Bovine Corpus Luteum Research Articles

Isolation of luteinising hormone receptor binding inhibitor from bovine corpus luteum

A luteinising hormone receptor binding inhibitor (LHRBI) has been purified from bovine corpus luteum (CL). Steroid-free extract of the CL was subjected to successive chromatographies on Sephadex G-50, Q-Sepharose, Orange A dye and metal chelate affinity columns followed by high performance-reverse phase and gel filtration columns. Purification was monitored by the ability of the fractions to inhibit the binding of 125I-human chorionic gonadotropin (hCG) to porcine granulosa cells in vitro. The final isolate showed an 8000-fold enrichment of activity. It was also capable of inhibiting porcine granulosa cell secretion of estradiol and progesterone (P) in vitro. Administration of LHRBI into follicle-stimulating hormone (FSH)-stimulated, immature rats strongly inhibited the ovarian ovulatory response to hCG as revealed by decreased P levels and the number of ova released. The M r of LHRBI as assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis was ca. 15 kDa and the p I was between 5.0 and 5.5.

Binding and action of insulin-like growth factors and insulin in bovine luteal tissue during the oestrous cycle.

The effect of insulin-like growth factors (IGFs) and insulin on the release of progesterone and oxytocin from bovine corpus luteum was investigated at early (days 5-7), mid- (days 8-12) and late (days 15-18) luteal phases of the oestrous cycle in an in vitro microdialysis system. The expression of specific receptors was evaluated in bovine corpora lutea of the respective luteal stages. A 30 min infusion of IGF-1, IGF-2 (1.3, 13 and 130 nmol l-1) or insulin (13, 130 and 1300 nmol l-1) caused a stimulation of the release of progesterone (P < 0.05). IGF-1 was most effective in releasing progesterone. Oxytocin release from corpora lutea was stimulated by insulin at all doses tested (13-1300 nmol l-1), whereas the IGFs were only effective at the highest dose (130 nmol l-1) applied. The high doses of IGFs (130 nmol l-1) and insulin (1300 nmol l-1) stimulated the release of progesterone and oxytocin throughout the luteal phase (P < 0.05). For all three peptides, greatest stimulation was seen during the late luteal phase (days 15-18 of the oestrous cycle) with the peak of progesterone release directly related to peptide infusion (P < 0.05). In addition, IGF-1 stimulated total release of progesterone (units in 4 h) after the beginning of the stimulation during this phase (P < 0.05). IGF-1 caused a gradual increase of progesterone even beyond the time of peptide perfusion, whereas IGF-2 and insulin stimulated progesterone release only during the peptide perfusion. Distinct receptors for IGF-1 and IGF-2 were present in corpora lutea membrane preparations at all stages investigated. Specific binding for insulin was also seen in all stages of the cycle without any cycle-dependent changes in the amount of binding. The displacement of labelled insulin by unlabelled IGF-1 and IGF-2 did not show the rank of order that has been described as typical for insulin receptors (i.e. insulin > IGF-1 > IGF-2), but comparable binding affinities were observed for the three unlabelled ligands. Specific binding of IGF-2 was markedly higher than that of IGF-1 or insulin throughout the cycle (1.9- and 4.9-fold higher compared with IGF-1 and insulin, respectively). Receptor specificity did not change during luteal development. Binding affinity and capacity of IGF-1 receptor was constant throughout the oestrous cycle. Specific IGF-2 binding increased and showed a positive co-operativity towards the end of the cycle. Specific binding of insulin was not significantly different in the three luteal stages examined.

Expression of thromboxane A2 receptor gene and thromboxane A2 synthase in bovine corpora lutea.

Studies were undertaken to investigate the expression of thromboxane (TXA2) receptor gene, from mRNA to functional receptor protein in terms of ligand binding, along with the cellular and subcellular distribution of the enzyme that catalyzes the formation of the ligand for the receptors. Bovine corpora lutea contained a single TXA2 receptor mRNA transcript of 2.8 kb. All the cell types in bovine corpora lutea contained immunoreactive TXA2 synthase, TXB2, TXA2 receptor transcripts, and receptor protein that bound the TXA2 antagonist 9,11-dimethylmethano-11,12-methano-16 (3-iodo-4-hydroxyphenyl)-13-14-dihydro-13-aza-15 alpha beta-omega-tetranor TXA2. The large luteal cells (20-35 microns) contained more receptor transcripts, receptor protein, and immunoreactive TXA2 synthase than did the small luteal cells (12-19 microns), luteal blood vessels, and nonluteal cells (7-12 microns). After correction for the cellular area differences, small luteal cells were seen to contain more receptor protein than did large luteal cells and nonluteal cells. All the cells showed an increase of TXA2 receptors and catalytically active TXA2 synthase from mid-luteal phase to early pregnancy, suggesting the possibility that TXA2 could be a luteotropic eicosanoid. Bovine lung homogenates (a positive control), bovine luteal plasma membranes-mitochondria-lysosomes fraction, rough-smooth endoplasmic reticulum-Golgi fraction, and highly purified nuclei contained 65-kDa immunoreactive protein, presumably representing TXA2 synthase. In addition, the luteal fractions, but not bovine lung, contained other small and large molecular-size immunoreactive proteins. Immunogold electron microscopy showed that immunoreactive TXA2 synthase was present primarily in plasma membranes, rough endoplasmic reticulum, nuclear membranes, and chromatin; and immunoreactive TXB2 was present primarily in different-size vesicles and nuclear chromatin. In summary, the present studies demonstrate for the first time that primarily small and large luteal cells and secondarily blood vessels and nonluteal cells in bovine corpora lutea express TXA2 receptor gene along with the functional TXA2 synthase. The presence of functional enzyme in luteal cell nuclei suggests that the enzyme and/or its product may have previously unrecognized functions in nuclei.

Modulation of luteinizing hormone-stimulated inositol phosphate accumulation by phorbol esters in bovine luteal cells.

The present studies were conducted to evaluate the effects of protein kinase C activators on the inositol phospholipid-phospholipase C second messenger system in isolated bovine luteal cells. This report describes the effects of phorbol esters on inositol phosphate accumulation in LH- and prostaglandin F2 alpha (PGF2 alpha)-stimulated bovine luteal cells. Corpora lutea of early pregnancy were dispersed with collagenase and luteal cells were prelabelled for 3 h with [3H]inositol. Inositol phosphates produced in response to LH or PGF2 alpha were analyzed by ion exchange column chromatography. The tumor promoter and protein kinase C activator 12-O-tetradecanolyphorbol 13-acetate (TPA) had no effect on basal levels of inositol phosphates but inhibited LH-stimulated accumulation of inositol mono-, bis-, and trisphosphates by 72%, 68%, and 65%, respectively. TPA reduced the response to maximally effective concentrations of LH and tripled the concentrations of LH required to evoke half-maximal accumulation of inositol mono-, bis-, trisphosphates. The inhibitory effects of TPA were rapid (5 min) whether added before or after treatment with LH. Treatment with TPA also reduced (58%) the initial phase of intracellular calcium mobilization in LH-treated cells. The inhibitory effects of TPA were not associated with acute reductions in [3H]inositol incorporation, [3H]inositol phospholipid levels, cAMP levels, or progesterone accumulation in control or LH-stimulated luteal cells. The effects of phorbol esters were concentration dependent and specific for active tumor promoters with 10-50 nM TPA producing maximal inhibitory effects. A synthetic diacylglycerol, 1-oleyl-2-acetylglycerol, mimicked the inhibitory effects of TPA. In contrast, pretreatment with a physiological activator of protein kinase C, PGF2 alpha, had no effect on LH-stimulated inositol phosphate accumulation. The inhibitory effects of TPA could not be explained by a generalized inhibition of phospholipase C or G-proteins since the accumulation of inositol phosphates in PGF2 alpha- and NaF-treated cells was not inhibited by TPA. These results demonstrate that tumor promoting phorbol esters modulate the inositol phospholipid-phospholipase C transmembrane signaling system in LH-stimulated bovine luteal cells. The results suggest that phorbol esters may alter the coupling of the LH-receptor complex to phospholipase C. These findings implicate protein kinase C in the regulation of transmembrane signaling in the bovine corpus luteum.

Cultured microvascular endothelial cells derived from the bovine corpus luteum possess NCAM-140

Previously, five phenotypically different, stable types of microvascular endothelial cells (MVE) were isolated from the bovine corpus and cultured successfully. We found that three out of these five types of MVE express the neural cell adhesion molecule (NCAM). As shown by immunocytochemistry, weak NCAM immunoreactivity occurred mainly in the perinuclear area of cell type 1. Monolayers of types 2 and 5 revealed heavy NCAM immunoreactivity, which was localized predominantly at the lateral cell surface outlining the contact zones of adjacent cells. In contrast, cell types 3 and 4 were not NCAM immunoreactive. Western blot analyses substantiated these results: While cell type 1 showed a weak immunoreactive band, cell types 2 and 5 displayed strong NCAM-immunoreactive bands of a molecular weight of approximately 140 kDa (NCAM-140), which was absent in cell types 3 and 4. These results reveal for the first time that NCAM can be expressed by cultured MVE and may serve in mediating endothelial cell contacts. Since luteal cells also express NCAM-140, this adhesion molecule could in addition be involved in the interactions of luteal cells with MVE.

Purification and characterization of a high-affinity binding protein for pancreatic-type phospholipase A 2

A high-affinity and specific binding site for mammalian group I phospholipase A 2 (PLA 2-I) was found on the membranes of bovine corpus luteum. Affinity labeling experiments revealed that PLA 2-I binds to a single polypeptide with a mass of 190–200 kDa. The PLA 2-I binding protein in the membranes was solubilized in an active form with n-octyl β- d-thioglucoside, and then purified approx. 16 000-fold. The purification procedures consisted of diethylaminoethyl-Sephacel chromatography, PLA 2-I-affinity gel chromatography and gel-filtration high-performanceliquid chromatography on a TSKgel G3,000SW XL column. The final preparation migrated as a single molecular species of 190 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and identification of the 190 kDa protein as the PLA 2-I binding protein was demonstrated by ligand blotting analysis. The purified protein possessed a binding capacity with high affinity and specificity for a mammalian mature type of PLA 2-I. Treatment of the purified material with N-glycosidase F resulted in increased mobility of the protein on SDS-PAGE as well as considerable abolition of the PLA 2-I binding activity, thus suggesting the requirement of the carbohydrate moiety of the PLA 2-I binding protein for receptor-ligand interactions.

Evidence for oxytocin receptors in cultured bovine luteal cells.

Specific receptors for oxytocin (OT) on intact luteal cells are demonstrated. Cultured cells from bovine corpora lutea (CL) at different stages (Days 3-5, 8-12, and 15-18 of the estrous cycle) were examined for OT receptors by a radioreceptor assay using the 125I-labeled OT antagonist [d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH2(9)] -vasotocin. Binding specificity was demonstrated in displacement studies with various related peptides. Scatchard analysis revealed the presence of a binding site with an association constant of Ka = 2.6 x 10(9) M-1 and a capacity of 5.9 fmol/micrograms DNA. Additionally, in 50% of the experiments (n = 6) two different binding sites were observed. The Ka of the high-affinity site was 2.6 x 10(10) M-1; its capacity was 0.73 fmol/micrograms DNA. The low-affinity site had an apparent Ka of 4.9 x 10(8) M-1 and a capacity of 8.8 fmol/micrograms DNA. Observation of one versus two binding sites related neither to the assay conditions nor to the state of the individual CL used for the cell culture and therefore appeared to reflect individual variation within the OT receptor population. Significant binding of OT was observed at all luteal stages. OT binding was maximal at the mid-luteal stage (Days 8-12). We conclude that a direct action of OT on the bovine CL is mediated by the OT receptor, supporting the hypothesis that luteal OT plays an important physiological role in the regulation of progesterone release and/or other luteal functions in a paracrine or autocrine fashion.

Heterogeneity of spindle structure in different microvessel endothelial cell types derived from bovine corpus luteum

Using an antibody against tyrosinated tubulin and epifluorescence microscopy, mitosis was studied in three different microvessel endothelial cell types recently isolated from bovine corpus luteum. Dividing cells were flat and at certain stages individual microtubules could be followed for considerable lengths. The structure of the spindle apparatus and the course of mitosis were conventional. Microtubule asters were small from prophase until metaphase in all three cell types. However, whereas in two cell types telophase asters remained inconspicuous, prominent asters, of mostly straight microtubules, formed in telophase cells of a third cell type. Thus, aster size is heterogeneous between different endothelial cell types. Large microtubule asters are not regularly found in dividing cultured mammalian cells. The microendothelial cell types present themselves as appropriate systems for spindle research and especially for the study of aster microtubule dynamics and function.

Differential adhesion of granulocytes to five distinct phenotypes of cultured microvascular endothelial cells

Adhesion of isolated human polymorphonuclear granulocytes (PMNs) to five different phenotypes of cultured microvascular endothelial cells derived from bovine corpora lutea was investigated by measuring the myeloperoxidase content of cell lysates. Untreated and interleukin 1 (IL-1) -pretreated confluent monolayers were overlaid with unstimulated and phorbol ester (PMA)-stimulated PMNs in the absence and presence of the monoclonal antibody IB4 recognizing and functionally blocking β 2 (CD18) of the leukocyte integrins. Unstimulated PMN adhesion was highest on type 4, followed by type 3 and 5 endothelial cells. This adhesion was not inhibited by treatment with IB4. IL-1 pretreatment of endothelial cells resulted in a significant increase of PMN adhesion on types 1, 2, and 4, most of which was also β 2 integrin-independent. PMA-stimulation of PMNs increased adhesion to maximal values on cell types 1 and 5, which was largely blocked by IB4. Type 2 endothelial cells supported significantly less PMA-stimulated PMN adhesion than all other types. In the presence of IB4, adhesion of PMNs to untreated and IL-1-pretreated type 3 and 4 endothelial cells was significantly reduced by PMA. This reduction of β 2 integrin-independent adhesion by PMA stimulation is compatible with possible shedding of the lectin-like leukocyte adhesion molecule, l-selectin, from PMNs. Differential PMN adhesion may reflect distinctive expression of endothelial adhesion molecules in different phenotypes of microvascular endothelial cells. Endothelial specialization within the microcirculation may have important functional consequences for the inflammatory response in vivo.

Role of frequency and amplitude of repetitive HCG stimulations for sustained progesterone secretion from the bovine corpus luteum in vitro.

This investigation aimed at evaluating a role for frequencies and amplitudes of repeated HCG stimulations for the optimal maintenance of progesterone (P4) secretion from the bovine corpus luteum in vitro. Slices (100-120 mg) of midluteal bovine corpora lutea were perifused with medium M199 (0.05% BSA, pH 7.2, 38.5 degrees C) and the perifusion effluent collected at 15 minute intervals for 20-29 hours. Unstimulated P4 release (n = 5) was distinctly pulsatile (by Pulsar pulse algorithm), with pulses occurring every 90 +/- 6 minutes (mean +/- SEM) and pulse amplitudes of 14.4 +/- 1.1 ng. Conversely, no pulses were detected in two control perifusions. Unstimulated P4 release increased during the first 5 perifusion hours (from 39.3 +/- 4.6 to 50.3 +/- 5.6 ng/15 min, p less than 0.01), but then appeared to decline (to 29.3 +/- 1.3 ng/15 min, p less than 0.05) towards the end of the perifusion periods. Hourly pulses of HCG (6.7 mM) did not change the P4 pulse amplitudes (16.6 +/- 2.0 ng), the pulse periodicities (105 +/- 15 min) and overall release rates (34.7 +/- 5.7 ng/15 min), nor did they prevent the decline in P4 secretion towards the end of perifusions (n = 5). In contrast, 2-hourly HCG stimulations maintained stable P4 release rates throughout the perifusion periods (34.7 +/- 6.8 ng/15 min), with P4 pulses of similar amplitudes (14.7 +/- 1.7 ng), but of lower periodicities (135 +/- 2 min, p less than 0.05) than during unstimulated conditions (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

COOH-terminally-extended processing forms of oxytocin in human ovary

Human granulosa cells synthesize and secrete the oxytocin hormone. We have already shown that oxytocin-Gly, the last post-translational maturation intermediate of pro-hormone, is largely secreted by cultured granulosa cells deprived of ascorbate (Plevrakis et al. (1990) J. Endocrinol. 124, R5–R8). Using a combination of high performance liquid chromatography and radioimmunoassay, the oxytocin-like material present in human granulosa cell extracts, in follicular fluid, in cultured granulosa cell supernatants and in corpora lutea extracts was identified. We have demonstrated the presence of oxytocin-Gly, oxytocin-Gly-Lys and oxytocin-Gly-Lys-Arg, the same post-translational maturation intermediates as those we identified in bovine corpus luteum secretory granules. Thus we conclude that post-translational maturation of pro-oxytocin/neurophysin in human ovary proceeds by the same proteolytic events as those we described in bovine post-pituitary gland and corpus luteum.

The interphase microtubule cytoskeleton of five different phenotypes of microvessel endothelial cell cultures derived from bovine corpus luteum

The interphase microtubule cytoskeleton of five different microvessel endothelial cell cultures, recently established from bovine corpus luteum, was analysed using anti-tubulin immunofluorescence. An antibody against acetylated microtubules detected four cell types each of which possessed a single cilia. The length of the cilia were up to 10 μm for cell types 1 and 2. Ciliary stubs had a length of up to 0.37 μm in cell types 4 and 5. Cilia were missing in cell type 3. Long and short cilia were located in the perinuclear region from where cytoplasmic microtubules radiated. Cell type 3 displayed straight microtubules rather than the wavy path seen in the other cell types. The amount of tyrosinated microtubules visualized by a specific antibody was consistently higher than that of posttranslationally acetylated microtubules. The latter were more apparent in cell types 4 and 5 than in the other cell types. We conclude: Differences in the cytoplasmic microtubule inventory of each microvessel endothelial cell type points at individual functions maintained in culture.

Production of heparin-binding angiogenic factor(s) by bovine corpora lutea during pregnancy.

Effects of luteal-conditioned media (LCM) on proliferation and migration of endothelial cells were used to assess angiogenic activity of corpora lutea (CL) obtained from cows on d 100 (n = 5), 150 (n = 6), 200 (n = 6), and 250 (n = 6) of gestation. Explants of CL (200 mg) were incubated for 6 h in 3 mL of serum-free media containing no hormone, LH (1 microgram/mL), prostaglandin F2 alpha (PGF2 alpha; 3 microM), or both hormones. Media from the four stages of gestation were subjected to the following procedures: 1) ultrafiltration, 2) high-salt (3.0 M NaCl) treatment and then ultrafiltration, 3) heat treatment, 4) heparin-Sepharose affinity chromatography, 5) immunoneutralization with specific antibodies against heparin-binding growth factor (HBGF)-1 and against HBGF-2, and 6) dot immunoblot assay for HBGF-2. Fractions from the first five procedures were evaluated in the endothelial cell proliferation bioassay. In addition, progesterone concentration of LCM was determined by RIA. Across all days of gestation and hormone treatments, LCM stimulated (P less than .05) proliferation and migration of endothelial cells, but activities did not differ among stages of gestation or hormone treatments. Both mitogenic and migration-stimulating fractions seemed to have Mr greater than 100,000. The mitogenic activity fraction had an apparent Mr greater than 100,000 even after treatment with high salt and was heat-labile. This endothelial mitogen was retained on heparin-Sepharose columns and was eluted with 2.0 M NaCl. Mitogenic activity was partly neutralized (P less than .05) by antibodies against HBGF-2 but not HBGF-1. Presence of HBGF-2 in LCM was detected by dot immunoblot assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of norepinephrine on the release of progesterone and ovarian oxytocin in cattle

Heifers had catheters inserted from the aorta abdominalis cranial to the origin of the ovarian artery on Day 10 of the oestrous cycle (in which oestrus is Day 0). On Day 11, four experiments were performed. In Experiment 1, norepinephrine (NE) was infused (0.3 μg kg −1 body weight min −1) for 30 min. This produced a rapid rise ( P < 0.01) in peripheral plasma oxytocin from the basal concentration (3–6 pg ml −1) to more than 100 pg ml −1 in 5 min. Oxytocin levels fell to the pretreatment values within 60 min of the end of infusion. Progesterone concentrations increased 3–5 ng ml −1 ( P < 0.01) above the pre-infusion level during drug infusion. The increase in levels of both hormones was either concomitant, or the oxytocin rise preceded that of progesterone. The changes in concentration and amounts of secreted oxytocin were similar ( P > 0.05) to those seen after injecting a luteolytic dose of an analogue of prostaglandin PGF 2α (Experiment 3). In Experiment 2, PGF 2α was given i.m. at the start of NE infusion. This did not alter the profile observed for oxytocin seen in Experiment 1 but the stimulatory effect of NE on progesterone secretion was reduced within 15 min. In Experiment 4, an i.m. injection of PGF 2α was given 1 h after the NE infusion. Each treatment produced a similar increase in oxytocin release, comparable with increases observed in Experiments 1 and 2 ( P > 0.05) respectively. In conclusion, NE stimulates the release of both ovarian oxytocin and progesterone, indicating some relationship between these hormones in the mid-luteal phase of the oestrous cycle in cattle. NE stimulated a release of oxytocin which was comparable with that produced by injected PGF 2α and did not deplete the corpus luteum (CL) of oxytocin. On this basis, it seems that NE can affect the components of the post-translational processing pathway of oxytocin synthesis in the bovine CL.

Diversity of ultrastructure in different phenotypes of cultured microvessel endothelial cells isolated from bovine corpus luteum

Five different types of cultured microvessel endothelial cells defined by use of light microscopy and scanning electron microscopy in a preceding study were investigated by transmission electron microscopy. Type-1 cells displayed a deep invagination of the cell membrane or a single cilium. Granules of low electron density were abundant. A perinuclear ring of intermediate filaments occurred. Cultures of type-2 cells were subdivided into phenotype A, reminiscent of cell-type 1, and into phenotype B, assumed to be vascular smooth muscle cells. Many highly electron-dense granules appeared in late postconfluent cultures of both phenotypes. Cell-type 3 was conspicuous because of a large intracytoplasmic vacuole. Lysosomes with curvilinear bodies were found in cell-types 3 and 4. Both cell types developed a peripheral regular network of microfilaments. Cell-type 5 showed vesiculation of the rough endoplasmic reticulum, lipid droplets and a peripheral felt-like belt of microfilaments. Tubular forms seen in late postconfluent cultures of cell-types 1 to 3 displayed a core of extracellular matrix. Pseudotubular forms of cell-type 4 contained apoptotic bodies. Thus, as seen at the ultrastructural level, different features are maintained by cultured microvessel endothelial cells, suggesting that they have different inherent properties.

Mapping of the Bovine Oxytocin Gene Control Region: Identification of Binding Sites for Luteal Nuclear Proteins in the 5′ Non‐Coding Region of the Gene

Abstract In view of the small number of hormone-producing cells, the factors regulating oxytocin gene expression in the classic site of synthesis, in the magnocellular neurons of the hypothalamus, have not yet been characterized. In the early bovine corpus luteum there is a tissue-specific oxytocin expression involving many more cells. This tissue therefore was chosen as a experimental system to identify deoxyribonucleic acid elements and nuclear proteins involved in the regulation of oxytocin gene expression. 3.2 kb from the 5'non-coding region of the bovine oxytocin gene have been sequenced and subcloned fragments used as probes for gel retardation and footprinting experiments. Binding sites for luteal as well as more ubiquitous proteins were detected in the oxytocin promoter region and in an artiodactyl-specific dispersed repeated deoxyribonucleic acid element. A binding site in the promoter region with a superficial similarity to an estrogen-responsive element (-159 to -152) was shown not to bind this steroid hormone receptor but to bind two nuclear proteins alternatively. One is a luteal protein, the other a more general transcription factor belonging to the steroid hormone receptor superfamily and similar, if not identical to the COUP protein. This alternative binding of a tissue- and phase-specifically expressed protein or an ubiquitous factor to the same site in the oxytocin promoter suggests a role for these two proteins in the transient up-regulation and subsequent down-regulation of the oxytocin gene during the differentiation of the bovine corpus luteum.

Cellular Distribution and Cycle Phase Dependency of Gonadotropin and Eicosanoid Binding Sites in Bovine Corpora Lutea

Bovine luteal functions are regulated by gonadotropins and eicosanoids. The specific binding sites that presumably mediate the actions of these regulatory agents have previously been characterized in bovine luteal tissue. However, the cellular distribution and/or the cycle phase dependency of these binding sites have never been investigated. In the present study, we investigated these parameters by using quantitative light microscope autoradiography. The results showed that both small and large luteal cells contained binding sites for LH/hCG, prostaglandin (PG)E2, PGF2 alpha, PGI2, and leukotriene (LT)C4. In addition, luteal blood vessels contained LH/hCG and LTC4 binding sites and luteal fibroblasts contained PGE2 binding sites. On a per cell basis, there were more binding sites for all ligands in large luteal cells as compared to small or nonluteal cells. After correction for the cellular area differences, small luteal cells contained more LH/hCG, PGE2, PGI2, and LTC4 binding sites, while large luteal cells contained more PGF2 alpha binding sites. The small and large luteal cell binding of hCG, PGE2, PGI2, and LTC4 increased from early to mid luteal phase, followed by a decline in the late luteal phase. PGF2 alpha binding, on the other hand, increased from early to late luteal phase. In contrast to luteal cells, binding of hCG and LTC4 to luteal blood vessels and binding of PGE2 to luteal fibroblasts did not change during the cycle. These results suggest that LH/hCG and eicosanoid regulation of luteal function is more complex than previously envisioned and it involves both small and large luteal cells and, in some cases, also nonluteal cells.

Expression of Major Histocompatibility Complex Antigens on the Bovine Corpus Luteum during the Estrous Cycle, Luteolysis, and Early Pregnancy1

Treatment of cultured bovine luteal cells with the cytokine, interferon-gamma, induces the expression of Class II major histocompatibility complex antigens (MHC Ags). To determine if Class II MHC Ags are present on the CL in vivo and if the degree of Ag expression changes during luteal life span, bovine corpora lutea were obtained on Day 6, Days 10-12, and Day 18 of the estrous cycle and MHC Ag expression was evaluated via indirect immunofluorescence. Flow cytometry was used to determine the percentage of MHC Ag-positive cells on cell populations distinguished by cell size and intracellular density. Minimal Class II MHC Ag expression was detected on Day 6 CL (approximately 25%), which consisted primarily of smaller cells. The midcycle and late CL consisted of these small cells (SC) and two populations of large cells that differed in intracellular density, or right-angle light scatter. In midcycle CL, few (less than 25%) SC or large, dense cells (LDC) expressed the Class II MHC Ag whereas a high percentage (75%) of the large, less-dense cells (LLDC) were Class II MHC Ag-positive. Class II MHC Ag expression remained negligible on the LDC of the Day 18 CL; however, there was an elevation in the percentage of SC and LLDC expressing Class II Ag (p less than 0.05). To determine if Class II MHC Ag expression also varied with different functional states of the CL, bovine CL were collected after prostaglandin (PG) F2 alpha-induced regression and on Day 18 of early pregnancy. When luteolysis was allowed to progress in vivo, the percentage of Class II MHC Ag-positive cells was increased in all cell populations (p less than 0.05). Class II MHC Ag expression was significantly lower (p less than 0.05) on the three cell populations comprising the CL of pregnancy as compared to the Day 18 cyclic CL. It is hypothesized that enhanced expression of Class II MHC Ags on the late CL and during PGF2 alpha-induced regression may potentiate immune response mechanisms for luteolysis.

Bovine corpus luteum function after removal of granulosa cells from the preovulatory follicle

Experiments were conducted to determine the effects of removing granulosa cells from bovine preovulatory follicles on subsequent corpus luteum (CL) function. Holstein heifers were assigned to three groups: untreated controls (n=6), removal of granulosa cells (n=9) and removal and return of granulosa cells (n=7). Surgery was performed 18–24 hr after the onset of estrus and in all cases after the preovulatory luteinizing hormone (LH) surge. Jugular venous blood was collected and estrous activity monitored twice daily. Corpora lutea were formed in six heifers in each group. Concentrations of plasma progesterone were reduced (P<0.05) on Days 7–17 in heifers from which granulosa cells were removed when compared to the other two groups. There were no differences in the lengths of the estrous cycles nor concentrations of LH in the three groups. In summary, these experiments support the concept that granulosa cells make a substantial contribution to the output of progesterone by the cyclic CL but may have a limited role in determining the functional lifespan of the CL. These experiments also establish the fact that granulosa cells develop into functional luteal cells after their removal and return to the preovulatory follicle.

Quantitative Cell Composition of Human and Bovine Corpora Lutea from Various Reproductive States1

The cell composition of human and bovine corpora lutea (CL) from various reproductive states was investigated by computerized video-based interactive Bioquant image analysis system IV and by light microscope immunocytochemistry. Human and bovine CL contained more nonluteal cells than luteal cells. Human CL contained a lower number of luteal and a greater number of nonluteal cells than bovine CL. Regardless of the reproductive state, human CL contained more small luteal cells than large luteal cells. In all reproductive states except in the late luteal phase, the bovine CL also contained more small luteal cells than large luteal cells. The average sizes of all the cells in human CL were smaller than in bovine CL. Human CL contained more vascular space than bovine CL during mid and late luteal phases. The number of luteal cells increased and nonluteal cells decreased from early to mid luteal phase, and then luteal cells decreased and nonluteal cells increased in late luteal phase and in degenerating human and bovine CL. While the change of number of small and large luteal cells first occurred from early to mid luteal phase in human CL, it did not take place until the late luteal phase in bovine CL. The average size of large luteal cells in humans and of small luteal cells in cattle did not change, whereas size of the other cells changed in different reproductive states in both human and bovine CL. The cell composition of term pregnancy human CL was similar to mid or late luteal phase, whereas the cell composition of early pregnancy bovine CL was similar to mid luteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)