Cytosol progesterone and 17 alpha-hydroxyprogesterone levels and luteinizing hormone and chorionic gonadotropin receptors in human corpora lutea.

Cytosol progesterone (P) and 17 alpha-hydroxyprogesterone (17-OHP) levels and luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors were measured in 27 corpora lutea and four corpora albicantia. Cytosol P concentrations were highest in corpora lutea (mean +/- SEM, 3.1 +/- 0.8 micrograms/g) during the midluteal phase (days 15 to 19) rather than the early (2.2 +/- 0.8 micrograms/g, days 20 to 25) and late luteal phases (1.8 +/- 0.8 micrograms/g, days 26 to 30). Cytosol 17-OHP concentrations also were 3.3 +/- 0.5, 4.3 +/- 0.6, and 3.3 +/- 1.0 micrograms/g in early, midluteal, and late luteal phases, respectively, and was significantly inversely correlated with occupied LH/hCG receptors in midluteal phase. Corpora albicantia had the lowest P (0.3 +/- 0.05 microgram/g) and 17-OHP (0.9 +/- 0.6 micrograms/g) concentrations. Cytosol P and 17-OHP may therefore reflect the balance between the luteal cell production and secretion, whereas the amount of occupied and unoccupied LH/hCG receptors may partially explain the relationship between LH and P secretion.

Ovarian failure and autoimmunity. Detection of autoantibodies directed against both the unoccupied luteinizing hormone/human chorionic gonadotropin receptor and the hormone-receptor complex of bovine corpus luteum.

We developed an ELISA system for the detection of human anti-ovarian antibodies. Bovine corpora lutea were extracted in PBS (pH 7.2) and fractionated by ultracentrifugation. Both the soluble fraction obtained after 80,000 g (S80) and the Triton-extracted membrane fraction (ST288) were used as antigens. Additionally, the luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor was isolated by affinity chromatography (wheat germ agglutinin and LH-Sepharose) and also used as an antigen. In 7 of 14 patients with primary sterility and endometriosis a positive reaction was observed. Similarly, 6 of 16 patients with secondary sterility and endometriosis were also positive. Patients being stimulated for in vitro fertilization and presenting either primary or secondary sterility were positive in 5 of 22 and 6 of 16 cases, respectively. In the S80 test 41 of 60 sera presented IgG2 antibodies, whereas in the ST288 test 38 of 60 belonged to the IgG1 subclass. Kappa and lambda chains were equally distributed. Some patients could recognize the unoccupied LH/hCG receptor as an antigen, while others recognized only the complex formed by the hormone plus the hormone receptor. The S80 and ST288 antigens were isolated by affinity chromatography. Gel permeation of the purified antigens revealed in each case the presence of an antigen complex. The apparent molecular weight was between 2,000 and 36,000 D. Cross-reactivity studies using affinity-purified antibodies demonstrated an antigenic relationship of the membrane, soluble, and extractable fractions. NAc-(beta-1----4)-D-glucosaminide and -D-galactopyranoside were the main terminal glycosides.

Western blot analysis of porcine corpus luteum heparin binding proteins using antibodies to acidic and basic fibroblast growth factor.

The 105,000 X g supernatant from homogenates of porcine corpora lutea was chromatographed on a heparin Sepharose affinity column. Bound protein was batch eluted and analyzed on Western blots using antibodies to acidic and basic fibroblast growth factor (FGF). The antibody to acidic FGF reacted specifically with at least four protein bands ranging from 21 to 50 kDa. Three antibodies to human basic FGF (145 residues) generated against either the N-terminal sequence (1-12), the internal sequence (33-43), or the C-terminal end (135-145) also reacted specifically with a total of four different bands. The apparent molecular weights ranged between 20 and 55 kDa. The luteal extract also expressed message for acidic FGF. The results show that there may exist a family of FGF-like molecules in the corpus luteum and demonstrates for the first time the presence of acidic FGF in that tissue.

Structure of rhesus monkey relaxin predicted by analysis of the single-copy rhesus monkey relaxin gene.

The gene encoding rhesus monkey relaxin has been investigated. A cDNA library was prepared using corpus luteal RNA from a pregnant rhesus monkey, cDNA clones encoding relaxin were isolated and the nucleotide sequence was determined. The amino acid sequence of rhesus monkey preprorelaxin, predicted from the cDNA, demonstrates that the sequence has not been strongly conserved when compared with that of man, although features characteristic of the relaxin molecule have been maintained. This structural information will allow production of rhesus monkey relaxin, leading to studies investigating the bioactivity of relaxin in a homologous primate system. Southern blot analysis indicated that there is only one relaxin gene in the rhesus monkey and baboon genomes. In this respect these primate genomes are different from the human genome which contains two relaxin genes.

Support for a physiological role of endogenous catecholamines in the stimulation of bovine luteal progesterone production.

To determine if catecholamines were present in bovine luteal tissue, corpora lutea (CL) were obtained during the mid-luteal phase (Days 10-12) and the concentration of dopamine (DA) and norepinephrine (NE) was determined by high-performance liquid chromatography. Both DA and NE were detected in luteal tissue at mean concentrations of 41.9 +/- 5.73 and 10.2 +/- 2.51 ng/g for DA and NE, respectively. These concentrations represented a luteal content of 306.6 +/- 66.88 ng/CL for DA and 70.5 +/- 16.88 ng/CL for NE. In vitro, DA at concentrations of 1.0 mM to 0.01 mM stimulated the production of progesterone (P4, p less than 0.05). The response to DA was inhibited by propranolol (a beta-adrenergic receptor antagonist, p less than 0.05) but not by phentolamine, phenoxybenzamine (alpha-adrenergic receptor antagonists), or haloperidol (a DA receptor antagonist, p greater than 0.05). Neither L-tyrosine nor L-dopa altered P4 production (p greater than 0.05). Inhibition of DA beta-hydroxylase, the enzyme that catalyzes the conversion of DA to NE by FLA-63 blocked the DA-induced increases in luteal P4 production (p less than 0.05). These results demonstrate the existence of DA and NE in bovine luteal tissue and indicate that exogenous DA can be converted to NE in luteal tissue. The results support a physiological role for catecholamines in the stimulation of bovine luteal function.

Direct luteotrophic effect of oestradiol-17 beta on pig corpora lutea.

Five gilts each received intraluteal implants releasing about 4.4 +/- 1.1 (Group 1), 15.0 +/- 1.1 (Group 2) or 22.4 +/- 1.0 (Group 3) micrograms oestradiol/day as determined by in-vitro incubation of implants of similar weight and oestradiol content. On Day 11 of the oestrous cycle (Day 0 = first day of oestrus), 3 CL in one ovary received oestradiol implants, 3 CL in the other ovary received vehicle implants, and all other CL in both ovaries served as uninjected control CL. An additional group of 6 animals served as controls and included 4 animals receiving bilateral vehicle implants (3 CL per ovary) on Day 11 and 2 unoperated gilts. All animals were slaughtered on Day 19 of their oestrous cycle, and the weight, progesterone content and concentration of each CL were determined. In Group 3 gilts, luteal weight, progesterone content and concentration were greater by 68.7 +/- 24.0 mg, 6.54 +/- 1.33 micrograms and 7.54 +/- 2.00 ng/ml respectively (P less than 0.01) in oestradiol-implanted CL than in vehicle-implanted CL, which appeared to be similar to uninjected control CL. No differences were seen between oestradiol-17 beta and vehicle-treated CL in CL from gilts in Groups 1 or 2. All CL of Group 3 gilts were heavier and contained a greater content and concentration of progesterone (P less than 0.01) than gilts in Groups 1 and 2, and in controls for which the values were similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-adrenergic receptor concentration and subtype in the corpus luteum of the adult pseudopregnant rat.

Luteal beta-adrenergic receptor concentration and subtype were determined in adult pseudopregnant rats during and after the period of the functional luteal phase. The specific beta-adrenergic receptor ligand (-)-3-[125I]iodocyanopindolol ([125I]ICYP) was used to determine the receptor concentration in corpora lutea of adult pseudopregnant rats. A 3-fold increase in beta-adrenergic receptor concentration was seen during the first 2-3 days of pseudopregnancy, whereafter the receptor concentration declined. During the functional luteal regression period (Day 12-15) the receptor levels were still low. In regressed (Day 16-22) corpora lutea a temporary increase in beta-receptor concentration was seen which may represent some role for beta-adrenergic mechanisms in the regulation of morphological regression in the corpus luteum. To determine the beta-adrenergic subtype, competition of [125I]ICYP-binding with selective beta 1- and beta 2-adrenergic antagonists was assessed in corpora lutea of different ages and in rat heart and uterus. The beta-adrenergic receptors in corpora lutea of adult pseudopregnant rats were shown to be solely of the subtype beta 2, regardless of the luteal age.

Human chorionic gonadotropin-induced heterologous desensitization of rabbit luteal adenylyl cyclase is associated with altered receptor and G-protein function.

We injected hCG into pseudopregnant rabbits on day 7 of pseudopregnancy and analyzed changes in the components of luteal adenylyl cyclase system in order to determine which components are responsible for altered hormonal responsiveness upon desensitization. hCG-induced desensitization was homologous (loss of responsiveness to LH) early (first 6 h), then became heterologous (partial loss of responsiveness to catecholamines) later (12-48 h). The total number of LH receptors was reduced approximately 30% 3 h after treatment at a time when LH stimulation of adenylyl cyclase activity was not altered. Total LH receptors remained at this level until 24 h, when total receptors were reduced by 88%. While total LH receptor number remained constant, LH-stimulated adenylyl cyclase activity was declining to 57% of the control value at 12 h. Available unoccupied LH receptors were reduced by 96% at 12 h. The affinity of the occupied receptors was reduced 4-fold before down-regulation. The changes in beta-adrenergic receptor number paralleled the changes in catecholamine responsiveness. hCG treatment also altered luteal G-protein function, as assessed by reconstitution of adenylyl cyclase activity in S49 cyc- lymphoma membranes and ADP ribosylation by cholera and pertussis toxins. Isoproterenol (ISO)-reconstituting activities of luteal Gs (the stimulatory G-protein of adenylyl cyclase) were depressed by 65% 12-48 h after hCG treatment, the same time as reduced catecholamine responsiveness. In contrast, NaF-reconstituting activities were at control levels at 12 h and reduced by 55% at 24 and 48 h. Pertussis toxin's ability to ADP ribosylate alpha i 40 was increased 3 and 6 h after treatment, while cholera toxin's ability to ADP ribosylate alpha s 45 was reduced throughout the study period. These studies demonstrate that hCG-induced heterologous desensitization results in a complex series of changes in beta-adrenergic and LH receptors as well as G-protein function, which account for the altered hormonal responsiveness.

Oxytocin receptors in the ovine corpus luteum.

There is inconclusive evidence that oxytocin acts directly on the corpus luteum and affects steroidogenesis. Since any such action would probably be mediated by oxytocin receptors, these should be present in luteal tissue. In this study, homogenates of corpora lutea from both pregnant and non-pregnant ewes were examined for oxytocin receptors by radioreceptor assay. Specific oxytocin binding was not observed in luteal tissue during the oestrous cycle. However specific binding was found in the corpora lutea of pregnant ewes; appearing at a fetal head length of approximately 0.65 cm (about 30 days of pregnancy) and persisting to a head size of 11 cm, the largest size examined in this study. The affinity (Kd) of the receptor was calculated as 2.9 +/- 0.3 nmol/l (S.E.M.; n = 9), a value similar to that obtained for the uterus. The receptor number ranged from a low of 8.7 +/- 3.2 fmol/mg protein (n = 6) at a head size of less than 0.65 cm, to a maximum of 40.1 +/- 6.5 fmol/mg protein (n = 25) at a head size of 2.5-3.75 cm. These values were lower than our estimate of 588 +/- 39 fmol/mg protein (n = 5) for the uterus. It is concluded that a direct action of oxytocin on the corpus luteum is possible but only after the first month of pregnancy and not in the corpus luteum of the oestrous cycle.

Localization of relaxin during formation of the porcine corpus luteum.

The presence and localization of relaxin (RLX) in luteal tissue during the estrous cycle of the pig have been studied using the avidin-biotin immunoperoxidase method and homologous antisera to purified RLX. Prepubertal gilts were induced to ovulate by treatment with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Ovaries were obtained at laparotomy during the periovulatory period and at specified times through Day 19 post-ovulation. Emphasis was placed on obtaining ovarian tissue at 12- and 24-h intervals up to 96 h after ovulation. RLX immunostaining was evident in theca interna (TI) cells before and at 6 h after ovulation. At 18 h after ovulation, RLX immunostaining comparable to that seen in TI cells was observed for the first time in luteinizing granulosa (G) cells. As luteinization progressed, it became difficult to identify the origin of the RLX immunostaining cells. However, the intensity of RLX immunostaining increased with corpus luteum (CL) development, with the staining becoming localized in the large luteal cells. By Day 19 after ovulation, RLX immunostaining was undetectable. These results indicate RLX is present in the CL during its formation and functional lifespan. Also, it would appear that the presence of RLX in G cells post-ovulation is associated with cell luteinization.

Inhibin mRNAs in ovine and bovine ovarian follicles and corpora lutea throughout the estrous cycle and gestation.

Follicles and corpora lutea were dissected from ovine and bovine ovaries and the RNA extracted and subjected to Northern blot analyses for alpha- and beta A-inhibin mRNAs, using bovine cDNA and cRNA probes. A cDNA probe detecting mRNA for cholesterol side-chain cleavage cytochrome P-450 (P-450scc) was used as a positive control. In cattle, alpha- and beta A-inhibin mRNAs were not detected in ovarian stroma, which could potentially have contained follicles up to 0.5 mm in diameter. Inhibin-alpha and -beta A mRNAs were detected in bovine antral follicles but after ovulation, the relative levels of alpha- and beta A-inhibin mRNAs declined and were undetectable in mature fully developed cyclic corpora lutea and in pregnancy corpora lutea from early to late gestation of the cow. In sheep, alpha- and beta A-inhibin mRNAs were detected in a pool of antral follicles but not in cyclic or pregnancy corpora lutea, which did contain P-450scc mRNA. It is concluded that in cattle and sheep, follicles and not mature corpora lutea are the ovarian source of inhibin.

Human corpus luteum: luteinizing hormone and chorionic gonadotropin receptors during the menstrual cycle.

To characterize and determine the concentration of LH/hCG receptors in human corpora lutea of the menstrual cycle, we measured occupied and unoccupied receptors and determined the association (Ka) and dissociation (Kd) constants individually in 23 corpora lutea (CL) and 4 corpora albicantia obtained at the time of tubal ligation from 25 normal cycling women. We found no [125I]hCG binding in any of the corpora albicantia. Scatchard plot analysis for each CL revealed a linear binding plot indicative of a single set of LH/hCG receptors. The mean concentration of unoccupied receptors was 36 +/- 10 (+/- SE) fmol/mg protein in the early luteal phase (days 15-19; n = 5), 64 +/- 11 fmol/mg protein in the midluteal phase (days 20-25; n = 13), and 42 +/- 19 fmol/mg protein in the late luteal phase (days 26-30; n = 5). The concentrations of occupied receptors were 56 +/- 8, 46 +/- 6, and 54 +/- 12 fmol/mg protein in the early, mid-, and late luteal phases, respectively. Total (occupied plus unoccupied) receptor concentrations reached maximum levels of 110 +/- 11 fmol/mg protein in the midluteal phase. Ka increased progressively from 12 +/- 4 X 10(9) mol/L-1 in the early luteal phase to 19 +/- 7 X 10(9) and 21 +/- 8 X 10(9) mol/L-1 in the mid- and late luteal phases. We conclude that in normal CL, 1) total and unoccupied LH/hCG receptor levels parallel progesterone secretion; 2) changes in the binding affinity may be important in sustaining and/or rescuing the CL; and 3) loss of LH/hCG receptors is probably related to luteolysis.

Developmental expression of tissue inhibitor of metalloproteinase (TIMP) RNA.

Single-stranded antisense RNA probes have been used to study the expression of the metalloproteinase inhibitor TIMP (tissue inhibitor of metalloproteinases), during mouse embryogenesis and in adult tissues. Using a sensitive RNase protection assay, low levels of transcript can be detected in a variety of tissues, including maternal deciduum, embryonic kidney, lung and amnion. Higher levels are seen in osteogenic tissues such as calvaria, while the highest level in any tissue is found in the ovary, though even here expression is an order of magnitude below that observed in growth factor-treated fibroblasts in vitro. Using the technique of in situ hybridization, TIMP transcripts can first be detected in osteogenic tissues in the head and limb at about 15.5 days post coitum, and increase in amount until birth. The high levels of TIMP RNA in the ovary are localized to cells of the corpora lutea.

Localization of transferrin and its receptor in ovarian follicular cells: morphologic studies in relation to follicular development.

Granulosa cells perform an essential role in ovarian follicle and ovum development. Proliferating cells have an absolute requirement for iron, which is delivered by transferrin with subsequent intracellular transport via the transferrin receptor. Because iron and transferrin concentration increase in follicular fluid with advancing follicular maturation, the authors studied the distribution of transferrin and its receptor in rat and human granulosa cells with light and electron microscopic immunohistochemistry. Intense cytoplasmic staining was found in granulosa cells, with immunostaining enhancement occurring with advanced follicle maturation, including the periovulatory period. Immunoelectron microscopy showed transferrin throughout the cytoplasm, often in proximity to polyribosomes and vesicular structures. When transferrin was absent in the culture medium used to maintain granulosa cells, diminished transferrin immunostaining was seen. Based on these findings, the authors conclude that follicular maturation is closely related to high levels of cellular transferrin and transferrin receptor. Acquisition of transferrin occurs primarily by either ultrafiltration or facilitated diffusion, whereas de novo local synthesis does not have a major role.

Assessing the early luteal phase in in vitro fertilization cycles: relationships between plasma steroids, endometrial receptors, and endometrial histology.

The corpus luteum-endometrial unit was investigated in in vitro fertilization (IVF) cycles using endocrine, morphologic, and biochemical measurements on the day normally scheduled for embryo transfer (day 16), in 12 stimulated and 4 natural cycles. Advanced endometrial histologic maturity was recorded in 9 of the 12 stimulated cycles. No in-phase endometria were seen when the preovulatory plasma estradiol (E2) was greater than 500 pg/ml or the day 16 plasma progesterone (P) greater than 10 ng/ml in natural or stimulated cycles. Significant negative correlations were noted between both preovulatory E2 and day 16 P and the concentration of cytosolic progesterone receptor (PRc). Advanced endometrial maturity tended to be associated with low concentrations of PRc. Regardless of endometrial maturity, the natural cycle was characterized by low cytosolic E2 receptors (ERc) and high PRc, whereas the concentration of both receptors was usually greatly reduced in stimulated cycles. It is concluded that the advanced endometrial maturation observed in stimulated IVF cycles is a consequence of the production of supraphysiologic levels of sex steroids by the corpus luteum that cause profound modifications of endometrial receptor dynamics.

Characterization of endometrial receptors for ovine trophoblast protein-1 during the estrous cycle and early pregnancy in sheep.

Scatchard analysis was used to determine the distribution, number, and affinity of unoccupied receptors for ovine trophoblast protein-1 (oTP-1) in endometrium of sheep throughout the estrous cycle and early pregnancy. In Experiment I, oTP-1 receptor characteristics were determined in membrane preparations of caruncular and intercaruncular regions of endometrium collected from uterine horns ipsilateral and contralateral to the ovary bearing the corpus luteum. Receptor concentrations and affinity constants for oTP-1 were not different (p greater than 0.1) between the four endometrial regions examined, suggesting that the expression of receptors for oTP-1 occurs uniformly throughout the endometrium. Endometrial receptor characteristics for oTP-1, luteal wet weights, and progesterone contents were determined throughout the estrous cycle and early pregnancy in Experiment II. Concentration of receptors and affinity constants for oTP-1 varied throughout the estrous cycle and early pregnancy (p less than 0.01), with the pattern of change differing between cyclic and pregnant ewes (p less than 0.01). Numbers of receptors for oTP-1 were maximal on Day 4 of the estrous cycle and declined progressively to Day 12 (p less than 0.05) in both cyclic and pregnant ewes. After Day 12, the quantity of unoccupied receptors for oTP-1 increased (p less than 0.05) gradually to Day 16 in cyclic ewes, but declined (p less than 0.05) further in the endometrium of pregnant ewes. The affinity constants of endometrial receptors for oTP-1 were similar in cyclic and pregnant ewes prior to Day 12, increasing threefold from Days 4 to 12 (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a monoclonal antibody which inhibits the biological activity of human chorionic gonadotrophin in human corpora lutea.

In the present study a murine monoclonal antibody (MCA), directed against human chorionic gonadotrophin (HCG), was investigated in vitro utilizing human corpora lutea (CL). The CL were excised from women of fertile age undergoing various gynaecological operations. The CL specimens were cut into pieces and incubated in standard medium for 2 or 4 h in the presence of HCG, luteinizing hormone (HLH) or prostaglandin (PG) E2 alone or in combination with the MCA directed against HCG. After incubation, the tissue levels of cyclic adenosine 3',5' monophosphate (cAMP) and the media contents of progesterone (P) were determined. HCG, HLH and PGE2 stimulated both cAMP and P formation in CL of all ages, while the MCA alone had no effect on these parameters. The MCA, however, effectively counteracted the stimulatory effect of HCG on both cAMP and P formation on a 1:1 molar basis. This antagonistic effect was clearly reversible and could be overcome by increasing the HCG concentrations. The stimulatory effect of HLH and PGE2, on the other hand, was not influenced by the antibody, even when administered at high concentrations. These in-vitro data show that the MCA studied effectively counteracts the stimulatory effect of HCG on human luteal tissue with high specificity.

Immunohistochemical localization of renin and angiotensin II in human ovaries.

Ovaries from six women with normal menstrual cycles, a follicle wall biopsy specimen from a gonadotropin-stimulated preovulatory ovary, and a corpus luteum of pregnancy were examined by immunohistochemistry for the presence of immunoreactive renin and angiotensin II. Both antisera densely stained thecal and stromal cells (interstitial complex) and luteal cells. Whereas granulosa cells in developing follicles were either unstained or lightly stained, the heavily luteinized granulosa cells of the preovulatory stimulated follicle were strongly positive for immunoreactive renin and angiotensin II. These anatomic findings are consistent with gonadotropin-stimulated local production of both renin and angiotensin II in the human ovary and support the functional roles proposed for the ovarian renin-angiotensin system in follicle development, ovulation, and luteal function and during pregnancy.

Effect of prolactin on ovarian steroidogenesis.

This review summarizes evidence that prolactin (PRL) is involved directly in the regulation of ovarian steroidogenesis. The scope of this paper will be limited to two areas. The first area involves the regulation of the corpus luteum function and the second the effect of PRL on steroidogenesis during the follicular phase of the oestrous cycle. Cyclic changes of plasma PRL levels and the ovarian receptors which bind PRL have been observed during the oestrous cycle of domestic animals. The luteotropic effect of PRL and its failure is also presented. PRL may exert a physiological effect on follicle steroidogenesis but its effect depends on the level of follicle maturation. The effect of PRL treatment on estrogen production by follicular cells in vivo and in vitro was studied. The results of many papers indicate suppression of estrogen secretion by the direct action of PRL at the ovarian level. However, there is abundant evidence, that PRL is involved directly in regulation of ovarian steroidogenesis, the precise mechanism remains to be discovered.

Cetacean relaxin. Isolation and sequence of relaxins from Balaenoptera acutorostrata and Balaenoptera edeni.

The tendency toward extremely high variability among relaxins derived from purportedly closely related species has come to an abrupt end with the discovery of quasi-porcine relaxin in the minke whale (Balaenoptera acutorostrata) and the Bryde's whale (Balaenoptera edeni). An aqueous abstract of the corpora lutea of the two baleen whales contained significant amounts of relaxin-like activity as determined by a mouse bioassay and by cross-reactivity with anti-pig relaxin antibodies. The activity could be isolated and purified to homogeneity. Sequence analysis revealed that both whale relaxins differed from each other by about 3 residues, whereas the relaxin of B. edeni differed at only one position from that of pig relaxin. The similarity appears to include even the chain length heterogeneity observed at the C-terminal end of the B chain in porcine relaxin which is produced by a peculiar mode of connecting peptide removal from the pro-hormone. This finding may well represent one of the better documented challenges to the current paradigm of molecular evolution.