Occurrence and properties of a prostaglandin F2alpha receptor in bovine corpora lutea.

Prostaglandin F2alpha was specifically bound by a particulate fraction from bovine corpora lutea. The rate constants for the association (7.5 X 10(3) M-1 S-1) and dissociation (2.1 X 10-4 S-1) reactions gave a dissociation constant of 2.8 X 10(-8) M which is similar to that determined from a Scatchard plot of binding data at equilibrium (5 X 10(-8) M). The receptor was stable for several hours at 23 degrees C but was rapidly destroyed at 37 degrees C. The pH optimum for the binding reaction was 6.3. The receptor had high specificity for prostaglandin F2alpha and had much lower affinities for other prostaglandins. Luteinizing and follicle-stimulating hormones had no effect on the prostaglandin F2alpha-receptor interaction.     

Molecular weight of detergent-solubilized prostaglandin-F2 alpha receptor from bovine corpora lutea

A prostaglandin F2 alpha receptor localized in plasma membranes of bovine corpus luteum cells was solubilized by treatment with Triton X-100. Sepharose chromatographies of ([3H]prostaglandin F2 alpha)-receptor complex gave a Stokes' radius of 630 nm. In the absence of detergent, aggregated forms of the receptor appeared. Sedimentation experiments of solubilized receptor in sucrose/H2O and sucrose/2H2O density gradients gave the following values: sedimentation coefficient (S20, w) 4.6 S; partial specific volume (VB) 0.78 cm3/g and frictional ratio (f/fo) 1.6. Based on the sedimentation coefficient and the Stokes' radius and assuming that the receptor is a non-glycosylated protein the molar mass of the receptor-(Triton X-100) complex was 144000 g/mol. The VB value indicated that ca. 26% of the weight represented bound detergent and that the molecular weight of the prostaglandin F2 alpha receptor is approximately 107000.     

Cytokeratin expression in bovine corpora lutea

Cytokeratin (CK)-positive cells were obtained from bovine corpora lutea. When cultured, these cells behave like CK-positive endothelial cells obtained from bovine large blood vessels. The origin of CK-positive cells has now been studied in 45 bovine corpora lutea of different estrous cycle stages. Additionally, 7 corpora lutea of pregnant cows were examined. The tissues were grouped into early stage (days 2 to 4), secretory stage (days 5 to 17) and late stage (days 18 to 21) according to gross morphology, wet weight and total progesterone content. One portion of a corpus luteum was used for immunohistochemistry, and another for Western blot analysis. Twenty-six of the 45 corpora lutea showed CK expression, as confirmed by immunostaining and Western blotting. Cytokeratin expression was found in all corporalutea from the early stage, in 14 of 26 corpora lutea from the secretory stage, and 3 of 10 from the late stage. Early stage corpora lutea displayed zonation such that a high number of CK-positive luteal cells occurred in the region of the previous granulosa layer and a very low number in the previous thecal layer. Secretory CK-positive corpora lutea showed uniformly distributed, predominantly large luteal cells. In secretory corpora lutea of group A, CK-positive cells and a distinct microvascular tree were seen, the latter visualized by factor VIII-related antigen immunolabelling of endothelial cells. Group B showed none or very few CK-positive cells. Corpora lutea of pregnant cows behaved like corpora lutea of group B. Roughly 1% of CK-positive cells closely associated with the capillary wall were sometimes reminiscent of endothelial cell sprouts.     

Quantitative echotexture analysis of bovine corpora lutea

A study was designed to evaluate the attributes of ultrasound images of bovine ovarian CL throughout the estrous cycle. The ovaries of 8 heifers were examined daily by transrectal ultrasonography for 2 interovulatory intervals (ovulation = Day 0). Ultrasonographic examinations of the ovaries were videotaped daily, and recorded images of the CL were digitized for computer analysis of echotexture (mean pixel value and heterogeneity). Blood samples were taken daily and to determine plasma progesterone concentrations. Corpora lutea were of 2 morphological types, those with a central fluid-filled cavity (n = 6) and those without (n = 9). No differences were detected between CL with or without a fluid-filled cavity; therefore, data were combined. Mean pixel values of ultrasound images of the CL changed (P = 0.0001) during the interovulatory interval; values decreased (P < 0.05) from Day 0 to Day 3 during early growth of the CL, reached a plateau when increases in luteal diameter ceased, and decreased (P < 0.05) to minimal levels at the onset of regression of the CL. The mean pixel value subsequently increased (P < 0.05) after Day 17 to values similar to those at the beginning of the interovulatory interval. A time-dependent effect was not observed for heterogeneity of images of the CL (P > 0.5). The results supported the hypothesis that quantitative changes in luteal echotexture are reflective of changes in the physiologic status of the CL.     

Receptors for gonadotropins and prostaglandins in lysosomes of bovine corpora lutea.

The total mitochondrial fraction of bovine corpus luteum specifically bound [³H]prostaglandin (PG) E₁, [³H] PGF_2α, and ¹²⁵I-labeled human lutropin (hLH) despite very little 5′-nucleotidase activity, a marker for plasma membranes. Since the total mitochondrial fraction isolated by conventional centrifugation techniques contains both mitochondria and lysosomes, it was subfractionated into mitochondria and lysosomes to ascertain the relative contribution of these fractions to the binding. Subfractionation resulted in an enrichment of cytochrome c oxidase (a marker for mitochondria) in mitochondria and of acid phosphatase (a marker for lysosomes) in lysosomes. The lysosomes exhibited little or no contamination with Golgi vesicles, rough endoplasmic reticulum, or peroxisomes as assessed by their appropriate marker enzymes. Subfractionation also re ulted in [³H] PGE₁, [³H] PGF_2α, and ¹²⁵I-labeled hLH binding enrichment with respect to homogenate in lysosomes but not in mitochondria. The lysosomal binding enrichment and recovery were, however, lower than in plasma membranes. The ratios of marker enzyme to binding, an index of organelle contamination, revealed that plasma membrane and lysosomal receptors were intrinsic to these organelles. Freezing and thawing had markedly increased lysosomal binding but had no effect on plasma membrane binding. Exposure to 0.05% Triton X-100 resulted in a greater loss of plasma membrane compared to lysosomal binding. In summary, the above results suggest that lysosomes, but not mitochondria, in addition to plasma membranes, intrinsically contain receptors for PGs and gonadotropins. Furthermore, lysosomes overall contain a greater number of PGs and gonadotropin receptors compared to plasma membranes and these receptors are associated with the membrane but not the contents of lysosomes.     

Gonadotropin and prostaglandins binding sites in nuclei of bovine corpora lutea.

Highly purified nuclei isolated from bovine corpora lutea showed marked enrichment of NAD pyrophosphorylase, a marker for this organelle. Rough endoplasmic reticulum and lysosomal markers were undetectable, whereas plasma membrane and Golgi markers were detectable but not enriched in nuclei. These highly puridied nuclei exhibited specific binding with 125I-labeled human choriogonadotropin, [3H]prostaglandin E1 and [3H]prostaglandin F2 alpha. However, these bindings were only 15.4% (human choriogonadotropin), 7.9% (prostaglandin E1) and 8.9% (prostaglandin F2 alpha) of the plasma membrane binding observed under the same conditions. Washing of nuclei and plasma membranes twice with buffer containing 0.1% Triton X-100 resulted in gonadotropin and prostaglandin F2 alpha binding site and 5'-nucleotidase (EC 3.1.3.5) losses from nuclei that were different from those observed for plasma membranes. More importantly, the washed nuclei exhibited 44% (human choriogonadotropin), 21--26% (prostaglandins) of original specific binding despite virtual disappearance of 5'-nucleotidase activity. The nuclear membranes isolated from nuclei, specifically bound 125I-labeled human choriogonadotropin and [3H]prostaglandin F2 alpha to the same extent or significantly more ([3H]prostaglandin E1, P less than 0.05) than nuclei themselves, despite the marked losses of chromatin. In summary, our data suggest that gonadotropin and prostaglandins bind to nuclei and that this binding was intrinsic and was primarily associated with the nuclear membrane.     

Progesterone synthesis and histology of postpartum bovine corpora lutea

In vitro progesterone (P(4)) synthesis by corpora lutea (CL) from the first, second or third ovulation after calving was compared and correlated with their histology and cytology. The CL were removed 7 to 12 days after ovulation, and luteal cells isolated by digestion with collagenase. The response of isolated cells to luteinizing hormone (LH) was determined. Hematoxylin-eosin stained tissues were used to study histology, and the distribution of cell types was estimated by stereological methods. Ovulation occurred within 25 days of calving and interovulatory intervals were short, 12.1 +/- 3.9 days and 12.6 +/- 4.8 days, respectively. The CL removed after first ovulation were smaller and contained fewer live cells than those obtained after subsequent ovulations. Stimulation by LH in vitro was independent of cycle number or day of cycle but was related to the histology of the tissue. The CL composed of large cells (> 24 mum) with vacuolated cytoplasm contained high amounts of P(4) but were not stimulated by LH. Conversely, CL composed of small and medium- sized cells (10 to 20 mum) and/or intact larger cells contained little P(4) but were stimulated by LH. These observations indicate that the response of postpartum CL to LH in vitro is dependent upon the structural integrity of the tissue at the time of removal. Furthermore, these observations suggest that the short life of CL during the postpartum period may not be due to the absence of luteotrophic support, but to the action of a luteolytic mechanism.     

Subcellular distribution of prostaglandin and gonadotrop in receptors in bovine corpora lutea.

The subcellular distribution of prostaglandin (PG) E₁, F₂α and gonadotropin receptors in bovine corpora lutea was critically examined by preparing various subcellular fractions, assaying for various marker enzymes to assess the purity and examining ³H-PGE₁, ³H-PGF₂α and ¹²⁵I-human lutropin (hLH) specific binding. The marker enzyme data suggested that subcellular fractions were relatively pure with little or no cross contamination. The binding of ³H-PGs and ¹²⁵I-hLH was markedly enriched in plasma membranes with respect to homogenate. The other subcellular fractions also exhibited binding despite very little or no detectable 5′-nucleotidase activity. If 5′-nucleotidase was assumed to lack sensitivity and reliability to detect minor contamination with plasma membranes and ³H-PGs or ¹²⁵I-hLH binding were used as sensitive plasma membrane markers, it was still difficult to explain binding in other fractions based on plasma membrane contamination. Therefore, these results lead to the inevitable conclusion that plasma membranes were primary (or one of the primary) but not exclusive sites for PGE₁, PGF₂α and gonadotropin receptors.     

Expression and regulation of mRNAs for insulin-like growth factor-I receptor and LH receptor in corpora lutea

Relationship between insulin-like growth factor-I receptor (IGF-IR) and luteinizing hormone receptor (LHR) mRNA expression as well as their regulation was determined in rat corpora lutea (CL) . In the CL of estrous cycle rat, LHR mRNA positive CL expressed high level of mRNA of IGF-IR. While the expression of LHR mRNA decreased on estrus, the CL still expressed relatively high level of IGF-IR mRNA. In pseudopregnant rat CL, the expression level of LHR mRNA was low on day 1, the most intense signals were detected on day 8, the signals of LHR mRNA became undetectable on day 14. In contrast to LHR expression, the high level of IGF-IR mRNA was observed in pseudopregnant CL of day 1, and thereafter its signals were detected from day 2 to day 14. Pregnant rat CL expressed both LHR and IGF-IR mRNAs. IGF-I stimulated LHR expression in CL. PGF2 inhibited expression of IGF-IR and LHR. PGE2 negated the inhibiting effects of PGF2. These data suggest that IGF-I may be involved in regulating CL function, and maintaining CL structure through changes in expression of its receptors. Inhibited expression of IGF-IR by PGF2 may be part of mechanisms for regression of CL.     

Expression and regulation of mRNAs for insulin-like growth factor-I receptor and LH receptor in corpora lutea

Relationship between insulin-like growth factor-l receptor (IGF-IR) and luteinizing hormone receptor (LHR) mRNA expression as well as their regulation was determined in rat corpora lutea (CL) . In the CL of estrous cycle rat, LHR mRNA positive CL expressed high level of mRNA of IGF-IR. While the expression of LHR mRNA decreased on estrus, the CL still expressed relatively high level of IGF-IR mRNA. In pseudopregnant rat CL, the expression level of LHR mRNA was low on day 1, the most intense signals were detected on day 8, the signals of LHR mRNA became undetectable on day 14. In contrast to LHR expression, the high level of IGF-IR mRNA was observed in pseudopregnant CL of day 1, and thereafter its signals were detected from day 2 to day 14. Pregnant rat CL expressed both LHR and IGF-IR mRNAs. IGF-I stimulated LHR expression in CL. PGF2ainhibited expression of IGF-IR and LHR. PGE2 negated the inhibiting effects of PGF2α. These data suggest that IGF-I may be involved in regulating CL function, and maintai     

Prostaglandin F2 alpha-induced release of oxytocin from bovine corpora lutea in vitro

Two experiments were conducted to study the in vitro effects of prostaglandins F2 alpha (PGF2 alpha), E2 (PGE2), and luteinizing hormone (LH) on oxytocin (OT) release from bovine luteal tissue. Luteal concentration of OT at different stages of the estrous cycle was also determined. In Experiment 1, sixteen beef heifers were assigned randomly in equal numbers (N = 4) to be killed on Days 4, 8, 12, and 16 of the estrous cycle (Day 0 = day of estrus). Corpora lutea were collected, an aliquot of each was removed for determination of initial OT concentration, and the remainder was sliced and incubated with vehicle (control) or with PGF2 alpha (10 ng/ml), PGE2 (10 ng/ml), or LH (5 ng/ml). Luteal tissue from heifers on Day 4 was sufficient only for determination of initial OT levels. Luteal OT concentrations (ng/g) increased from 414 +/- 84 on Day 4 to 2019 +/- 330 on Day 8 and then declined to 589 +/- 101 on Day 12 and 81 +/- 5 on Day 16. Prostaglandin F2 alpha induced a significant in vitro release of luteal OT (ng.g-1.2h-1) on Day 8 (2257 +/- 167 vs. control 1702 +/- 126) but not on Days 12 or 16 of the cycle. Prostaglandin E2 and LH did not affect OT release at any stage of the cycle studied. In Experiment 2, six heifers were used to investigate the in vitro dose-response relationship of 10, 20, and 40 ng PGF2 alpha/ml of medium on OT release from Day 8 luteal tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotropin and prostaglandins binding sites in nuclei of bovine corpora lutea

Highly purified nuclei isolated from bovine corpora lutea showed marked enrichment of NAD pyrophosphorylase, a marker for this organelle. Rough endoplasmic reticulum and lysosomal markers were undetectable, whereas plasma membrane and Golgi markers were detectable but not enriched in nuclei. These highly purified nuclei exhibited specific binding with ¹²⁵I-labeled human choriogonadotropin, [³H]prostaglandin E₁ and [³H]prostaglandin F_2α. However, these bindings were only 15.4% (human choriogonadotropin), 7.9% (prostaglandin E₁) and 8.9% (prostaglandin F_2α) of the plasma membrane binding observed under the same conditions. Washing of nuclei and plasma membranes twice with buffer containing 0.1% Triton X-100 resulted in gonadotropin and prostaglandin F_2α binding site and 5′-nucleotidase (EC 3.1.3.5) losses from nuclei that were different from those observed for plasma membranes. More importantly, the washed nuclei exhibited 44% (human choriogonadotropin), 21–26% (prostaglandins) of original specific binding despite virtual disappearance of 5′-nucleotidase activity. The nuclear membranes isolated from nuclei, specifically bound ¹²⁵I-labeled human choriogonadotropin and [³H]prostaglandin F_2α to the same extent or significantly more ([³H]prostaglandin E₁, P < 0.05) than nuclei themselves, despite the marked losses of chromatin. In summary, our data suggest that gonadotropin and prostaglandins bind to nuclei and that this binding was intrinsic and was primarily associated with the nuclear membrane.     

Purification and properties of human chorionic gonadotropin/lutropin receptor from plasma-membrane and soluble fractions of bovine corpora lutea.

Plasma-membrane and soluble fractions containing human chorionic gonadotropin/lutropin receptor were prepared from bovine corpora lutea by ultracentrifugation. The plasma-membrane and soluble fractions were studied for physicochemical properties, salts and gangliosides. The receptor preparations obtained from the plasma-membrane purified individually by sucrose-density-gradient centrifugation, which resulted in a partial dissociation of the hormone-binding subunit from the intact functional receptor unit, which consists of both hormone-binding (regulatory) and adenylate cyclase-associated (catalytic) subunits. The fractions containing the functional receptor unit were further purified by gel filtration on Sepharose-6B and chromatography on concanavalin A-Sepharose. The 'receptor' was finally purified by affinity chromatography on a column of controlled-pore glass covalently coupled to hu man chorionic gonadotropin. The purified receptor from the plasma-membrane and the soluble fractions contained binding capacities of 901000 and 87000 fmol of human chorionic gonadotropin/mg of protein. Yields of 0.02 and 0.22mg of protein were obtained from 250 g of bovine corpora lutea, which represents a 10000- and 1000-fold increase respectively in the specific binding with 125I-labelled human chorionic gonadotropin. Immunization of rabbits with a partially purified receptor fraction generated antibodies that specifically inhibited the binding of the 125I-labelled human chorionic gonadotropin to the receptor.