF－1对卵巢黄体发育过程dgVEGF依赖性血管新生的调控作用,为进一步研究哺乳动物卵巢黄体发育过程中血管增生的分子调控机制提供坚实的理论基础。     

超声在诊断卵巢黄体破裂中的应用

目的探讨超声对卵巢黄体破裂的诊断价值。方法回顾性分析35例经超声诊断卵巢黄体破裂的声像特点及临床表现。结果35例均有不同程度的腹盆腔积液。24例手术治疗并病理证实,11例保守治疗,超声表现为盆腔积液,卵巢囊肿,附件区包块。结论超声对卵巢黄体破裂的早期快速诊断和制定治疗方案及愈后,有极其重要价值和意义。     

Formation and fate of the corpus luteum in the Dusky leaf monkey (Presbytis obscura)

The formation and fate of the corpus luteum have been described for a previously un researched species of South-east Asian colobine, the Dusky leaf monkey, Presbytis obscura. Histological material from 44 wild female monkeys collected at various stages of the menstrual cycle, pregnancy and lactation over a 12-month period was available for study. The corpus luteum of the menstrual cycle was a cystic structure and consisted of a thin rim of luteal tissue surrounding a central cavity filled with a meshwork of fibrin. At the end of the luteal phase the corpus luteum either degenerated into a corpus albicans, or became transformed into a corpus aberrans. Corpora aberrantia have previously only been described in the ovaries of the rhesus monkey, where they may persist for many months. Ultimately the corpus aberrans may also degenerate into a corpus albicans. Small corpora lutea atretica were observed during early pregnancy but there was no evidence of corpora lutea accessoria. Anovulatory cycles were common amongst the females included in this study and may play a role in limiting the growth of troops in their natural environment. Comparisons have been drawn between the findings presented here and those published for other species of catarrhine primate.     

Effect of prostaglandins on the in vitro steroidogenesis in human ovarian tissues

Although prostaglandins are luteolytic in some species, in $\text{in vitro}$ conditions they stimulate progesterone production in the corpus luteum (1). Apart from this effect prostaglandins may also stimulate other steps in the steroidogenic sequence e.g. corticosteroidogenesis in superfused rat adrenal glands (2) and aromatization of testosterone by perfused human placenta (3). With this possibility in view and also because of paucity of data on the effect of prostaglandins on steroidogenesis in human ovarian tissues we have been studying under $\text{in vitro}$ conditions the effect of prostaglandins on progesterone formation in human corpora lutea and on the utilization of C₂₁ steroids by the luteal and follicular compartments of the ovary. These studies are still in progress. However, the data obtained so far indicates that in addition to stimulating progesterone synthesis in the corpus luteum prostaglandins may also affect other steps in steroidogenesis in human ovarian tissues. We wish to report here in brief these preliminary results.     

Assessment of possible luteolytic effect of intra-ovarian injection of prostaglandin F2α in the human

A group of five patients awaiting laparoscopic tubal diathermy were followed by daily assay of luteinising hormone (LH) and progesterone. Between five and eight days after the LH peak, prostaglandin F_2α (PGF_2α) was injected into either the corpus luteum or the ovarian stroma. Doses of 100 μg into the corpus luteum, 1000 μg into the adjacent stroma and 500 μg into an indeterminate ovarian structure had no effect on peripheral plasma progesterone levels or uterine bleeding.An injection of 500 μg or 1000 μg given unequivocally into the corpus luteum produced a rapid and profound fall in plasma progesterone levels, the nadir coinciding with the onset of uterine bleeding which commenced 24 hours after the injection and persisted for more than seven days. Injection of 100 μg in the same volume of saline had no such effect. Despite continued bleeding plasma progesterone levels returned to normal luteal levels for three days and then fell again.     

A role for interferons in early pregnancy

In order to survive, the developing conceptus must interrupt the normal ovarian cycle of the mother and extend the production of progesterone by the corpus luteum. An unusual Type 1 interferon (IFN), related structurally to the IFN-alpha molecule and produced in massive amounts for only a few days by the first epithelium (trophectoderm) of the preimplantation conceptus, has been implicated as the antiluteolytic agent in sheep and cattle. IFN-alpha therapy during this critical period can also improve pregnancy success in sheep. It remains unclear, however, whether the trophoblast IFN have specialized biological properties or whether they are unique merely in the timing, magnitude and site of their expression.     

超声在诊断卵巢黄体破裂中的应用

目的探讨超声对卵巢黄体破裂的诊断价值。方法回顾性分析35例经超声诊断卵巢黄体破裂的声像特点及临床表现。结果35例均有不同程度的腹盆腔积液。24例手术治疗并病理证实,11例保守治疗,超声表现为盆腔积液,卵巢囊肿,附件区包块。结论超声对卵巢黄体破裂的早期快速诊断和制定治疗方案及愈后,有及其重要价值和意义。     

Fundamental cryobiology of reproductive cells and tissues

During the last half of the 20th century there have been considerable advancements in mammalian reproductive technologies, including in vitro production of pre-implantation embryos and embryo sexing, and even cloning in some species. However, in most cases, management of non-cryopreserved reproductive cells (i.e., spermatozoa or oocytes) and tissues (i.e., testicular tissue or ovarian tissue) is problematic due to difficulties in donor-recipient synchronization and the potential for transmission of infectious pathogens, which cumulatively limits widespread application of these techniques. Therefore, there is an urgent need for the development of optimum cryopreservation methods for reproductive cells and tissues from many species. Today frozen-thawed spermatozoa and embryos have become an integral component of animal agriculture, laboratory animal genome banking, and human sperm banking and infertility programs. However, although widely implemented, the protocols currently used to cryopreserve bull sperm, for example, are still suboptimal, and cannot readily be extrapolated to other species' sperm. Similarly, embryo-freezing protocols successfully used for mouse and cattle have yielded little success when applied to some other species' embryos, or to a related cell type, oocytes. To date, with the exception of mouse oocytes, almost all mammalian species' oocytes studied have proven very difficult to successfully cryopreserve. Currently, there is a growing interest to understand the underlying cryobiological fundamentals responsible for these low survival rates in an effort to develop better cryopreservation methods for oocytes. Additionally, there is growing interest in developing technologies for the optimal isolation and cryopreservation of the earliest stage of male (spermatogonia, spermatids) and female (primordial follicle) germ cells, with subsequent maturation to the desired stage in vitro. Female gamete maturation, fertilization, and embryo development entirely under in vitro conditions from primordial follicles has been achieved in mice, however techniques for this and other species are still very early in their development. Furthermore, with the recent advances made in intracytoplasmic sperm injection (ICSI), and gamete isolation and maturation, close attention has been given to cryopreservation of gametes in the form of gonadal tissue (i.e., testicular tissue and ovarian tissue) containing various developmental stages of male (spermatogonia, spermatids, and spermatozoa) and female (primordial, secondary) germ lines.     

Demonstration of luteinizing hormone/human chorionic gonadotrophin receptor-binding inhibitor in aqueous extracts of frozen human corpus luteum

Aqueous extracts of frozen human corpora lutea were tested for the presence of an inhibitor of luteinizing hormone-receptor site binding (LHRBI) and for the subsequent effect on the stimulatory response of luteinizing hormone (LH) on progesterone synthesis by sheep ovarian cells. In the presence of human corpus luteum extract of normal menstrual cycle (30,000-g supernatant), the binding of 125I human chorionic gonadotrophin (hCG) to granulosa and luteal cells of sheep ovaries was markedly reduced, but the ability of rat testicular LH receptors to bind labelled hCG was less affected. However, extracts of corpora lutea of the first trimester of pregnancy appeared to be less inhibitory on the binding of LH/hCG to ovarian cells and had no effect on the binding of rat testicular cells compared to those of normal menstrual cycle. Addition of both extracts separately inhibited the LH-stimulated in vitro progesterone synthesis by granulosa cell cultures and by incubated sheep corpus luteum slices. These findings provide evidence for the presence of LHRBI in human corpus luteum.