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Chorionic gonadotropin, chorionic somatomammotropin, and prolactin in the uterine vein and peripheral plasma of pregnant rhesus monkeys.

Seven adult female rhesus monkeys were laparotomized at days 22, 42, and 157 of pregnancy and blood was collected from the uterine vein and peripheral circulation. Plasma samples were analyzed for monkey chorionic gonadotropin (mCG), monkey chorionic somatomammotropin (mCS), and prolactin by radioimmunoassay. Levels of mCG at day 22 of pregnancy were approximately 250 ng/ml; however, during the later stages of gestation mCG was either nondetectable or less than 0.7 ng/ml. There was no statistical difference in prolactin concentrations between days 22 and 42 of pregnancy, mean levels being between 176-424 ng/ml, but by day 157 prolactin levels of greater than 2,000 ng/ml were recorded. No statistical difference existed between peripheral and uterine vein concentrations of either mCG or prolactin at any of the stages of gestation examined. At day 22, mCS was not detectable; however, at day 42 of gestation mCS titers averaged 1.5 micrograms/ml and 2.3 micrograms/ml in the peripheral and uterine vein plasma, respectively. A statistically significant mCS increase occurred by day 157, levels in the periphery and uterine vein averaging 11.0 micrograms/ml and 16.3 micrograms/ml, respectively. Uterine vein titers of mCS were significantly higher than peripheral titers at both days 42 and 157. Thus, the highest levels of mCG were present during early pregnancy, whereas the highest levels of mCS and prolactin were present during late pregnancy.

Animals

Preliminary studies on the indium slide immunoassay for estimation of human chorionic gonadotropin and antihuman chorionic gonadotropin antibody.

Human chorionic gonadotropin (hCG) is synthesized and secreted as early as 170 hr after fertilization and has been used as an index for pregnancy. Neutralization of hCG with a beta-subunit hCG vaccine(s) has been proposed as a contraceptive technique. To monitor the duration of effectiveness of the vaccine, it will be necessary to monitor the anti-hCG antibodies, especially those responsible for inhibiting the hCG bioactivity. We report a simple, rapid technique using an indium slide immunoassay for the qualitative estimation of hCG and to monitor a bioeffective anti-hCG antibody. The sensitivity of the indium slide assay to measure hCG ranged from 1 microgram/ml to 1 ng/ml, depending on the format of the assay. The indium slide assay also detected anti-hCG antibodies generated against a specific determinant on hCG recognized by a neutralizing monoclonal antibody (P3W80) in women immunized with a contraceptive vaccine.

Antibodies

Chorionic gonadotropin receptors and immunoreactive chorionic gonadotropin in implantation of the rabbit blastocyst.

To determine the temporal relationship between immunoreactive chorionic gonadotropin, chorionic gonadotropin receptors, and implantation of the rabbit blastocyst, (1) immunoreactive chorionic gonadotropin in plasma, uterine flushings and blastocysts; (2) chorionic gonadotropin receptors on blastocysts (day 5 and 6) and embryonic and interembryonic segments of the uterus (day 7); and (3) chorionic gonadotropin receptors on the endometrium and myometrium (day 1 through 6) were measured. Binding of I125-labeled beta-subunit of human chorionic gonadotropin (hCG) by cell membranes from blastocysts increased significantly from 6.0 +/- 1.1 fmol/mg of protein (mean +/- SE) on day 5 (N = 6) to 16.1 +/- 1.3 fmol/mg of protein on day 6 (n = 6) (P less than 0.001). Immunoreactive chorionic gonadotropin levels in blastocyst fluid increased from 0.3 ng/ml of day 5 to 0.65 ng/ml on day 6. Specific binding of I125-labeled hCG was absent in endometrial and myometrial cell membranes before implantation (days 1 to 6) but was found in decidual cells from embryonic segments on day 7. Plasma immunoreactive chorionic gonadotropin or chorionic gonadotropin--like material increased from 6 ng/ml of day 1 to 52 ng/ml on day 7. Uterine flushings had chorionic gonadotropin levels of 0.4 ng/ml of day 2, which increased slowly to 1.0 ng/ml on day 7. Intrauterine instillation of hCG into nonpregnant uterine horns demonstrated transuterine absorption of hCG with plasma hCG levels showing a dose-related response. Our findings demonstrate that (1) immunoreactive chorionic gonadotropin or chorionic gonadotropin--like material is detectable in plasma, uterine flushings, and blastocyst fluid before implantation, (2) chorionic gonadotropin receptors are present on the blastocyst cells before implantation, and (3) the uterus can absorb chorionic gonadotropin from its lumen.

Absorption

Is deglycosylated human chorionic gonadotropin an antagonist to human chorionic gonadotropin? Characterization of deglycosylated human chorionic gonadotropin action in two testicular interstitial cell fractions.

In order to determine the significance of carbohydrate residues of human chorionic gonadotropin (hCG) in receptor interaction and signal transduction leading to steroidogenesis, the effect of deglycosylated hCG (DG-hCG) was studied in vitro with two different hCG-responsive purified testicular interstitial cell fractions. Fraction I light cells, previously found to bind 125I-labeled hCG with high affinity without producing testosterone, also bound 125I-labeled DG-hCG with high affinity (Kd 7.2.10(-10) M) without stimulating testosterone production. Fraction IV heavier cells, which produced testosterone in response to hCG without detectable high-affinity hCG-binding sites, neither bound DG-hCG nor sufficiently produced cAMP and testosterone in response. With the addition of intact hCG, DG-hCG inhibited cAMP levels, although not sufficiently to inhibit testosterone production. This observation was contrary to previous studies in which DG-hCG was shown to be an antagonist to hCG action. We conclude that: (a) DG-hCG retains its binding activity in light cells and this high-affinity binding is unrelated to steroidogenesis; (b) DG-hCG does not bind to heavier cells with high affinity and loses its biological activity as result of deglycosylation; (c) DG-hCG actions in this study strengthen the concept of two different hCG-responsive cells in the rat interstitium which, if not separated, will yield misleading data supporting the coexistence of hCG high-affinity binding and biological response in the same cell; and (d) DG-hCG partially antagonizes the activation of adenylate cyclase but does not block testosterone production, thus questioning the usefulness of this analogue in antagonizing the action of native hCG in rat testis.

Animals

[Pregnancies caused by in vitro fertilization in cycles, stimulated with human hypophyseal gonadotropin and chorionic gonadotropin].

In a program of in vitro fertilization, laparoscopies for oocyte aspiration were performed on 35 patients receiving human pituitary gonadotropin (hPG) and human chorionic gonadotropin (hCG). Of the 122 preovulatory oocytes which were recovered from these patients, 44 (36%) were fertilized and cleaved, and were transferred. 144 immature oocytes were collected, and attempts were made to mature these in vitro. 40 oocytes (28%) could be fertilized and cleaved, and were transferred. Eight pregnancies resulted from 32 embryo transfers (25%), and 22,9% from laparoscopies, respectively. Of the eight pregnancies, there were three preclinical abortions and two clinical abortions, and three patients are well along in the pregnancy. Among the three patients there is one twin pregnancy.

Adult

Observations on the combination of clomiphene citrate-human menopausal gonadotropin-human chorionic gonadotropin in the management of anovulation.

To study the effects of the addition of clomiphene citrate to human menopausal gonadotropin (hMG) and human chorionic gonadotropin (hCG), 23 cycles of clomiphene citrate-hMG-hCG (CHH) were administered to 10 anovulatory women. Of these 10 women, 5 also received 15 cycles of hMG-hCG (HH). Although there was a significant increase in the ovulation rate in the CHH group (p less than 0.01), there was no difference in the pregnancy rate (p less than 0.05). Patients in the CHH group required significantly less hMG for ovulation induction than the HH group (p less than 0.01). Patients receiving CHH who had low serum levels of follicle-stimulating hormone required significantly more hMG than those with normal values. The luteinizing hormone releasing hormone test may predict those patients who will require less hMG during CHH therapy. The relative safety of the CHH treatment is discussed.

Anovulation

The association between preovulatory serum 17 beta-estradiol pattern and conception in human menopausal gonadotropin-human chorionic gonadotropin stimulation.

One hundred forty-four women suffering from obstructive tubal disease underwent laparoscopy for in vitro fertilization and embryo transfer (IVF-ET). Ovulation was induced by a human menopausal gonadotropin/human chorionic gonadotropin (hMG/hCG) protocol. The patients were divided according to their estradiol (E2) response to hMG/hCG administration: group A, an increase of E2 levels on the day after hCG administration (day +1); group B, a decrease of E2 levels on the day after hCG administration. Each of these groups was further subdivided into high responders (E2 greater than 500 pg/ml on day 0), designated A-1 and B-1, respectively, and low responders (E2 less than 500 pg/ml on day 0), designated A-2 and B-2, respectively. Patients responding in an A-1 pattern had significantly higher E2 levels from day 0 to day +8. Fertilization and cleavage rates did not differ among the groups, nor did luteal-phase progesterone. Twenty-one pregnancies were achieved from 126 transfers (17%), of which 16 (75%) occurred in women responding in an A pattern. We recommend that laparoscopy be deferred in women responding with a B pattern, because this preselection may increase pregnancy rates per IVF-ET cycle.

Chorionic Gonadotropin

Gonadotropin-releasing hormone effects on placental hormones during gestation: I. Alpha-human chorionic gonadotropin, human chorionic gonadotropin and human chorionic somatomammotropin.

The release of alpha-human chorionic gonadotropin (alpha hCG), gonadotropin human chorionic gonadotropin (hCG) and human chorionic somatomammotropin (hCS) in vitro from placentas of different gestational ages was studied. In addition, the effect of gonadotropin-releasing hormone (GnRH) on these hormonal releases, as related to the gestational age of the placenta cultured and the dose of GnRH, was determined. The basal release of alpha hCG and hCG was greatest at 9-13 wk of gestation (1000-1500 ng/mg and 250-350 ng/mg, respectively). Lowest release rates were at term (28 ng/mg and 20 ng/mg, respectively). Hormonal release declined with extended culture, except from the cultures of 13- and 15-wk placentas, in which the initially high release continued throughout the 8 days of culture. The initial release of hCS was low at 6 wk, increased to maximum rates by 15 wk, and was similar to the initial rate of release at term. Gonadotropin-releasing hormone stimulated the release of alpha hCG and hCG most dramatically in cultures of 16-wk and 17-wk placentas, where as much as a 400- and 250-fold increase, respectively, on Day 6 of culture was observed (p less than 0.0001). In term placenta cultures after 6 days in vitro, a 20-fold stimulation of alpha hCG and a 10-fold increase of hCG was effected by GnRH (p less than 0.001). The largest responses of alpha hCG and hCG to GnRH were observed when estrogen levels were low. Dose-related responses were observed in some placentas, yet in some instances, maximal effects were attained with all doses utilized in these studies (0.2 to 50 micrograms/ml). These data demonstrate that human placentas of different gestational ages have varying hormonogenic capabilities in vitro. The data also establish that synthetic GnRH is capable of stimulating alpha hCG and hCG production, but the degree and pattern of response to GnRH stimulation are related to the gestational age of the placental tissue and its time in culture. The most responsive period to exogenous GnRH stimulation of alpha hCG and hCG release was on Days 5 and 6 of culture, when basal estrogen release was very low. These data support the hypothesis that hCG release might be controlled by a chorionic GnRH stimulation and suggest that local steroid levels may modulate the hCG response to GnRH stimulation.

Chorionic Gonadotropin

Reduction of testicular human chorionic gonadotropin receptors by human chorionic gonadotropin in infertile men.

We measured testicular human chorionic gonadotropin (hCG) receptors in 30 infertile men either before or 1, 3, 5, 7, or 14 days after a single administration of 5000 IU of hCG. For 5 days the administration of hCG significantly reduced the testicular binding of 125I-hCG compared with that of the testes before administration. From the 7th day the binding capacity began to increase and returned to the preadministration level 14 days after the treatment. Occupied hCG binding sites accounted for about half of the reduction in binding sites on the day after administration of hCG. After this time, however, the occupancy did not contribute so much to the reduction in binding sites. These findings suggest that the reduction in testicular hCG binding sites after a single administration of 5000 IU of hCG is due to not only occupancy but also down-regulation of the binding sites.

Binding Sites

Reduction of testicular human chorionic gonadotropin receptors by human chorionic gonadotropin in vivo and in vitro.

Changes in rat and human testicular human chorionic gonadotropin (hCG) binding sites induced by hCG were estimated in vivo and in vitro. After a single administration of hCG, the specific 125I-hCG bindings were significantly reduced for 7 and 5 days in rat and human testes, respectively. Thereafter, 125I-hCG bindings had recovered to pretreatment values by the 14th day after the administration. Occupied hCG bindings accounted for about half of the reduced bindings on the day after administration of hCG. After this time, however, the occupancy did not contribute so much to the reduction of the bindings. In experiments in vitro using the organ culture technique, an exposure to hCG for 24 h induced a dose-related significant loss of the specific 125I-hCG bindings for 7 and 5 days in rat and human testes, respectively. Thereafter, the loss was gradually recovered. These patterns of changes in 125I-hCG bindings in vitro were similar to those in vivo. These findings suggest that the reduction in hCG binding sites by hCG is due to not only occupancy but also downregulation of the binding sites and that the testicular organ culture method used in the present study is useful to study hormonal regulation of testicular function, especially in human testes.

Animals

Gonadotropin-induced up- and down-regulation of ovarian follicle-stimulating hormone (FSH) receptor gene expression in immature rats: effects of pregnant mare's serum gonadotropin, human chorionic gonadotropin, and recombinant FSH.

The actions of gonadotropins on ovarian differentiation are associated with dynamic changes in gonadotropin receptor content, presumably due to modulation of receptor gene expression. The present studies used a reverse transcription-polymerase chain reaction to obtain a rat FSH receptor cDNA fragment, followed by synthesis of a labeled cRNA probe to examine the regulation of FSH receptor mRNA levels during follicular maturation, ovulation, and luteinization. Northern blot analysis of ovarian RNA with the FSH receptor probe revealed two predominant hybridization signals of 7.0 and 2.5 kilobases (kb) as well as minor signals of 4.2 and 1.8 kb. Treatment of immature rats with PMSG (10 IU) to induce follicular development resulted in increased FSH receptor mRNA levels 24 h after treatment, with a further increase at 52 h, coincident with increased [125I]FSH binding. Subsequent treatment with an ovulatory dose of hCG decreased FSH binding and receptor mRNA levels by 6 h, with a maximal inhibition at 24 h after hCG. In luteinized ovaries obtained 3 and 5 days after hCG treatment, the 7.0-kb FSH receptor mRNA increased again, but no concomitant elevation of [125I]FSH binding was detected. We recently demonstrated that FSH treatment alone is capable of inducing follicular growth and ovulation, thus providing a unique model to evaluate the effects of FSH on regulation of its receptor gene. Immature hypophysectomized estrogen-treated rats were implanted with an osmotic minipump delivering recombinant human FSH (rcFSH; 4 IU/day) to stimulate follicle growth, followed 52 h later with a single injection (20 IU) of rcFSH to induce ovulation. Stimulation of follicular growth with rcFSH increased both FSH receptor binding and mRNA levels. In contrast, the ovulatory dose of rcFSH decreased FSH binding and receptor message levels within 12 h. Thus, gonadotropin regulation of ovarian FSH receptor content during follicular growth, ovulation, and luteinization is associated with similar changes in FSH receptor message levels. Also, studies using rcFSH demonstrate that both up- and down-regulation of FSH receptor gene expression can be induced by the homologous hormone at different stages of follicle development.

Animals

[Testicular follicle-stimulating hormone receptors and effectiveness of human menopausal gonadotropin-human chorionic gonadotropin treatment in idiopathic male infertility].

To investigate the relationship between testicular follicle stimulating hormone (FSH) receptors and the effectiveness of human menopausal gonadotropin (hMG-hCG) treatment in idiopathic male infertility, 48 infertile men were examined. Most of the 14 patients without detectable testicular high affinity FSH receptors did not show any increase in sperm count after the hMG-hCG treatment, whereas 23 of the 34 patients with FSH receptors responded to the treatment. In patients with FSH receptors, patients with a middle or high Johnsen's score count responded more than those with a low score count did. From the above results, it seems that both the presence or absence of testicular FSH receptors and the histological appearance of spermatogenesis predict responsiveness to hMG-hCG treatment in infertile men.

Adult

Thymosin gene expression is modulated by pregnant mare's serum gonadotropin, human chorionic gonadotropin, and prostaglandin F2 alpha in the immature rat ovary.

We have investigated thymosin beta 10 mRNA levels in the PMSG/hCG-treated immature rat ovary. Thymosin beta 10 mRNA was constitutively expressed as a single (greater than 600-nucleotide) abundant transcript in the immature rat ovary. Administration of a single dose (50 IU/rat) of PMSG to immature rats resulted in a gradual increase in steady state ovarian thymosin beta 10 mRNA content detectable as early as 12 h and maximal (2-to 3-fold stimulation above preinjection levels) 48 h after PMSG treatment. Ovarian thymosin beta 10 mRNA levels declined thereafter. In separate experiments treatment of PMSG (50 IU)-primed rats with hCG (25 IU) precipitated a dramatic (80%) inhibition of ambient ovarian thymosin beta 10 protein and thymosin beta 10 mRNA; both parameters remained suppressed for the duration of the hormone-induced pseudopregnancy (15 days). HPLC analysis also indicated the presence in the ovary of thymosin beta 4, a variant member of the same protein family; in general, ovarian thymosin beta 4 levels fluctuated in a manner reciprocal to that exhibited by thymosin beta 10. A luteolytic dose of prostaglandin F2 alpha (500 micrograms/rat) had little impact on ovarian thymosin beta 10 gene expression. These findings show that 1) PMSG stimulation of ovarian thymosin beta 10 biosynthesis involves increased expression of the thymosin beta 10 gene; 2) decreased expression of thymosin beta 10 is associated with luteinization, while increased thymosin beta 4 levels characterize this process; and that 3) thymosin beta 4 and beta 10 are coexpressed in this tissue and may play a significant but, as of yet, undefined role(s) in the ovary.

Animals