The Mechanisms of Angiogenesis and Apoptosis During the Functional Formation and Regression of the Corpus Luteum in the Ovarian Reproductive Endocrine System

The reproductive tracts of 404 female white-tailed deer (Odocoileus virginianus) were studied for information on size, shape, and coloration of corpora lutea. Corpora lutea in fawns are regarded as regressing if they are less than 4.0 mm in diameter at the time when the rut for that age-class is nearing its end. In all age-groups of does where fetal membranes or embryos are present, corpora lutea smaller than 6.0 mm are regarded as regressing. These small ones are regarded as corpora lutea of an earlier heat and not as corpora lutea of the current pregnancy. Regressing corpora lutea of a previous ovulation also appear to have an irregular surface shape. Color is not a dependable criterion for differentiation of the two types of corpora lutea. During an investigation of reproduction in white-tailed deer in Iowa, information was obtained on the physical characteristics of their corpora lutea. In attempting to correlate fertilization rate with ovulation rate, difficulty has been encountered in differentiating corpora lutea of ovulation of a previous heat from corpora lutea of pregnancy. In sheep, corpora lutea of ovulation are formed in ruptured follicles within 4 days following ovulation (Asdell 1946:366-367) and are fully developed by the eighth day following ovulation (Harrison 1962:169). In deer, corpora lutea of ovulation begin regressing after about 14-15 days (Cheatum 1949:285) if fertilization does not occur. When fertilization and implantation take place, corpora lutea of ovulation persist as corpora lutea of pregnancy (Amoroso and Finn 1962:454). The corpus luteum has a glandular function, secreting progesterone hormone which is essential for implantation and early development of the fertilized ovum. This hormone also suppresses further follicular development and ovulation. After parturition, corpora lutea of pregnancy regress and become corpora albicantia. Ability to identify corpora lutea of ovution of a previous heat from those of pregnancy of a later heat would eliminate a possible source of error when using corpora lutea counts as an index to reproductive rates of deer. Golley (1957) found the use of the corpora lutea dependable for determining the ovulation incidence of the black-tailed deer (Odocoileus hemionus columbianus) but detected an error when using the corpora albicantia to indicate ovulation. Golley used histological techniques to determine the differences between the two types of tissue. This paper will discuss gross size, shape, and color variations of corpora lutea in white-tailed deer ovaries as a possible means for distinguishing the two types. The assistance of Eldie Mustard, former Iowa State Conservation Commission Biologist, is appreciated. Several of Iowa's Conservation Commission officers and biologists also assisted materially by collecting reproductive tracts from wild deer. The cooperation of deer hunters who saved tracts for the study was indispensable. Calvin Rayburn, Ronald Schara, Harold Prince, and David Bolton, students 1Journal paper J-4604 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa. A contribution from the Iowa Cooperative Wildlife Research Unit, which is jointly sponsored by Iowa State University of Science and Technology, U. S. Bureau of Sport Fisheries and Wildlife, the Iowa State Conservation Commission, and the Wildlife Management Institute. Financial support for Trauger was provided by the National Science Foundation Undergraduate Research Participation Program.

Corpus luteum (CL) formation involves dramatic tissue remodeling and angiogenesis. To determine the functional roles of the plasminogen activator and matrix metalloproteinase (MMP) systems in these processes, we have studied CL formation and function in plasminogen (plg)-deficient mice, with or without treatment with the broad-spectrum synthetic MMP inhibitor galardin. Both the adult pseudopregnant CL model and the gonadotropin-primed immature mouse model were used. We found that CL formed normally not only in plasminogen-deficient mice and in galardin-treated wild-type mice, but also in galardin-treated plg-deficient mice, suggesting that neither of the plasminogen activator and MMP systems is essential for CL formation. Nevertheless, in plg-deficient mice, serum progesterone levels were reduced by approximately 50%, and the progesterone levels were not reduced further by galardin treatment. When CL from plg-deficient mice were stained for several molecular markers for CL development and regression, they appeared healthy and vascularized, and were indistinguishable from CL from wild-type mice. This implies that the reduced progesterone levels were not caused by impaired CL formation. Taken together, our data suggest that neither plasmin nor MMPs, alone or in combination, are required for CL formation. Therefore, the tissue remodeling and angiogenesis processes during CL formation may be mediated by redundant protease systems. However, the reduced serum progesterone levels in plg-deficient mice suggest that plasmin, but not MMPs, plays a role in maintenance of luteal function. This role may be performed through proteolytic activation of growth factors and other paracrine factors.

The effect of the uterus on corpus Iuteum function was studied in adult multiparous alpacas subjected to different surgical treatments. Removal of the left uterine horn from females with a corpus luteum in the left ovary significantly prolonged luteal function in 3 out of 4 females, as evidenced by high peripheral plasma progesterone levels, large corpora lutea and lack of sexual receptivity. Removal of the right uterine horn in females with a corpus luteum in the right ovary also prolonged luteal function, but the effect in this case was much less marked. Only 1 animal, out of a total of 4, maintained a large and functional corpus luteum up to Day 70 and she rejected the male at all times. Removal of the oviduct ipsilateral to the ovary containing the corpus luteum or the contralateral uterine horn and oviduct, had no effect on the lifespan of the corpus luteum, regardless of whether this was located in the right or the left ovary; early regression of the corpus luteum, as indicated by a marked decline in plasma progesterone levels by Day 12 and sexual receptivity occurred in all of these animals. These results were similar to those of the control, sham operated females. In females with a corpus luteum in each ovary results were as follows: a) removal of both uterine horns and oviducts caused maintenance of both corpora lutea up to Day 70; b) removal of the left uterine horn and oviduct prolonged the lifespan of the corpus luteum in the left ovary, but the one in the right ovary showed early regression; c) removal of the right uterine horn and oviduct failed to exert any effect on the corpora lutea as evidenced by early regression of both. Results indicate that the luteolytic activity of the right uterine horn in the alpaca is local only, while that of the left uterine horn, has both a local effect and a systemic effect.

sRal interacting protein of 76 kDa (RLIP76) is multifunctional protein localized and distributed in the plasma membrane, cytosol, and nucleus of the cell. In tumorigenesis, RLIP76 emerges as a common feature for the solid tumor growth. RLIP76 is ubiquitously expressed in various tissues including the ovary. Interestingly, the similar physiological events in obtaining an adequate supply of nutrient by gaining access to the host vascular system are required either for corpus luteum formation or tumor development. In addition, the identical angiogenesis modulators were found in neoplastic and normal ovaries. Our previous study involving RLIP76−/− mice implanted with melanoma or carcinoma cell conclusively demonstrated that RLIP76 is necessary for angiogenesis and neovascularization of primary solid tumors. RLIP76 plays an essential role in tumor angiogenesis through the regulation of pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1 (HIF-1). In certain previous studies, those pro-angiogenic factors were found significantly to be upregulated during the corpus luteum formation. To that, the following review will discuss the likelihood of RLIP76 role in ovarian corpus luteum.

Tumors and the ovarian corpus luteum have complex mechanisms in the growth microenvironment. Angiogenesis is especially important for demonstrating the molecular mechanism of dynamic cellular function in tumors and corpus luteum. Angiogenesis in tumors and corpus luteum seems to have a similar function, and Ral-interacting protein 76 (RLIP76) and vascular endothelial growth factor (VEGF) are expressed in the tissues of tumors and ovarian corpus luteum. RLIP76 is a potential factor with VEGF in the tumor and corpus luteum angiogenesis. RLIP76 regulates a small GTPase (R-Ras) in cell survival, spreading, and migration. VEGF activates angiogenic functions in tumor and endothelial cells. Hypoxia-inducible factor-1 (HIF-1) is important in tumor growth, tumor angiogenesis, and corpus luteum. VEGF and HIF-1 regulate the angiogenic function of RLIP76, and RLIP76 controls vascular growth in endothelial and tumor cells. RLIP76, R-Ras, VEGF, and HIF-1 may be useful in the research of corpus luteum and cancer therapy and the study of mechanisms of tumor and corpus luteum angiogenesis. This review will help to elucidate the roles of RLIP76 and VEGF in tumor and corpus luteum angiogenesis, tumorigenesis, and the specific regulation of RLIP76 and VEGF. Thus, we reviewed the potential role of RLIP76 and VEGF in the angiogenesis of the tumor and corpus luteum in the tumor and ovarian microenvironment.

In the domestic dog (Canis familiaris), the corpus luteum (CL) is the only source of progesterone (P4) in non-pregnant and pregnant animals. The progesterone secretion profiles are almost identical in both conditions until the last third of the luteal phase when the gradual P4 decline turns into a steep drop in pregnant bitches, indicating the onset of parturition. Consequently, the length of the CL-phase in non-pregnant dogs exceeds the luteal lifespan in pregnant animals. The canine CL-function is regulated by many species-specific regulatory mechanisms, the most intriguing of which is the reported independence of gonadotropic support during the first third of dioestrus. Recently, PGE2 has been proposed as one of the most important luteotropic factors acting locally during this time, but afterwards prolactin (PRL) appears to be the main luteotropic factor. Luteal regression/luteolysis occurs, however, in spite of an increased gonadotropic support. Lately, by demonstrating the expression of PRL-receptor (PRLr), a new insight into possible regulatory mechanisms has indicated that the supply of P4 could be controlled upstream of the steroidogenic machinery at the level of PRLr expression and/or function, subsequently leading to the functional suppression of the steroidogenic machinery. An endogenous source of a luteolytic agent is apparently lacking, implicating the luteal regression in non-pregnant bitches as a passive, degenerative process even if the PGF2α-receptor is constitutively expressed in canine CL. This is in contrast to pregnant dogs in which prepartum luteolysis seems to be an active process of CL destruction by PGF2α of utero/placental origin targeting the luteal PGF2α-receptor.

This study evaluated the structural changes in the reproductive tract of Asiatic black bears using serial transrectal ultrasonography. In addition, the ultrasonographic observations were compared with the results of vaginal cytology and hormonal analyses. The collection of blood for hormonal analysis, vaginal cytology and transrectal ultrasonography was performed in two bears (Bears 1 and 2) from June 2011 to August 2013 without mating and in a third bear (Bear 3) from April to December 2012, allowing natural mating. Serial ultrasonographic observations showed cyclic changes in ovarian structures (e.g. emergence of small follicles, growth and ovulation of dominant follicles and corpus luteum (CL) formation) during the reproductive cycles of the three bears. The diameter of the uterine horns remained similar throughout the reproductive cycle in Bears 1 and 2, and it remained similar from April until October, but an enlargement containing foetuses was observed in Bear 3 in December. The ultrasonographic observations were consistent with the data obtained through vaginal cytology and progesterone analysis during the reproductive cycle. An average of 4.0 (±0.4) dominant follicles was observed during the oestrous stage (May-August), during which the superficial cells accounted for >90% of the total vaginal cells. In addition, the detection of an average of 2.6 (±0.2) CL was associated with increased plasma progesterone concentrations (3.0 ± 0.4 ng/ml) between June and December (near hibernation). In conclusion, serial transrectal ultrasonography demonstrated yearly oestrous (ovulation) cycles via follicular dynamics and CL formation on ovaries, accordingly with vaginal cytology and hormonal level in the Asiatic black bear.

Serum hormones, corpus luteum (CL) progesterone, and CL LH receptors were characterized and interrelated to ovarian follicle and CL number and preimplantation embryo quality/survival in the cat. Blood samples were collected from queens ovariohysterectomized at 64 (n = 7), 76 (n = 11), 100 (n = 8), 124 (n = 7), 148 (n = 6), or 480 (n = 8) h after first copulation (3-times-daily matings on Days 2 and 3 of estrus). Ovarian CL were enucleated, weighed, and bisected; one hemi-CL was assayed for progesterone and the other for LH receptors. Serum was assessed for estradiol-17 beta and progesterone. Serum estradiol-17 beta concentrations did not return (p > 0.05) to baseline approximately 20 pg/ml) until 124 h after first copulation, whereas serum progesterone began to increase (> 1 ng/ml) by 76 h, was elevated (p < 0.05) by 124 h, and continued to rise thereafter. Serum progesterone was highly correlated with CL mass and LH receptor and progesterone concentration (range, r = 0.69-0.82; p < 0.01). CL progesterone and LH receptor concentrations were similar (p > 0.05) at 64 and 76 h, and both increased (p < 0.05) at subsequent time intervals and were correlated closely (r = 0.65; p < 0.01). Although ovarian CL were distinct and well organized by 64 h, pronounced elevations in serum progesterone and CL LH receptors and progesterone did not occur until at least 36 h later. The rapid increase in serum progesterone concentrations between 100 and 148 h was related to accelerated LH receptor synthesis and CL progesterone production and not entirely to enhanced CL growth.(ABSTRACT TRUNCATED AT 250 WORDS)

There is evidence suggesting that the mere presence of a hormone-responsive adenylyl cyclase system in a tissue may not be sufficient for desensitization to occur since phosphorylation reactions might also be involved. The purpose of this study was to determine if luteal tissue in the absence of other ovarian tissues would desensitize to human CG (hCG). One or both ovaries were removed from rabbits 5 h before hCG-induced ovulation and the periovulatory follicles were transplanted underneath the kidney capsule where they formed ectopic corpus luteum [or corpora lutea (CL)]. Rabbits which were bilaterally ovariectomized received estradiol implants at the time of ovariectomy to maintain control serum estradiol concentrations. On day 7 of pseudopregnancy, the rabbits were injected with saline (control) or with 75 IU hCG and were killed 24 h later at which time ovarian and ectopic CL progesterone content and adenylyl cyclase activity were assessed. As expected, in ovarian CL there was decreased LH-responsive adenylyl cyclase (69% relative to control) and a correspondingly decreased luteal progesterone content (40% relative to control). In the same rabbits, the ectopic CL showed much the same pattern of response as the ovarian CL but perhaps to a slightly lesser extent (decreases relative to control of 59% in adenylyl cyclase response to LH and 29% in progesterone content). However, in rabbits with ectopic CL only, the luteal tissue showed no change either in hormone-responsive adenylyl cyclase activity or in progesterone content. Similarly, binding of radiolabeled hCG to luteal membranes 24 h after hCG was almost totally absent in ovarian CL, was decreased by 50% in ectopic CL with one ovary present, and was unaltered in ectopic CL of bilaterally ovariectomized rabbits. These data suggest that nonluteal ovarian tissue may be required for the induction in CL of the appropriate protein kinases for the proposed phosphorylations involved in adenylyl cyclase desensitization.

During the luteinization after ovulation in mammalian ovary, the containing cells undergo an energy consuming function re-determination process to differentiate into luteal cells under avascular environment. Previous evidences have delineated the contribution of autophagy to the cell differentiation and the catabolic homeostasis in various types of mammalian cells, whereas few interest had been focused on the involvement of autophagy in the luteinization of granulosa cells during the formation of early corpus luteum. Herein, the present study investigated that expression and contribution of autophagy during granulosa cell luteinization and early luteal development through in vivo and in vitro experiments. The results clearly demonstrated that HIF-1α/BNIP3-mediated autophagy plays a vital role in the luteinization of granulosa cells during the early luteal formation in vivo and in vitro. In the neonatal corpus luteum, HIF-1α up-regulated BNIP3 expressions, which contributed to the autophagic initiation by disrupting beclin1 from Bcl-2/beclin1 complex and protected cells from apoptosis by curbing the skew of mitochondria balance under avascular niche. Notably, Inhibition of HIF-1α activity by echinomycin enhanced the levels of cytoplasmic cytochrome c and cell apoptosis in the nascent corpus luteum. These findings revealed that HIF-1α/BNIP3-mediated autophagy enabled the process of granulosa cell luteinization and protected the granulosa-lutein cells from further apoptosis under hypoxia niche. To our knowledge, the present study firstly clarified that HIF-1α/BNIP3-mediated autophagy contributes to the luteinization of granulosa cells during the formation of pregnant corpus luteum, which will help us further understanding the luteal biology and provide us new clues for the treatment of luteal insufficiency.

Unilateral ovariectomy was performed on 20 rats 4-5 wk before mating (ULO-B group), and 18 rats 8 days after mating (ULO-A group); 16 rats were maintained as controls. The effects on growth of individual corpora lutea (CL) in the remaining ovary and peripheral blood progestagen concentrations were examined. CL hypertrophy was observed by Day 13 in the ULO-B group and by Day 16 in the ULO-A group. Progesterone and 20 alpha-dihydroprogesterone concentrations were double control values at Day 16 in the ULO-B group, but half the control values in the ULO-A group. The CL hypertrophy and increased progestagen concentrations in the ULO-B group occurred despite crowding due to a doubling of the normal number of CL within the remaining ovary.

Ovarian follicle ovulation and subsequent luteinization have been described as a controlled inflammatory event, comprising tissue damage and repair. To elaborate this further in cattle, the contribution of immune cells to dominant follicle luteinization, ovulation, and corpus luteum formation was investigated. Ovulation in beef heifers was synchronized using an 8-day progesterone-based synchronization program. Heifers were slaughtered at a local abattoir at 5 timepoints (T): (T1) 24h before ovulation (n=10); (T2) 2h before ovulation (n=9); (T3) 6h after ovulation (n=10); (T4) 24h after ovulation (n=10); (T5) 72h after ovulation (n=10), and ovarian tissue was collected and returned to the laboratory on ice. Follicular fluid, theca, granulosa, and corpus luteum (CL) tissues were recovered by dissection and processed for analysis. The concentrations of a panel of cytokines were measured using an antibody-conjugated magnetic bead immunoassay. The abundance of T-lymphocytes, mast cells, neutrophils, eosinophils, monocytes, macrophages, and dendritic cells was determined by immunohistochemistry. The mRNA relative abundance of candidate genes, including angiogenic growth factors, adhesion factors, chemokines and cytokines, was determined by quantitative real-time PCR analysis. The resulting datasets were analysed using the linear mixed model procedure of SAS and data are presented as least squares means; reported differences were deemed significant at P≤0.05. The cytokines IFNy, IP-10, IL-10, IL-36RA, MCP-1, MIP-1a, MIP-1b, and VEGF-A were detected in follicular fluid. The concentrations (pg mL−1) of IL-10 and VEGF-A were significantly higher in T1 follicular fluid samples compared with T2 (7.70 vs. 0.86 and 2193.33 vs. 293.93, respectively). Although dendritic cells were the most abundant cells in bovine ovulatory follicular and early corpus luteum tissue at all time points (P&amp;lt;0.05), their numbers peaked in ovulatory (T2) thecal tissue (261.5 cells/mm2). The greatest number of neutrophils was identified in thecal tissue at T1 (45/mm2); thereafter, their numbers declined to 1.1/mm2 in CL tissue by T5. Similarly, the numbers of T-lymphocytes, mast cells, monocytes, and macrophages declined in CL tissue at T4 and T5. Candidate gene mRNA expression profiles appeared to be time- and tissue-specific; for example, IFNA was highest in the preovulatory granulosa tissue (T1), IL8 was highest in peri-ovulatory thecal tissue (T2), VEGFA and MMP9 were highest in the early CL tissue (T4 and T5), MMP1, TIMP1, and VCAM1 expression was highest in theca, granulosa, and CL tissue collected on or after ovulation (T2, T4, T5), expression of the prostaglandin-related genes PTGES and PTGS2 was lowest in CL tissue, and that of PTGIS was highest. The current findings support the hypothesis that ovulation in heifers is characterised by an initial proinflammatory cascade followed by a dramatic switch to tissue repair, growth, and remodelling, all occurring within a 72-h period and commencing with the LH surge. Our results highlight the roles of neutrophils, dendritic cells, and macrophages as the key actors in this process.

The corpus luteum is an endocrine organ (Fig. 11-1) that develops from the ruptured follicle at ovulation. During its functional life span it grows, and then regresses to a small connective tissue scar (corpus albicans) within the ovarian stroma. The corpus luteum secretes the steroid hormone progesterone. In primates, the corpus luteum is also a source of estrogens and prostaglandin PGF2α, and in the human, pig, and rat it also secretes relaxin (see also Chapter 12). Progesterone has a number of biological effects on the reproductive organs of the female during the estrous cycle, and all or part of pregnancy, depending on the species. Inadequate development and growth, and hence function, of the corpus luteum accounts for significant loss of mammalian embryos during early pregnancy, and so there is much interest in understanding mechanisms influencing the formation, growth, and function of the corpus luteum. An understanding of these mechanisms might also identify new approaches to regulate fertility. This chapter summarizes quantitative morphological information on corpus luteum development and growth that has accumulated over the past decade. Particular attention is focused on ultrastructural-functional relationships in the luteal cell. This information is due to the recent description [1] and use of stereolo- gical methods to quantitate biological structures. Most of the description here relates to work completed on the pregnant rat, as this is the most common experimental animal used and such quantitative data are not yet available, nor probably possible, from human studies.

This study analyzed the effect of corpus luteum (CL) formation during weeks 3-5 postpartum on the subsequent reproductive performance of dairy cows. Factors contributing to CL formation during the postpartum period were also determined. Data were collected from 1524 Holstein dairy cows on 18 farms using a single ultrasonographic examination to determine the presence or absence of a CL during weeks 3-5 postpartum. The dates of calving, AI, conception and cow parity were also collected. Data were acquired for a subset of 475 cows on five farms related to peripartum reproductive events and the body condition score (BCS) during weeks 3-5 postpartum. The hazard of first postpartum insemination by 150 days in milk (DIM) was higher for cows with a CL compared with herd mates without a CL during week 3 (hazard ratio [HR]: 1.40, P=0.007), week 4 (HR: 1.28, P=0.004) and week 5 postpartum (HR: 1.43, P<0.0001). Furthermore, the pregnancy hazard was also higher by 210 DIM for cows with a CL compared with cows without a CL during week 3 (HR: 1.56, P=0.0009), week 4 (HR: 1.28, P=0.006) and week 5 postpartum (HR: 1.20, P=0.04). Cows calved during autumn were more likely to have a CL than cows calved during spring (odds ratio [OR] =2.32, P=0.003). Primiparous cows were less likely to have a CL than multiparous cows (OR=0.63, P=0.03). Cows with a BCS < 3.00 were less likely to have a CL than cows with a BCS ≥ 3.00 (OR=0.51, P=0.0013). In conclusion, CL formation during weeks 3-5 postpartum was related to subsequent improved reproductive performance when compared with herd mates without a CL.

Angiogenesis and angioregression gene expression analyses in swine corpus luteum