Luteal regression in the primate: different forms of cell death during naturaland gonadotropin-releasing hormone antagonist or prostaglandin analogue-induced luteolysis

Morphological changes in the corpus luteum following natural and induced luteolysis in the marmoset were investigated by light and electron microscopy. Functional corpora lutea were studied in the mid and late luteal phase, naturally regressed corpora lutea in the early and late follicular phase, and corpora lutea induced to regress by administration of GnRH antagonist or prostaglandin F(2alpha) analogue in the midluteal phase. Natural luteolysis was associated with lutein cell atrophy, condensation of cytoplasmic inclusions and organelles, and accumulation of lipid. GnRH antagonist treatment resulted in aggregations of smooth membranes and myelin-like bodies in the cytoplasm of the lutein cells together with complex aggregations of degenerative cells. After prostaglandin treatment, the lutein cells contained numerous small and large vesicles; as the degenerative changes advanced, these vesicles coalesced into alveolar-type vacuoles, and nuclei involuted. These results show that in the marmoset, natural luteolysis and the two luteolytic treatments reveal different forms of luteal degeneration and cell death, none of which fit the ultrastructural criteria for apoptosis. More emphasis needs to be placed on understanding these predominant nonapoptotic forms of cell death in order to elucidate the process of luteolysis in the primate.     

Expression of monocyte chemoattractant protein-1 and distribution of immune cell populations in the bovine corpus luteum throughout the estrous cycle.

This study characterizes the expression of monocyte chemoattractant protein-1 (MCP-1) and the relative distribution of immune cell populations in the bovine corpus luteum throughout the estrous cycle. Immunodetectable MCP-1 was evident in corpora lutea of cows at Days 6, 12, and 18 postovulation (Day 0 = ovulation, n = 4 cows/stage). Day 6 corpora lutea contained minimal MCP-1 that was confined primarily to blood vessels. In contrast, relatively intense staining for MCP-1 was observed in corpora lutea from Days 12 and 18 postovulation. MCP-1 was again most evident in the cells of the vasculature, but it was also observed surrounding individual luteal cells, particularly by Day 18. An increase in immunohistochemical expression of MCP-1 on Days 12 and 18 postovulation corresponded with increases in MCP-1 mRNA and protein in corpora lutea as determined by Northern blot analysis and ELISA. Monocytes and macrophages were the most abundant immune cells detected in the bovine corpus luteum, followed by CD8+ and CD4+ T lymphocytes. In all instances, Day 6 corpora lutea contained fewer immune cells than corpora lutea from Days 12 and 18. In conclusion, increased expression of MCP-1 was accompanied by the accumulation of immune cells in the corpora lutea of cows during the latter half of the estrous cycle (Days 12-18 postovulation). These results support the hypothesis that MCP-1 promotes immune cell recruitment into the corpus luteum to facilitate luteal regression. These results also raise a provocative issue, however, concerning the recruitment of immune cells several days in advance of the onset of luteal regression.     

Immune cell populations in the equine corpus luteum throughout the oestrous cycle and early pregnancy: an immunohistochemical and flow cytometric study

Recent evidence indicates that the cells of the immune system and their large network of secretory products, or cytokines, play an active role in the ovary throughout the oestrous cycle. In the present study, immune cell populations (T and B lymphocytes, macrophages, granulocytes and eosinophils) and expression of major histocompatibility complex (MHC) class II were investigated in corpora lutea from mares in early (days 2-4), mid- (days 7-10) and late (days 12-14) dioestrus, the post-luteolytic phase (days 16-17) and early pregnancy. The number of T lymphocytes within the corpus luteum increased in the late luteal phase. CD4+ cells did not increase until day 16, whereas the number of CD8+ cells increased before functional luteolysis; an apparently selective luteal infiltration of CD8+ cells was observed. MHC class II expression by non-steroidogenic cells was increased in samples from days 16-17, as was the number of infiltrating macrophages. Flow cytometry revealed very low expression of MHC class II by large luteal cells at all stages of the oestrous cycle. In early pregnancy, the number of CD4+ and CD8+ cells and macrophages decreased, as did MHC class II expression, compared with mid-dioestrous samples. B cells were present in very small numbers in all samples examined. Eosinophils were similarly sparsely distributed and numbers decreased further in pregnancy. After exogenous PGF2 alpha administration, populations of CD4+ cells and non-specific esterase staining cells were significantly smaller than after natural luteolysis, whereas eosinophil numbers were increased compared with samples from days 16-17. However, the number of CD8+ and CD5+ cells and MHC class II expression were not significantly different from those observed after natural luteolysis. These findings indicate that populations of immune cells in the equine corpus luteum vary during its lifespan. The selective increase in CD8+ cells before functional luteolysis indicates that they have a physiological role in the regression of the corpus luteum.     

A light and electron microscopic study of the periparturient bovine corpus luteum

Corpora lutea were obtained surgically from fifteen mature Angus crossbred cows representing three experimental groups of five cows each. Cows in Group A were 180 days of gestation, cows in Group B had recently experienced parturition (相似文献   

Determination of cell type specificity and estrous cycle dependency of monocyte chemoattractant protein-1 expression in corpora lutea of normally cycling rats in relation to apoptosis and monocyte/macrophage accumulation

In regressive corpora lutea, apoptosis of luteal cells, expression of monocyte chemoattractant protein-1 (MCP-1), and accumulation of monocytes/macrophages occur. However, whether these three events are correlated and what cell type expresses MCP-1 have yet to be determined. To clarify these issues, we performed histochemical examinations to determine the localization and the numbers of MCP-1 mRNA-containing cells, apoptotic cells, and monocytes/macrophages in corpora lutea of normally cycling rats. We found that the Mcp-1 gene is expressed in nonapoptotic steroidogenic luteal cells. Corpora lutea that contained MCP-1 mRNA-expressing cells increased in number at estrus together with those containing apoptotic luteal cells. When individual corpora lutea at estrus were analyzed, those with many MCP-1-expressing cells contained few apoptotic cells, and vice versa. These results collectively suggest the following pathway for apoptosis- and MCP-1-dependent regression of the corpus luteum: 1) luteal cells are induced to undergo apoptosis at estrus, and the activation of Mcp-1 gene expression follows in nonapoptotic luteal cells; 2) monocytes/macrophages are chemoattracted by MCP-1 toward corpora lutea containing apoptotic luteal cells; and 3) monocytes/macrophages invade corpora lutea and eliminate apoptotic luteal cells by phagocytosis.     

Effect of prostaglandin E2 alpha on the hCG-stimulated progesterone production by human corpora lutea

The effect of prostaglandin PGF2 alpha on the hCG stimulated and basal progesterone production by human corpora lutea was examined in vitro. hCG (40 i.u./ml) stimulated progesterone formation in corpora lutea of early (days 16-19 of a normal 28 day cycle), mid (days 20-22) and late (days 23-27) luteal phases. This stimulation was inhibited by PGF2 alpha (10 micrograms/ml) in corpora lutea of mid and late luteal phases. PGF2 alpha alone did not show a consistent effect on basal progesterone production. The inhibition of hCG stimulated progesterone production by PGF2 alpha at times corresponding to luteolysis indicates a role for that prostaglandin in the process of luteolysis in the human corpus luteum.     

Canine corpus luteum regression: apoptosis and caspase-3 activity

The present study evaluated the occurrence of apoptosis and caspase-3 activity in the canine corpus luteum during the period of luteal regression in eight pregnant and nine nonpregnant diestrus bitches. Intact luteal cells were obtained from corpora lutea in both peripartum pregnant bitches and nonpregnant diestrus bitches at approximately 65 d (range 63-68) after estrus, but not at days 75 and 85 in nonpregnant bitches. In all bitches, apoptotic cells were rarely detected and when present, those cells were more easily detected using the hematoxylin and eosin technique than using the critical electrolyte concentration technique. The luteal structures at 75 and 85 d of diestrus had histological characteristics similar to a corpus albicans. Caspase-3 activity was detected in morphologically normal corpora lutea from both pregnant and diestrus bitches around day 65, and also in the later structures considered corpus albicans tissue. These results suggested that apoptosis may not be the major mechanism involved in canine functional luteal regression, and that caspase-3 participated in both functional and morphological luteolysis and in the tissue reorganization involved in corpus albicans formation.     

The corpus luteum of the guinea pig. III. Cytochemical studies on the Golgi complex and GERL during normal postpartum regression of luteal cells, emphasizing the origin of lysosomes and autophagic vacuoles

The postpartum involution of corpora lutea was examined by electron microscope cytochemistry of guinea pig ovaries previously fixed by vascular perfusion, a method which produces optimal preservation of steroid-secreting cells and yet maintains enzyme activity. The intracellular digestive apparatus was identified through the localization of two acid hydrolases, acid phosphatase (ACPase) and arylsulfatase. Other marker enzymes localized were thiamine pyrophosphatase (in Golgi cisternae) and alkaline phosphatase (along plasma membranes). Prolonged osmication was used to mark the outer Golgi cisterna. The results demonstrate that luteal cell regression is characterized by a striking increase in the number of lysosomes and the appearance of numerous, double-walled autophagic vacuoles. Both lysosomes and the space between the double walls of autophagic vacuoles exhibit ACPase and arylsulfatase activity. In contrast to earlier periods, just before and during regression, Golgi complex-endoplasmic reticulum-lysosomes (GERL) is markedly hypertrophied, displaying intense acid hydrolase activity. On the basis of various criteria, GERL is proposed to function in the formation of lysosomes and autophagic vacuoles. Lysosomes seem to develop from GERL as focal protuberances of varying size and shape, which detach from the parent structure. Double- walled autophagic vacuoles, often large and complex in structure, initially are produced as GERL cisternae envelop small areas of cytoplasm. Lytic enzymes, perhaps furnished by the engulfing membranes and trapped lysosomes, presumably bring about digestion of the contents of these vacuoles, producing first aggregate-type inclusions, then, as the contents are further degraded, myelin figure-filled residual bodies. ACPase activity occasionally appears within smooth endoplasmic reticulum tubules and cisternae in advanced regression, possibly suggesting that lytic enzymes utilize this membrane system as an access route to GERL. These data indicate that cellular autophagy is a prominent mechanism underlying luteal cell involution during normal postpartum degeneration of guinea pig corpora lutea. Furthermore they suggest that in regressing luteal cells GERL is responsible for packaging acid hydrolases into lytic bodies.     

Localization of relaxin in human gestational corpus luteum

Summary Corpora lutea from 12 pregnant women were prepared for immunohistochemical localization of relaxin using a highly specific antiserum. A positive response is given by luteal cells that are diffusely distributed throughout the corpus luteum. These cells do not form a distinctive group in any particular area. A negative response is seen in the adjacent ovarian tissue, and also in nongestational corpora lutea in an early luteal phase.     

Evidence for a switch in the site of relaxin production from small theca-derived cells to large luteal cells during early pregnancy in the pig

The presence of immunoreactive relaxin was studied in corpora lutea of sows during the oestrous cycle and early pregnancy by immunohistochemistry and radioimmunoassay using three different anti-relaxin sera. Sections were immunostained using the peroxidase-anti-peroxidase or the immunogold-silver technique. Before Day 14, staining in corpora lutea from non-pregnant and pregnant animals was indistinguishable. With all antisera, no immunostaining was seen on Day 3, but was detected on Days 5-7 in cells from the theca interna. In non-pregnant animals, this immunostaining decreased and by Day 15 only an occasional large cell in the centre of the corpus luteum was stained. No staining was seen by Day 22. The relaxin content of corpora lutea measured by radioimmunoassay remained low throughout the luteal phase. In contrast, the amount of immunoreactive relaxin in corpora lutea rose dramatically (140-fold) between Days 11 and 14 of pregnancy and by Day 14 of pregnancy immunostaining was seen in the majority of large luteal cells. By Day 20 of pregnancy the concentrations of immunoreactive relaxin had further increased. Histochemical staining for alkaline phosphatase suggested that, while the relaxin-immunoreactive cells seen in the early luteal phase may be theca-derived, those during early pregnancy may be derived from the granulosa. The results are compatible with the suggestion that relaxin is produced by theca-derived cells during the early luteal phase and that between Days 11 and 14 there is a switch in the site of relaxin synthesis from theca-derived cells to granulosa-derived large luteal cells. In the absence of luteolysis, as during pregnancy, this switch is accompanied by a dramatic increase in relaxin synthesis.     

Effect of exogenous prostaglandin and/or estrogen on luteolysis after electrocauterization of the largest follicles at the end of the bovine estrous cycle

In cows, electrocauterization of large follicles at the end of the luteal phase, lengthened the life span of corpora lutea. Injection of 5 mg of estradiol valerate or of 1 mg of an analogue of prostaglandin F(2alpha) induced luteolysis; however, the injection of estrogen was associated with precocious estrus without either ovulation or corpus luteum growth. Injection of both estradiol valerate and prostaglandin analogue gave the same results as estradiol valerate alone. Deferred luteolysis, observed after electrocauterization of large follicles, seemed to be due to the withdrawal of estrogens and the consequent lack of prostaglandin F(2alpha) production.     

Concentration of prostaglandins PGE and PGF, estrone, estradiol, and progesterone in human corpora lutea

The concentrations of prostaglandins PGE and PGF, estrone, estradiol and progesterone in human corpora lutea were measured by radioimmunoassay at various stages of the luteal phase of the menstrual cycle. The concentrations of PGF were found to be significantly higher in both the mid and late luteal phases than in the early luteal phase. In the mid luteal phase there was a concomittant increase in PGE levels, but these levels had declined in the late luteal phase. Steroid concentrations were generally lower in the late luteal phase.

It has been postulated that in the human corpus luteum locally produced prostaglandins may be responsible for luteolysis. Our data on the concentrations of PGF and PGE in corpora lutea at various stages of the luteal phase support such a possibility.     

Actions of prostaglandin F2alpha and prolactin on intercellular adhesion molecule-1 expression and monocyte/macrophage accumulation in the rat corpus luteum

Expression of intercellular adhesion molecule-1 (ICAM-1) and the accumulation of monocytes/macrophages are inflammatory events that occur during PRL (PRL)-induced regression of the rat corpus luteum. Here we have compared the ability of prostaglandin F2alpha (PGF) and PRL to induce, in rat corpora lutea, inflammatory events thought to perpetuate luteal regression. Immature rats were ovulated with eCG-hCG and then hypophysectomized (Day 0), which resulted in a single cohort of persistent, functional corpora lutea. On Days 9-11, the rats received twice daily injections of saline, PGF (Lutalyse, 250 microg/injection), or PRL (312 microg/injection) to induce luteal regression. Surprisingly, luteal weight and plasma progestin concentrations (progesterone and 20alpha-dihydroprogesterone) did not differ between PGF-treated rats and controls; whereas both luteal weight and plasma progestins declined significantly in PRL-treated rats. Furthermore, corpora lutea of PGF-treated rats and controls contained relatively minimal ICAM-1 staining and few monocytes/macrophages. In contrast, but as expected, corpora lutea of PRL-treated rats stained intensely for ICAM-1 and contained numerous monocytes/macrophages. In an additional experiment, there was no indication that luteal prostaglandin F2alpha receptor mRNA diminished as a result of hypophysectomy. These findings suggest that prolactin, not PGF, induces the inflammatory events that accompany regression of the rat corpus luteum.     

First postpartum ovulations and corpora lutea in ewes which lamb in the breeding season

Two experiments involving crossbred ewes which lambed during the breeding season were performed to determine whether: (a) the interval to first postpartum ovulation could be reduced by weaning or mastectomy; (b) there are differences in luteal structure and luteinizing hormone (LH) receptor concentration between first postpartum corpora lutea induced with GnRH and normal cycling corpora lutea and (c) pretreatment of postpartum ewes with progesterone would affect luteal LH receptor concentration and luteal phase serum progesterone concentration.In experiment I, the mean interval (±SEM) to the first postpartum ovulation was 22.3 ± 1.1 days and was not significantly altered by weaning or mastectomy. More than half of the ewes had small, short-lived peaks of serum progesterone associated with short-lived corpora lutea prior to the normal luteal phase rise of serum progesterone. In experiment II, 2 h after GnRH injection on day 18 postpartum, serum LH concentrations were higher in ewes which received progesterone treatment on days 13 and 14 than in control ewes. Progesterone treatment did not affect mean corpus luteum weight (157 mg) or concentration of LH receptors (0.95 fmol/mg) in first postpartum corpora lutea, but progesterone-treated ewes had significantly higher endogenous serum progesterone concentrations on days 21–24. GnRH-induced corpora lutea from postpartum ewes were lighter in weight, paler in color, had lower LH receptor concentrations and had a more regressed histological appearance than corpora lutea of a similar age from normal, cycling ewes.     

Immunocytochemical evidence for the presence of oxytocin and neurophysin in the large cells of the bovine corpus luteum

Summary The presence of neurophysin, oxytocin and vasopressin in the bovine corpus luteum was examined immunocytochemically. Tissue blocks of corpora lutea from pregnant and non-pregnant animals were fixed with glutaraldehyde/paraformaldehyde fixative and immunostained by the peroxidase-antiperoxidase (PAP) method. The simultaneous presence of immunoreactive oxytocin and immunoreactive oxytocin-neurophysin was demonstrated in large luteal cells of non-pregnant animals, while no staining for vasopressin or vasopressin-neurophysin was observed. None of the peptides were detected in the corpus luteum of pregnant animals. The small luteal cells were not found to be stainable at any time.     

Characterization of large luteal cells and their secretory granules during the estrous cycle of the cow.

Large steroidogenic cells of the bovine corpora lutea were evaluated for morphological changes on Days 3, 7, 11, 14, 17, and 19 of the estrous cycle. Large cells were readily identified by size (25-50 microns diameter), numerous mitochondria, and the presence of dense secretory granules (150-300 nm in diameter). These granules were found in a discrete cluster and were not dispersed throughout the cytoplasm. Only 3% of the large cells contained a cluster of granules on Day 3. The percentage was highest during midcycle (Day 7, 84%; Day 11, 64%), dropped on Day 14 (26%), and was lowest on Days 17 (16%) and 19 (8%). Electron microscopic immunocytochemistry showed that oxytocin and neurophysin were co-localized in these granules on all days evaluated. As early as Day 14, large cells were observed with characteristics typical of regressing corpora lutea, i.e., a reduction in cells with secretory granules, large cytoplasmic lipid droplets, and swollen mitochondria with dense inclusions. However, since this was a time of the cycle when plasma concentrations of progesterone were very high, this corpus luteum is referred to as involutive rather than regressive. Our results may be summarized as follows: 1) from Day 7 to Day 14 there was a 69% decline in the number of large cells containing oxytocin-laden secretory granules. This occurred prior to the rise in uterine oxytocin receptors and the large luteolytic pulses of prostaglandin that reportedly occur after Day 14. The role of this apparent early release of oxytocin is not known. 2) Large steroidogenic luteal cells of the estrous cycle have morphological characteristics similar to those of large luteal cells during pregnancy. However, large luteal cells of the estrous cycle contain oxytocin whereas those of pregnancy are devoid of oxytocin.     

Immunohistochemical localization of estrogen receptors in the ovine corpus luteum throughout the estrous cycle

Using immunohistochemistry and Western blot analysis we attempted to identify the estrogen receptors in ovine luteal cells at different stages of the estrous cycle. Monoclonal antibody against estrogen receptors was used for immunolocalization of estrogen receptor-alpha in corpora lutea sections. Generally, the most intense cytoplasm staining was present in large luteal cells. On the 6th day of the estrous cycle, weak immunostaining of estrogen receptors was observed in large luteal cells as well as in the connective tissue. Luteal cells from regressing corpora lutea expressed the weakest immunostaining. The most intense immunoreactivity for estrogen receptors was found in sections of corpora lutea collected on the 9th day of the cycle. Both, cytoplasmic and nuclear localization was observed depending on cell types in the ovine corpus luteum. Our studies demonstrated the presence of the estrogen receptor-alpha in the luteal cells and suggested an autocrine/paracrine role of estrogen in the regulation of estrous cycle in sheep.     

Effects of prostaglandin F2 alpha-induced luteolysis on the populations of cells in the ovine corpus luteum

Receptors for prostaglandin (PG) F2 alpha in the ovine corpus luteum are localized on large steroidogenic luteal cells. Therefore, it was hypothesized that during luteolysis, the first demonstrable effects of PGF2 alpha would occur in the population of large luteal cells. To test this hypothesis, the numbers and sizes of large and small luteal cells, fibroblasts, capillary endothelial cells, and pericytes were determined in corpora lutea collected 12, 24, or 36 h (6 animals/group) following administration of PGF2 alpha on Day 10 postestrus and from untreated ewes on Days 10 and 12 postestrus. The numbers and sizes of luteal cells were determined after enzymatic dissociation of the luteal tissue into single cell suspensions and by morphometric analysis of luteal slices. Serum levels of progesterone decreased (p less than 0.05) within 12 h of treatment, indicating that luteolysis was induced. Recovery of the two types of steroidogenic luteal cells following enzymatic dissociation was different (p less than 0.05). Recovery of both steroidogenic cell types decreased with time after PGF2 alpha treatment, suggesting that they had become more fragile. As determined by morphometry, the number of large luteal cells was not different at any time point examined; however, by 36 h after treatment, the average diameter of large luteal cells had decreased (p less than 0.05). In contrast, by 24 h after treatment, there was a decrease in the number of small luteal cells (p less than 0.05) but no change in their diameter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Luteal regression in the normally cycling rat: apoptosis, monocyte chemoattractant protein-1, and inflammatory cell involvement

In hypophysectomized rats, prolactin induces regression of the corpora lutea. Luteal regression is accompanied by infiltration of monocytes/macrophages, declines in luteal mass and plasma progestins, and increased staining for monocyte chemoattractant protein-1 (MCP-1). We investigated whether similar events are induced during the estrous cycle, after the proestrous prolactin surge. Rats were killed on proestrus or on estrus, and one ovary was frozen for immunohistochemical detection of MCP-1, monocytes/macrophages (ED1-positive), and differentiated macrophages (ED2-positive) and for in situ detection of apoptotic nuclei. Corpora lutea of the current (proestrus) or preceding (estrus) cycle were dissected from the ovaries of additional rats and frozen for the same analyses and for determination of total protein content. In sections of whole ovaries, intensity and distribution of MCP-1 staining were increased in corpora lutea of multiple ages on estrus as compared to proestrus, as were numbers of differentiated macrophages and apoptotic nuclei per high-power field. Sections of isolated corpora lutea showed these increases on estrus, and the number of monocytes/macrophages per high-power field was also significantly increased. Accompanying these inflammatory/immune events, the corpora lutea on estrus showed decreased weight and total protein per corpus luteum, as compared to corpora lutea on proestrus. These changes are consistent with a proposed role for prolactin in the initiation of luteal apoptosis and of a sequence of inflammatory/immune events that accompany regression of the rat corpus luteum during the normal estrous cycle.