Morphometric analysis of cell types in the ovine corpus luteum throughout the estrous cycle

The cellular composition of ovine corpora lutea obtained during the early (Day 4), mid (Days 8 and 12), and late (Day 16) stages of the estrous cycle was determined by morphometric analysis. Individual corpora lutea were collected via midventral laparotomy from a total of 19 ewes. A center slice from each corpus luteum was processed for electron microscopy and subsequent morphometric analysis of the numbers and sizes of steroidogenic and nonsteroidogenic cells. Luteal weight progressively increased throughout the estrous cycle (p less than 0.05). Corpora lutea collected on Day 16 were assigned to one of two subgroups on the basis of gross appearance and weight: nonregressed (NR, 542 +/- 25 mg) or regressed (R, 260 +/- 2 mg). There were no significant changes in the proportion of the corpus luteum occupied by small luteal cells (19 +/- 2%) or large luteal cells (36 +/- 1%) throughout the estrous cycle. The total number of steroidogenic cells per corpus luteum increased from 21.8 +/- 3.7 (X 10(6)) on Day 4 to 61.7 +/- 5.4 (X 10(6)) on Day 8 (p less than 0.05) and remained elevated thereafter. The number of small luteal cells was 10.0 +/- 2.7 (X 10(6)), 39.7 +/- 1.4 (X 10(6)), 46.1 +/- 5.8 (X 10(6)), 49.0 +/- 13.7 (X 10(6)), and 29.9 +/- 8.6 (X 10(6)) on Days 4, 8, 12, 16 (NR), and 16 (R), respectively (p less than 0.05, Day 4 vs. Days 8, 12, 16 NR). In contrast, the number of large luteal cells was 11.8 +/- 1.5 (X 10(6)) on Day 4 and did not vary significantly during the remainder of the estrous cycle. The numbers of nonsteroidogenic cell types increased (p less than 0.05) from Day 4 to Day 16 (NR) but were decreased in regressed corpora lutea (Day 16 R). Regression was characterized by a 50% decrease (p less than 0.05) in the total number of cells per corpus luteum from 243 +/- 57 ( X 10(6)) on Day 16 (NR) to 125 +/- 14 ( X 10(6)) on Day 16 (R) (p less than 0.05). Small luteal cells remained constant in volume throughout the entire estrous cycle (2520 +/- 270 microns 3), whereas large luteal cells increased in size from 5300 +/- 800 microns 3 on Day 4 to 16,900 +/- 3300 microns 3 on Day 16 (NR) (p less than 0.05). In summary, small luteal cells increased in number but not size throughout the estrous cycle, whereas large luteal cells increased in size but not number.     

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.     

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.     

Size distribution of ferret luteal cells during pregnancy

Steroidogenic cells in the corpus luteum of the ferret (Mustela putorius) during early (Days 6 and 13) to midpregnancy (Day 24) were characterized using electron microscopy, immunocytochemical localization of neurophysin, and smears of dispersed cells obtained by dissociating luteal cells with collagenase. The latter were stained for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity, and the diameters of the cells were determined with an ocular micrometer. Very small cells (less than 12 microns) stained negative for 3 beta-HSD, occurred in clumps of 5-50 cells, and were presumed to be primarily endothelial cells. 3 beta-HSD-positive cells covered a wide spectrum of sizes ranging from 14 to 56 microns and did not exist as two discrete populations. The ratio of small (less than 25 microns) to large (greater than 25 microns) cells was 1.86:1.0 on Day 6, with the 17- to 20-microns cell size class predominating. On the day of implantation (Day 13), about 75% of the cells ranged from 26 to 50 microns, with the 29-microns size predominating. By Day 24, the ratio of small-to-large cells had declined to 0.15. Nearly 90% of the cells were in the 26- to 56-microns range, the predominant size being 35 microns. All size classes of luteal cells stained negative for neurophysin on all 3 days of pregnancy studied. Luteal cells obtained on Days 6, 13, and 24 of pregnancy failed to reveal any evidence of mitosis after in vivo or in vitro colchicine treatment. We interpret these results as indicating that the 3 beta-HSD-positive luteal cells of ferrets progressively increase in size as small luteal cells complete their differentiation from granulosa cells and ultimately form larger luteal cells with somewhat different ultrastructural characteristics.     

Isolation and characterization of cell subpopulations from the monkey corpus luteum of the menstrual cycle

This study was designed to test the hypothesis that the corpus luteum of primate species consists of cell subpopulations that differ in physical characteristics, function, and regulation by endocrine and paracrine factors. The corpus luteum (n = 25) was removed from rhesus monkeys at the mid-luteal phase of the menstrual cycle (Days 7-8 after the surge of luteinizing hormone, LH) and enzymatically dispersed. Freshly dispersed cells were analyzed and sorted on the basis of their forward and 90 degrees light scatter (FLS and 90LS, respectively) properties using an EPICS C flow cytometer. Freshly dispersed and sorted cells were fixed, stained histochemically for the presence of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and measured to determine their diameters. Freshly dispersed (MIX) and sorted cells from corpora lutea during the early (Days 4-5 after the LH surge; n = 4) and mid-luteal phases of the cycle were incubated in vitro and steroid production was assessed. The size distribution of dispersed cells revealed four peaks that corresponded to small (10-15 microns in diameter) 3 beta-HSD-negative, and small, medium (16-20 microns), and large (greater than 20 microns) 3 beta-HSD-positive cells. Analysis of dispersed cells for FLS and 90LS demonstrated two continua (C alpha and C beta). C alpha contained single cells and cell clusters; 99.7 +/- 0.3% (n = 3) of the cells were less than or equal to 15 microns in diameter and 96.7 +/- 0.3% were 3 beta-HSD-negative. C alpha cells produced low levels of progesterone (0.2 +/- 0.1 ng/ml per 5 x 10(4) cells; n = 3) in vitro under basal conditions. C beta consisted of single cells from 10 microns to 40 microns in diameter and contained the lipid-filled and 3 beta-HSD-positive cells. Two regions (R1 and R3) of C beta were defined and their cells separated. In R1, 96 +/- 2% (n = 3) of the cells had diameters of less than or equal to 15 microns, whereas 82 +/- 4% (n = 3) of those in R3 were greater than or equal to 20 microns. Basal progesterone production by R3 cells from early luteal phase of the cycle was 12 times greater than that by R1 cells (n = 3 per group).(ABSTRACT TRUNCATED AT 400 WORDS)     

Prostaglandin metabolism in the ovine corpus luteum: catabolism of prostaglandin F(2alpha) (PGF(2alpha)) coincides with resistance of the corpus luteum to PGF(2alpha)

To examine possible mechanisms involved in resistance of the ovine corpus luteum to the luteolytic activity of prostaglandin (PG)F(2alpha), the enzymatic activity of 15-hydroxyprostaglandin dehydrogenase (PGDH) and the quantity of mRNA encoding PGDH and cyclooxygenase (COX-2) were determined in ovine corpora lutea on Days 4 and 13 of the estrous cycle and Day 13 of pregnancy. The corpus luteum is resistant to the action of PGF(2alpha) on Days 4 of the estrous cycle and 13 of pregnancy while on Day 13 of the estrous cycle the corpus luteum is sensitive to the actions PGF(2alpha). Enzymatic activity of PGDH, measured by rate of conversion of PGF(2alpha) to PGFM, was greater in corpora lutea on Day 4 of the estrous cycle (P < 0.05) and Day 13 of pregnancy (P < 0.05) than on Day 13 of the estrous cycle. Levels of mRNA encoding PGDH were also greater in corpora lutea on Day 4 of the estrous cycle (P < 0. 01) and Day 13 of pregnancy (P < 0.01) than on Day 13 of the estrous cycle. Thus, during the early estrous cycle and early pregnancy, the corpus luteum has a greater capacity to catabolize PGF, which may play a role in the resistance of the corpus luteum to the actions of this hormone. Levels of mRNA encoding COX-2 were undetectable in corpora lutea collected on Day 13 of the estrous cycle but were 11 +/- 4 and 44 +/- 28 amol/microgram poly(A)(+) RNA in corpora lutea collected on Day 4 of the estrous cycle and Day 13 of pregnancy, respectively. These data suggest that there is a greater capacity to synthesize PGF(2alpha), early in the estrous cycle and early in pregnancy than on Day 13 of the estrous cycle. In conclusion, enzymatic activity of PGDH may play an important role in the mechanism involved in luteal resistance to the luteolytic effects of PGF(2alpha).     

Immunocytochemical localization of relaxin in corpora lutea of sows throughout the estrous cycle

Porcine relaxin has been sought by localization in the corpus luteum of sows on Days 3, 7, 9, 11, 12, 15, 18, 19, and 21 of the estrous cycle, using the avidin-biotin immunoperoxidase method and an antiserum to purified porcine relaxin. Simultaneous localization of relaxin in corpora lutea from sows on Days 108 and 113 of pregnancy was used to compare the intensity of immunostaining with that of corpora lutea of cyclic animals. However, the antiserum dilution necessary for optimal localization differed considerably in these two states (1:10,000 in pregnancy and 1:750 in the cycle), suggesting that lower levels of antigen are present in the luteal cells of the cycle. Relaxin immunostaining was undetectable on Day 3 of the cycle but became evident by Days 7 and 9. At Day 11 staining intensity increased and persisted through Day 15. On Day 18 some stain was still evident, but by Days 19, 20, and 21 there was complete absence of immunostain. Relaxin immunostaining appeared to be located throughout the cytoplasm of the luteal cell, as clear areas in the nuclear region were often observed. The results suggest that relaxin is produced in low amounts by the luteal cells of the cyclic sow and that the levels fluctuate with stage of the cycle. Lack of evidence from radioimmunoassay for a surge of relaxin secretion into the systemic circulation prior to luteolysis in the pig estrous cycle suggests that the relaxin localized in the luteal cells of the cycle may have an intraovarian function.     

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)     

Morphometric quantification of mitochondria in the two steroidogenic ovine luteal cell types

Progesterone secretion is regulated by different mechanisms in large and small steroidogenic ovine luteal cells. Large cells secrete approximately 7-fold more progesterone in an unstimulated state than small cells. Since cholesterol side-chain cleavage, which is catalyzed by an inner mitochondrial membrane enzyme complex, is a major rate-limiting step in progesterone synthesis, mitochondrial components were quantified in the two steroidogenic cell types throughout the estrous cycle. Corpora lutea collected on Days 4 (n = 4), 8 (n = 4), 12 (n = 5), and 16 (n = 6) of the estrous cycle were prepared for electron microscopy. Volume densities of cell types within corpora lutea and mitochondrial densities within cell types were estimated by point-counting; nuclear and cytoplasmic volume densities were estimated by planimetric analysis. A total of 570 micrographs (magnification 5300 X) were analyzed. Large cell volume density was unchanged during the cycle (35 +/- 1%) while small cell volume density increased (p less than 0.05) from 13 +/- 1% on Day 4 to 20 +/- 3% on Day 12. Large cell mitochondrial volume density increased (p less than 0.05) from 13 +/- 1% on Day 4 to 23 +/- 1% on Day 16 accompanied by an increase in cytoplasmic volume density such that nuclear to cytoplasmic ratio increased (p less than 0.05) from 1:14 to 1:34 between Days 4 and 16. Small cell mitochondrial volume density increased from 11 +/- 1% on Day 4 to 14 +/- 1% (p less than 0.05) for the rest of the cycle while the nuclear to cytoplasmic ratio remained at 1:14.(ABSTRACT TRUNCATED AT 250 WORDS)     

A morphometric analysis of the corpus luteum of the cow during the estrous cycle and early pregnancy

Corpora lutea were collected from cows on Days 6, 8, 10, 12, 14, 16, 18 and 19 of the estrous cycle and early pregnancy (n=2/d) and were examined by light microscopy. Mean lutein cell diameter was significantly (P<0.05) greater in pregnant than in cyclic cows on Days 6, 8, 10, 12, 16, 18 and 19 (cyclic versus pregnant: Day 6: 13.9 +/- 0.22 vs 14.9 +/- 0.24; Day 8: 13.8 +/- 0.20 vs 15.4 +/- 0.2; Day 10: 14.8 +/- 0.24 vs 17.4 +/- 0.24; Day 12: 13.2 +/-0.25 vs 17.9 +/- 0.31; Day 16: 13.9 +/- 0.28 vs 16.5 +/- 0.31; Day 18: 13.0 +/- 0.22 vs 16.5 +/- 09.36, and Day 19: 15.0 +/- 0.23 vs 17.6 +/- 0.33 mum, respectively). The distribution of cell sizes was leptokurtotic throughout the estrous cycle and the first 10 d of pregnancy, but tended towards bimodality after Day 14 of pregnancy. The proportion of lutein cell cytoplasm occupied by vacuoles was lower in pregnant than in cyclic cows from the 12th day post estrus, but there was a marked (P<0.05) increase in vacuolation of cells from cows undergoing luteolysis. Stainable intercellular collagen was also less abundant in pregnant than cyclic cows from the 12th day post estrus. The higher rate of progesterone secretion of pregnant, compared with cyclic cows may be attributed to the greater numbers and greater contribution to luteal mass of large lutein cells in the corpus luteum of pregnancy.     

Bovine membrane-type 1 matrix metalloproteinase: molecular cloning and expression in the corpus luteum

Matrix metalloproteinase-2 (MMP-2) is produced as a zymogen, which is subsequently activated by membrane-type 1 metalloproteinase (MT1-MMP). The objectives of the present study were to clone bovine MT1-MMP and to investigate its expression in the corpus luteum. Corpora lutea were harvested from nonlactating dairy cows on Days 4, 10, and 16 of the estrous cycle (Day 0 = estrus; n = 3 for each age). The bovine MT1-MMP cDNA contained an open reading frame of 1749 base pairs, which encoded a predicted protein of 582 amino acids. Northern blotting revealed no differences (P > 0.05) in MT1-MMP mRNA levels between any ages of corpora lutea. Western blotting demonstrated that two species of MT1-MMP, the latent form ( approximately 63 kDa) and the active form ( approximately 60 kDa), were present in corpora lutea throughout the estrous cycle. Active MT1-MMP was lower (P < 0.05) in early stages of the corpus luteum than the mid and late stages, where MMP-2 activity, as revealed by gelatin zymography, was also elevated. Furthermore, immunohistochemistry revealed that MT1-MMP was localized in endothelial, large luteal, and fibroblast cells of the corpus luteum at different stages. Taken together, the differential expression and localization of MT1-MMP in the corpus luteum suggest that it may have multiple functions throughout the course of the estrous cycle, including activation of pro-MMP-2.     

Size distribution and hormonal responsiveness of dispersed rabbit luteal cells during pseudopregnancy

Due to the evidence for two distinct steroidogenic cell types in corpora lutea of large domestic animals, cells of the rabbit corpus luteum were characterized with respect to cell diameters, relative abundance, steroidogenic capacity and responsiveness to hormones. Pseudopregnancy was induced in New Zealand rabbits by injection of 30-160 IU pregnant mare's serum gonadotropin (PMSG) followed in 2-4 days by an i.m. injection of 20-35 micrograms gonadotropin-releasing hormone (GnRH). Corpora lutea were obtained 2, 5 and 9 days after injection of GnRH and dissociated into single cell suspensions. Suspended steroidogenic cells were incubated (2 h, 37 degrees C) in medium 199 alone or in medium containing ovine luteinizing hormone (oLH) (100 ng/ml), or isoproterenol (100 microM). Media were collected and assayed for progesterone content. Secretion of progesterone (means +/- SE, n = 4) was stimulated (p less than 0.05) by oLH on each day: Day 2 = 1.7 +/- 0.2-fold; Day 5 = 3.5 +/- 0.4-fold; and Day 9 = 3.1 +/- 0.6-fold stimulation above controls. Isoproterenol also stimulated (p less than 0.05) secretion of progesterone by suspended luteal cells on Days 2 and 9. Microscopic examination of cell suspensions stained for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity provided identification of cells with steroidogenic capacity. The diameters (means +/- SE) for steroidogenic cells increased (p less than 0.05) from Days 2 to 9 (Day 2 = 15.2 +/- 0.2 micron; Day 5 = 22.4 +/- 0.4 micron; Day 9 = 28.3 +/- 1.6 micron). The large cell to small cell ratio increased from 0.01 on Day 2 to 2.03 on Day 9.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Size distribution of luteal cells from pregnant and non-pregnant marmoset monkeys and a comparison of the morphology of marmoset luteal cells with those from the human corpus luteum

The size distribution of marmoset luteal cells was determined on Days 6, 14 and 20 after ovulation in non-pregnant cycles and in early pregnancy. Image analysis was used to estimate the cell diameter of dispersed cells prepared from the marmoset corpus luteum (CL). Steroidogenic cells showed a size distribution consistent with one population of cells. There was a significant increase in mean cell diameter (P less than 0.05) from Day 6 to Day 14 in pregnant and non-pregnant animals with no further increase on Day 20. Micrographs of marmoset luteal tissue showed cells of greater than 10 micron containing the organelles typical of steroid-producing cells, and smaller non-steroidogenic cells surrounding the steroid-producing cells. On the basis of microscopy, there were no areas within the CL where cell composition was noticeably different. In contrast, micrographs of human luteal tissue showed two types of steroidogenic cell; most cells were similar to those in the marmoset CL but a smaller population of smaller cells could be distinguished around the periphery and along vascular septa. It is likely that these smaller and larger types of steroidogenic cells are of theca and granulosa cell origin respectively, the two cell populations differing in the degree of electron density and amount of rough endoplasmic reticulum. A distinguishing feature between marmoset and human luteal cells was the shape of the mitochondrian which were considerably rounder in marmoset luteal cells. The origin of steroidogenic cells in the marmoset CL is unclear, although in marmosets and man the luteal cell types display morphological characteristics distinct from the large and small luteal cells described for CL of the domestic ungulates.     

Changes in lipid contents and fatty acid compositions in ovine corpora lutea during the estrous cycle and early pregnancy

Changes in lipid contents and fatty acid compositions of each lipid fraction were examined in corpora lutea from 34 unmated ewes between Days 8 and 16 of the estrous cycle and from 6 ewes at Day 16 of pregnancy. Four patterns were observed during advancement of the estrous cycle. Luteal concentrations of free cholesterol and triglyceride (neutral lipids) increased between Days 14 and 16, during luteal regression, in a manner approximated by exponential functions of time, whereas luteal concentrations of phospholipid (polar lipids) increased and then decreased between Days 8 and 16 in a manner approximated by a sin function of time. Likewise, within the various lipid class component fatty acids, changes in palmitic acid weight percentages were approximated by sin functions of time, whereas arachidonic acid weight percentages increased between Days 14 and 16 in a manner approximated by exponential functions of time. Pregnancy either inhibited or reversed the changes in luteal lipid profiles, especially arachidonic acid percentages, between Days 14 and 16 of the estrous cycle. Luteal lipid profiles of corpora lutea from Day 16 pregnant sheep approximated lipid profiles of corpora lutea recovered from sheep between Days 12 and 14 of the estrous cycle. Comparison of luteal lipid profiles after tissue incubations at either 0 or 37 degrees C for 2 h revealed an effect of reproductive status on fatty acid metabolisms at Day 16. Changes observed in luteal lipid contents and fatty acid compositions during advancement of the estrous cycle represent aspects of lutein cell maturation and impending senescence that can be inhibited or reversed by pregnancy.     

Effect of luteinizing hormone and human chorionic gonadotropin on cell populations in the ovine corpus luteum

Two experiments were conducted to examine the effect of treatment with human chorionic gonadotropin (hCG) or ovine luteinizing hormone (LH) on the number and size distribution of steroidogenic luteal cells. In Experiment I, 27 ewes were assigned to one of three groups: 1) hCG (300 IU, i.v.) administered on Days 5 and 7.5 of the estrous cycle (Day 0 = Estrus); 2) LH (120 micrograms, i.v.) administered at 6-h intervals from Days 5 to 10 of the cycle; 3) saline (i.v.) administered as in the LH treatment group. Blood samples were drawn daily from the jugular vein for quantification of progesterone. On Day 10, corpora lutea were collected, decapsulated, weighed, and dissociated into single cell suspensions. Cells were fixed, stained for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity, and the size distribution of 3 beta HSD-positive cells was determined. Treatment with hCG, but not LH, increased (p less than 0.05) concentrations of progesterone in serum and the weight of corpora lutea. Treatment with either hCG of LH increased the proportion of cells greater than 22 micron in diameter and decreased the proportion of cells less than or equal to 22 micron (p less than 0.01). The ratio of small to large luteal cells decreased after treatment with either hCG or LH (p less than 0.05). In Experiment II, 9 ewes were assigned to one of two groups: 1) LH (120 micrograms, i.v.) administered at 6-h intervals from Days 5 to 10 of the estrous cycle, and 2) saline (i.v.) administered as in the LH treatment group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in rat luteal ultrastructure and P450scc mRNA and protein content after in vivo treatment with a gonadotropin-releasing hormone agonist

Previous studies have demonstrated that plasma progesterone levels decrease in pregnant rats treated in vivo with a gonadotropin-releasing hormone agonist (GnRH-Ag), without changes in testosterone or estradiol levels in ovarian vein plasma. The objective of this study was to determine the loci of GnRH-Ag disruption of progesterone synthesis by examining luteal mitochondria, lipid droplets, cellular composition, and P450 side-chain cleavage (P450scc) enzyme and mRNA content in the pregnant rat. On Day 7 or 11 of pregnancy, osmotic minipumps containing GnRH-Ag were implanted into 5-7 rats. Sham operations were performed on 5-6 controls at each time period. Five micrograms per day of GnRH-Ag were released for about 24 h, after which corpora lutea and jugular vein plasma were collected. The corpora lutea were prepared for microscopy or analyzed for P450scc enzyme and mRNA content. Plasma progesterone levels were measured by RIA. In those rats treated with GnRH-Ag, progesterone levels had decreased, and within the luteal cells, there was an increase in the number of lipid droplets and a decrease in the number of tubular cristae within the mitochondria. Concomitantly, P450scc enzyme and mRNA content decreased on both Day 8 and Day 12 of pregnancy. Also, GnRH-Ag treatment decreased the ratio of large to small steroidogenic luteal cells on Day 8 of pregnancy, but did not alter cellular ratios on Day 12 of pregnancy. These observations suggest that treatment with GnRH-Ag inhibits progesterone synthesis by decreasing the amount of P450scc mRNA and enzyme content, which may alter the mitochondrial cristae structure on Day 8 and Day 12 of pregnancy. The reduction in tubular cristae and P450scc enzyme in the mitochondria may account for the increase in lipid droplets, as less cholesterol is converted to pregnenolone. An additional mechanism of inhibition may be the reduction in the number of large steroidogenic luteal cells, which appear to be the major source of progesterone in the rat corpus luteum on Day 8 of pregnancy.     

Progesterone production by monkey luteal cell subpopulations at different stages of the menstrual cycle: changes in agonist responsiveness.

Small (less than or equal to 15 microns diameter) and large (greater than 20 microns diam.) luteal cells of the rhesus monkey have been separated by flow cytometry based on light scatter properties. To determine whether the steroidogenic ability and agonist responsiveness of luteal cell subpopulations vary during the life span of the corpus luteum, small and large cells were obtained at early (Days 3-5), mid (Days 7-8), mid-late (Days 11-12), and late (Days 14-15) luteal phase of the cycle. Cells (n = 4 exp./group) were incubated in Ham's F-10 medium + 0.1% BSA for 3 h at 37 degrees C with or without hCG (100 ng/ml), prostaglandin E2 (PGE2; 14 microM), dibutyryl-cAMP (db-cAMP; 5 mM), or pregnenolone (1 microM). Basal progesterone (P) production by large cells was up to 30-fold that by small cells depending on the stage of the cycle. HCG stimulated (p less than 0.05) P secretion by both small (1.8 +/- 0.2-fold) and large (3.7 +/- 0.7-fold) cells in the early luteal phase. HCG responsiveness declined during the luteal lifespan; P production by small cells was not significantly enhanced by hCG by mid luteal phase, whereas that by large cells was stimulated 1.7 +/- 0.2-fold (p less than 0.05) even at late luteal phase. Cell responses to db-cAMP were similar to those for hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Macrophage migration inhibitory factor in the bovine corpus luteum: characterization of steady-state messenger ribonucleic acid and immunohistochemical localization

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine produced by T cells and macrophages. A number of tissues also produce MIF during states of active differentiation and/or proliferation. The purpose of this study was to determine whether MIF is present in the corpus luteum (CL). The steady-state mRNA for MIF was examined in CL by Northern analysis on Day 5, Days 9-12, and Day 18 of the estrous cycle and at 0.5, 1, 4, 12, 24, and 36 h after a luteolytic injection of prostaglandin F(2alpha) (PGF(2alpha)) (n = 4 CL per time point). The greatest amount of MIF mRNA was observed in Day 5 CL compared with midcycle and Day 18 CL. Messenger RNA for MIF in CL collected 0.5 h post-PGF(2alpha) was greater than in midcycle and all other regressing CL. Immunohistochemical analysis (n = 4) revealed that MIF was present in the bovine CL throughout the estrous cycle and appeared to be localized to large luteal cells. It was concluded that MIF is produced within the bovine CL, mRNA expression is maximal in the early CL, and the protein is primarily localized to large luteal cells. The functional significance of MIF remains to be determined.     

Effects of luteinizing hormone on luteal cell populations in hypophysectomized ewes

To examine the effect of purified LH on development and function of luteal cells, 27 ewes were assigned to: (1) hypophysectomy plus 2 micrograms ovine LH given i.v. at 4-h intervals from Days 5 to 12 of the oestrous cycle (oestrus = Day 0; Group H + LH; N = 7); (2) hypophysectomy with no LH replacement (Group N-LH; N = 6); (3) control (no hypophysectomy) plus LH replacement as in Group H + LH (Group S + LH; N = 7); (4) control with no LH treatment (Group S-LH; N = 7). Blood samples were collected at 4-h intervals throughout the experiment to monitor circulating concentrations of LH, cortisol and progesterone. On Day 12 of the oestrous cycle corpora lutea were collected and luteal progesterone concentrations, unoccupied receptors for LH and number and sizes of steroidogenic and non-steroidogenic luteal cell types were determined. Corpora lutea from ewes in Group H-LH were significantly smaller (P less than 0.05), had lower concentrations of progesterone, fewer LH receptors, fewer small luteal cells and fewer non-steroidogenic cells than did corpora lutea from ewes in Group S-LH. The number of large luteal cells was unaffected by hypophysectomy, but the sizes of large luteal cells, small luteal cells and fibroblasts were reduced. LH replacement in hypophysectomized ewes maintained luteal weight and the numbers of small steroidogenic and non-steroidogenic luteal cells at levels intermediate between those observed in ewes in Groups L-LH and S-LH. In Group H + LH ewes, luteal and serum concentrations of progesterone, numbers of luteal receptors for LH, and the sizes of all types of luteal cells were maintained. Numbers of small steroidogenic and non-steroidogenic cells were also increased by LH in hypophysectomized ewes. In Exp. II, 14 ewes were assigned to: (1) sham hypophysectomy with no LH replacement therapy (Group S-LH; N = 5); (2) sham hypophysectomy with 40 micrograms ovine LH given i.v. at 4-h intervals from Day 5 to Day 12 of the oestrous cycle (Group S + LH; N = 5); and (3) hypophysectomy plus LH replacement therapy (Group H + LH; N = 4). Experimental procedures were similar to those described for Exp. I. Treatment of hypophysectomized ewes with a larger dose of LH maintained luteal weight, serum and luteal progesterone concentrations and the numbers of steroidogenic and non-steroidogenic luteal cells at control levels.(ABSTRACT TRUNCATED AT 400 WORDS)