Early embryonal culture of the cynomolgus monkey (Macaca fascicularis)

Ovaries of ten female cynologus monkeys (Macaca fascicularis) were superstimulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). During this treatment, follicular development was monitored by ultrasonography. After three to four injections of PMSG, ovaries began to show an increase in their size. After seven to ten injections of PMSG and a hCG injection, a total of 183 oocytes (18.3±14.1/ animal, mean±SD) were recovered from these females by laparotomy. Five nulliparous females produced 24.2±18.2 oocytes per animal, and the remaining five parous females produced 12.5±5.46 oocytes per animal. In total, 133 (72.6%) of the 183 oocytes were classified morphologically as matured, and 130 of them were inseminated with cynomolgus monkey sperm for 18 hours in vitro. The second polar body was observed in 92 (71%) of the inseminated oocytes, and 54 (59%) of the 92 oocytes showed cleavage division at least once. When these oocytes were fertilized and cultured in serum-free media, they stopped their development at 4-cell stage. In contrast to this condition, about a half of fertilized oocytes in serum-free media were able to develop to morula stage 4 days after insemination by transferring them into serum-containing media after 2-cell stage.     

Superovulation in immature and mature Chinese hamsters

Mature female Chinese hamsters ovulate an average of 8.8 ± 1.0 (mean ± SD) eggs per female in each estrous cycle. Superovulation can be induced in both immature and mature females by subcutaneous or intraperitoneal injections of pregnant mare serum gonadotropin (PMSG) and either human chorionic gonadotropin (hCG) or pituitary luteinizing hormone (PLH). The best superovulation in immature females was induced by the administration of 15 IU of PMSG followed 72 hr later by injection of 15 IU of hCG (about 25 eggs per female) or 0.2 mg (200 IU) PLH (about 46 eggs per female). Ovulation started about 13–15 hr after administration of hCG (or PLH) and was completed during the next 5–6 hr. Superovulation in mature females could be induced by injecting PMSG any day of the estrous cycle, but the best superovulation (about 39 eggs per female) was induced by injecting 15 IU of PMSG on day 1 (day of ovulation) followed by the injection of 0.4 mg of PLH 72 hr later. When immature females treated with the best superovulatory protocol were mated on the evening of PLH injection, only 5% of the eggs were found fertilized 50 hr after PLH administration. On the other hand, about 60% of the eggs were found fertilized in mature females mated following treatment with the best superovulatory protocol. The majority (83–85%) of superovulated eggs obtained from both immature and mature females were normally fertilized in vitro.     

Selective action of chorionic gonadotropin on lymphocyte subpopulations

Influence of chorionic gonadotropin on induction and formation of specific T-suppressors, TDTH-effectors and spontaneous killer cells, depending on its doses and schema of injections was studied. It is shown that chorionic gonadotropin has a stimulating effect on precursors of T-suppressors, not affecting the mature specific T-suppressors, causes damage to the TDTH-effectors and spontaneous killer cells, that permits chorionic gonadotropin to be recognized as an immunomodulating agent.     

Effect of super-ovulatory doses of pregnant mare serum gonadotropin and human chorionic gonadotropin in blastocyst implantation in golden hamsters

The effect of super-ovulatory dose of pregnant mare serum gonadotropin and human chorionic gonadotropin on ovulation, advancement of ovulation, subsequent embryo development and implantation were studied in the hamster. Groups of hamsters received pregnant mare serum gonadotropin injection on day 1 of the estrous cycle followed by human chorionic gonadotropin injection either at 56 or 76 h later, pregnant mare serum gonadotropin alone on day 1 or human chorionic gonadotropin alone on day 3.The combination therapy (pregnant mare serum gonadotropin and human chorionic gonadotropin) resulted in super-ovulation (an average of 40 mature ova/animal) while human chorionic gonadotropin alone yielded an average of 10 mature ova/animal. Ovulation was advanced by 24 h by giving human chorionic gonadotropin at 56 h instead of 76 h after pregnant mare serum gonadotropin. Subsequent embryo development and implantation occurring under different hormonal regimens were studied. The ova obtained by giving human chorionic gonadotropin injection at 56 h were poorly fertilizablein vivo and hence the pregnancy rate was low (6 %). These ova however, were fertilizablein vitro, suggesting that the low fertilization rate and developmental failure may be due to inhibition of sperm capacitation/transport because of premature human chorionic gonadotropin administration. In the group receiving human chorionic gonadotropin alone on day 3 there was fertilization and cleavage, but no implantation occurred due to failure of functional corpora lutea. However, administration of progesterone and estrone from day 2 of gestation resulted in 80% implantation and sustenance of pregnancy. On the other hand, the pregnant mare serum gonadotropin and human chorionic gonadotropin combination therapy resulted in super-pregnancy. The number of fetuses present at term was higher in the group receiving pregnant mare serum gonadotropin alone than in the group receiving the combination therapy. Embryo resorption however was higher (37%) in the latter group compared with the former (9.5%). However, preimplantation embryos were found to be viable as evidenced by fluorescein diacetate staining.     

Effects of intrafollicular administration of gonadotropins in two species of nonhuman primates using laparoscopy.

Direct administration of 0.5 IU of human chorionic gonadotropin (HCG) into the matured ovarian follicles of adult female Saimiri sciureus resulted in ovulation in 45% of the animals treated. When purified ovine luteinizing hormone (LH) was administered, ovulation was observed in 55% of the animals while intrafollicular injection of saline had no effect. Similar injections of HCG into matured follicles of Macaca fascicularis would not cause ovulation but did result in a 55% increase in menstrual cycle length. These results indicate that the necessity of a primate source of LH for inducing ovulation may be due to an extrafollicular mechanism.     

Ovarian and immunological responses to alternating exogenous gonadotropin regimens in the ocelot (Leopardus pardalis) and tigrina (Leopardus tigrinus)

Exogenous gonadotropins frequently are used to stimulate ovarian follicular growth and ovulation in mammalian species, including felids. However, repeated exogenous gonadotropin treatment can result in decreased ovarian responsiveness due to antibody formation. In this study, our objectives were to assess the effectiveness of alternating gonadotropin regimens on ovarian responses in ocelots and tigrinas, and investigate the humoral immune responses to these gonadotropins in each species. Females were treated four to six times with alternating equine chorionic gonadotropin (eCG)/human chorionic gonadotropin (hCG) and porcine follicle stimulating hormone (pFSH)/luteinizing hormone (pLH) regimens at 4‐month intervals. With each treatment, the females were evaluated laparoscopically to assess ovarian follicular development and recover oocytes from mature follicles. Blood was collected before each treatment and at laparoscopy. Overall, the ocelots averaged more (P<0.05) follicles and corpus luteum (CL) (6.8±0.8; mean±SEM) per stimulation than the tigrinas (2.3±0.4), but the percentage of mature oocytes (mean range=54–55%) did not differ (P<0.05). Within species, both gonadotropin regimens were equally effective (P>0.05) in inducing follicular growth and oocyte maturation. The total number of ovarian structures and oocyte maturation percentages did not decrease (P<0.05) in either species with sequential stimulations. Although the percentage of blood samples containing anti‐gonadotropin immunoglobulins increased (P<0.05) with sequential treatment, the presence of positive titers did not cause a decrease (P<0.05) in ovarian responsiveness. In summary, the female ocelots and tigrinas continued to respond to these alternating ovarian stimulation protocols after repeated use, despite the formation of anti‐gonadotropin antibodies in some of the females. These findings suggest that the use of alternating gonadotropin regimens may permit more intensive reproductive management of these endangered cat species for conservation. Zoo Biol 00:1–14, 2005. © 2005 Wiley‐Liss, Inc.     

Fecal steroid hormone analysis as an indicator of reproductive function in the cheetah

Techniques were developed and validated to measure fecal estrogen and progesterone concentrations of the female cheetah. Fecal samples were collected from seven mature females. Cheetahs were monitored before mating and continued until parturition. Four females had normal pregnancies, one conceived but the pregnancy resulted in spontaneous abortion, one was mated but apparently did not conceive and one was treated with gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. Vaginal superficial cells increased with increasing estrogen concentrations. Peak estrogen occurred one day postcopulation. Increases in fecal progesterone concentrations, indicative of ovulation, occurred after copulation and hormonally induced ovulation. For the first time reproductive function can be monitored in the cheetah using noninvasive sample collection. © 1994 Wiley-Liss, Inc.     

GnRH agonist Lupron (leuprolide acetate) pre-treatments prevent ovulation in response to gonadotropin stimulation in the clouded leopard (Neofelis nebulosa)

In many species, controlling the ovary prior to induction of ovulation improves the success of ovarian response and artificial insemination (AI). We assessed the impact of suppression of estrus with the GnRH agonist, Lupron, on ovarian sensitivity to equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) in the clouded leopard. Seven female clouded leopards were given two injections of Lupron (3.75 mg IM) 23 d apart, followed 44 d later by eCG and hCG. Daily fecal samples were collected from 60 d before Lupron to 60 d after hCG. Fecal metabolites of estrogen (E) and progesterone (P) were measured by radioimmunoassay. Lupron decreased (P < 0.05) the number of E peaks during Lupron treatment compared to pre-Lupron. All females had baseline E and six of seven (86%) had nadir P on day of eCG. Exogenous gonadotropins induced E elevations in all females. However, mean E in the gonadotropin-provoked estrus was decreased (P < 0.05) compared to pre-Lupron estrous periods. Only one of seven (14%) females ovulated after eCG/hCG. In conclusion, estrous cycle control with Lupron resulted in predictable ovarian suppression prior to gonadotropin stimulation but altered ovarian sensitivity by an as yet unknown mechanism so that ovulation was inhibited, even when using a proven exogenous gonadotropin protocol.     

Response of large oocytes of Xenopus laevis to progesterone in vitro in relation to oocyte size and time after previous HCG-induced ovulation.

The injection of Xenopus laevis females with human chorionic gonadotropin (HCG) leads to ovulation (and maturation) of oocytes whose diameters are 1.2 mm or larger. However, when Xenopus oocytes are removed from their follicular investments by manual dissection and exposed to the steroid, progesterone, in vitro, they exhibit maturation down to about 0.90 mm in diameter with the majority larger than 1.0 mm showing a positive response. Within each female the larger of the oocytes undergo maturation earlier than smaller ones.The response of oocytes also was shown to depend on the length of time since females were last stimulated to ovulate. Similar-sized oocytes from recently ovulated (stimulated) females matured much faster than those of untreated, unstimulated females. Indeed, even the smaller oocytes from stimulated females often matured before the largest oocytes of females without previous HCG injection.The experiments demonstrate that the physiological state of an oocyte cannot be accurately deduced solely from its size nor response to gonadotropins; unresponsiveness presumably being due to inability of follicular elements to respond to the trophic hormones or transfer the stimulus to the oocyte via the appropriate steroid.     

Maternal-restraint stress increases oocyte aneuploidy by impairing metaphase I spindle assembly and reducing spindle assembly checkpoint proteins in mice

Studies in both humans and animals suggest detrimental effects of psychological stress on reproduction. Although our recent study shows that maternal-restraint stress diminishes oocyte developmental potential, the mechanism behind this effect is unknown. This prompted us to study the potential role of maternal-restraint stress in the genesis of aneuploidy during meiosis I. At 24 h after equine chorionic gonadotropin injection, mice were subjected to restraint stress for 24 h. After the restraint, some mice were killed to recover immature oocytes for in vitro maturation, while others were injected with human chorionic gonadotropin to recover in vivo matured oocytes. Analysis on chromosome complements of both mature oocytes and parthenotes confirmed that maternal restraint increased aneuploidy in both in vivo and in vitro matured oocytes and that the percentage of aneuploid oocytes were three times higher in the earlier matured oocytes than in the later matured ones. Further observations indicated that maternal restraint 1) impaired metaphase I (MI) spindle assembly while inhibiting MAPK activities, 2) accelerated progression of anaphase I while down-regulating the expression of spindle assembly checkpoint (SAC) proteins, and 3) induced intraoocyte oxidative stress. The following possible model was proposed to explain the results. Maternal-restraint stress increased oocyte aneuploidy by impairing MI spindle assembly and decreasing the SAC. Whereas abnormal spindles would affect centromere attachments, a reduction in SAC would accelerate the anaphase I progression. Failure of centromere attachment, together with the hastened anaphase, would result in nondisjunction of the unattached chromosomes. Furthermore, maternal-restraint stress might also impair spindle assembly and SAC function by inducing intraoocyte oxidative stress, which would then reduce MAPK activity, a critical regulator of microtubule assembly and the establishment and maintenance of the SAC during oocyte maturation.     

Endogenous steroid production in cellular and subcellular fractions of rat testis after prolonged treatment with gonadotropins.

Steroid production and enzyme activities were examined in preparations of whole testis tissue, isolated interstitial tissue and seminiferous tubules obtained from adult rats with intact pituitaries receiving daily subcutaneous injections of 100 I.U. human chorionic gonadotropin for 5 days and from control animals. After human chorionic gonadotropin administration testosterone concentrations were increased in total homogenates of whole testis tissue, interstitial tissue and seminiferous tubules. The testosterone production from endogenous precursors was enhanced only in total homogenates of whole testis tissue and interstitial tissue obtained from testes of human chorionic gonadotropin-treated rats. The production of testosterone in the corresponding homogenates of isolated seminiferous tubules was very low. The specific activity of 3 beta-hydroxysteroid dehydrogenase was increased in total homogenates of whole testis tissue, isolated interstitial tissue and seminiferous tubules. No effect was observed on the specific activities of marker enzymes such as cytochrome c oxidase, monoamine oxidase, steroid sulfatase and lactate dehydrogenase, whereas the specific activities of carboxyl esterase were decreased in homogenates of whole testis tissue and interstitial tissue. Total activity of monoamine oxidase was increased in homogenates of interstitial tissue of tests from human chorionic gonadotropin treated rats. After the same prolonged human chorionic gonadotropin treatment the concentration of pregnenolone was increased in mitochondrial fractions of whole testis tissue, interstitial tissue and seminiferous tubules, and the amount of protein isolated in the mitochondrial fraction of interstitial tissue increased by 40%. Steroid production (estimated as pregnenolone) from endogenous precusors by mitochondrial fractions of whole testis tissue and interstitial tissue were increased after human chorionic gonadotropin treatment, for whole testis from 580 pmol/mg mitochondrial protein per h to 1420 pmol/mg per h; and for interstitial tissue from 2665 pmol/mg per h to 7050 pmol/mg per h. The production of pregnenolone in mitochondrial fractions obtaine from isolated seminiferous tubules was very low and contributed hardly at all to the total pregnenolone production in mitochondrial fractions of whole testis tissue from normal rats as well as from human chorionic gonadotropin-treated rats.     

Reduced intrafollicular androstenedione and estradiol levels in early-treated prenatally androgenized female rhesus monkeys receiving follicle-stimulating hormone therapy for in vitro fertilization

Five early-treated and four late-treated prenatally androgenized and five normal female rhesus monkeys were studied to determine whether prenatal testosterone propionate exposure beginning Gestational Days 40-44 (early-treated) or 100-115 (late-treated) affects follicular steroidogenesis during recombinant human FSH (rhFSH) treatment. All monkeys underwent rhFSH injections, without human chorionic gonadotropin administration, followed by oocyte retrieval. Serum FSH, LH, estradiol (E2), progesterone (P), 17alpha-hydroxyprogesterone (17 OHP), androstenedione (A4), testosterone, and dihydrotestosterone were measured basally during rhFSH therapy and at oocyte retrieval. Follicle fluid (FF) sex steroids, oocyte fertilization, and embryo development were analyzed. Circulating FSH, E2, 17 OHP, A4, and dihydrotestosterone levels increased similarly in all females. Serum LH levels decreased from basal levels in normal and late-treated prenatally androgenized females but were unchanged in early-treated prenatally androgenized females. Serum P levels at oocyte retrieval were comparable with those before FSH treatment in all females. All prenatally androgenized females showed reduced FF levels of A4 and E2 but not P or dihydrotestosterone. Intrafollicular T concentrations also were significantly lower in late-treated compared with early-treated prenatally androgenized females or normal females. In early-treated prenatally androgenized females, but not the other female groups, intrafollicular A4 and E2 levels were reduced in follicles containing oocytes that failed fertilization or produced zygotes with cleavage arrest before or at the five- to eight-cell embryo stage. Therefore, in monkeys receiving rhFSH therapy alone without human chorionic gonadotropin administration, early prenatal androgenization reduced FF concentrations of E2 and A4 in association with abnormal oocyte development, without having an effect on P, testosterone, or dihydrotestosterone concentrations.     

Relationships between follicular fluid steroid hormone concentrations, oocyte maturity, in vitro fertilization and embryonic development in the rhesus monkey

Oocytes and matched samples of follicular fluid (FF) were obtained from 70 follicles of five rhesus monkeys stimulated with either pregnant mare serum gonadotropin or human menopausal gonadotropin. Follicular aspiration was performed 30-32 h after human chorionic gonadotropin administration. The concentrations of estradiol (E2), progesterone (P), testosterone (T), and dihydrotestosterone (DHT) in FF were measured. Twenty-six percent of oocytes were classified as mature (M), 41% matured in vitro (Miv), 13% were dysmature, and 20% atretic. M oocytes were associated with significantly higher levels of P and a higher P:E2 ratio. There were no differences in hormone levels associated with fertilized and nonfertilized oocytes. Thirty-five embryos developed to the six- to eight-cell stage in vitro, of which 13 exhibited optimal cleavage rates. Significantly lower levels of E2 and higher P:E2 ratios were associated with the more rapidly cleaving embryos. Proportionally more embryos showing optimal cleavage rates developed from M compared to Miv oocytes, and only embryos derived from M oocytes developed to blastocysts in culture. Optimal cleavage rates to the six- to eight-cell stage in vitro, rather than fertilization rates, are a better indicator of (subsequent) developmental capacity, and, in this study, embryonic development was closely associated with the maturity of the oocyte at recovery.     

Modification of sialyl residues of gonadotropic hormones by reductive amination. Influence on biological activity and circulating half-life

The sialic acid residues of human chorionic gonadotropin, human lutropin and human follitropin were quantitatively modified by introduction of an amino compound. In radioreceptor assays, the modified chorionic gonadotropin, lutropin and follitropin saturated the receptors. However, in the low nanogram range, the gonadotropic binding was higher for the control compared to the modified sample.The hormonal activity of the chorionic gonadotropin was testedin vitro. The modified preparations were four- to thirteen-fold less stimulatory compared to the control but elicited the same maximal response. The biological activity of follitropin was determinedin vivo. In this case, the modified preparations were four- to five-fold less stimulatory than the control. Both the modified chorionic gonadotropin and follitropin preparations were found to act as agonists. Modification of the gonadotropin hormones did not significantly alter the immune recognition of these glycoproteins.The apparent circulating half-life in rats of the modified chorionic gonadotropin and follitropin was increased six- to nine-fold compared to that of native hormones; this might be a consequence of resistance of the modified sialyl residues to sialidases and the resultant slower exposure of terminal galactosyl residues; the plasma half-life of modified lutropin remained the same as that of the native hormone.Abbreviations hCG human chorionic gonadotropin - hLH human lutropin or luteinizing hormone - hFSF human follitropin or follicle stimulating hormone - mala methyl ester of alanine - hCG(ala, mala, etc.) human chorionic gonadotropin modified on sialicacid by reductive amination with alanine, methyl ester of alanine, etc. - IRP-HMG intact rat prostrate-human menopausal gonadotropin     

Hormonal Induction of Male Courtship Behavior in the Japanese Newt, Cynops pyrrhogaster

In the breeding season, the sexually mature male newt, Cynops pyrrhogaster, vibrates the tail in front of the female at an early stage of courtship. Effects of prolactin (PRL), gonadotropin (GTH), and sex steroids on this behavior were investigated in the male paired with the female receiving PRL and GTH. The behavior was elicited in the sexually inert male by injections of PRL of bovine, ovine, or bullfrog origin and human chorionic gonadotropin or bullfrog LH and FSH in combination. The effect of PRL or GTH alone was less marked than that of PRL plus GTH, especially in terms of frequency of the behavior. In the hypophysectomized male, combination of PRL and GTH significantly increased both the incidence and frequency of the behavior. However, PRL alone was not effective, and the effect of GTH alone was less pronounced than that in the intact animal receiving GTH injections. The effect of GTH was nullified by castration. In the PRL-treated castrated animal, testosterone or dihydrotestosterone, but not estradiol, was effective in inducing the behavior.     

Ovulation and fertilization in the vole, Pitymys subterraneus

The females of Pitymys subterraneus bred in laboratory conditions have an irregular sexual cycle and induced ovulation. The first freshly ovulated eggs, surrounded by dense cumulus cells, appear in oviducts 10 h after copulation. Administering exogenous gonadotropins: pregnant mare's serum (PMS), human chorionic gonadotropin (hCG) or luteinizing hormone releasing hormone (LHRH), also induces ovulation in mature females of Pitymys subterraneus. In these experimental conditions females ovulate a similar number of eggs as after copulation. Dual stimulation (PMS and hCG plus copulation) does not result in the ovulation of a large number of normal ova, however, it does cause a release of degenerated oocytes.     

The histological picture of the hypothalamus (the nucleus preopticus) and hypophysis in male Anguilla anguilla L. treated with hormones

Studies were conducted on the effect of human chorionic gonadotropin (HCG), testosterone and desoxycorticosterone acetate (DOCA) on the histological picture of the nucleus preopticus (NPO), hypophysis and gonads of male European eels. It was ascertained that none of the injections used changed the content of the Gomori-positive material within the NPO cells. HCG and testosterone injections altered the histological picture of the hypophysis and injections of HCG influenced the gonads by accelerating their maturity. The injections of DOCA did not change the histological picture of the hypophysis and gonads in male eels.     

Subcellular localization of intracellular forms of human chorionic gonadotropin in first trimester placenta

To determine the subcellular sites for synthesis and processing of human chorionic gonadotropin subunits in cells, first trimester placental cells were fractionated subcellularly on sucrose density gradients. Analysis of the subcellular fractions by immunobinding techniques revealed that the rough endoplasmic reticulum-rich fraction contained only intermediates having high-mannose oligosaccharides, but the Golgi-rich fraction contained not only intermediates but also mature forms which were resistant to endoglycosidase H but sensitive to neuraminidase. These results show that human chorionic gonadotropin subunits are synthesized in the rough endoplasmic reticulum as forms containing high-mannose oligosaccharides, and their maturation occurs in the Golgi apparatus by trimming with endogenous glycosidases. They are then modified by addition of complex oligosaccharides and terminal sialic acid through glycosyltransferases.     

Recognition of the beta-subunit of human chorionic gonadotropin and sub-determinants by target tissue receptors.

The beta-subunit of human chorionic gonadotropin, purified immunochemically to eliminate undissociated human chorionic gonadotropin, induced testosterone production by mouse Leydig cells at concentrations 400-fold higher than human chorionic gonadotropin. Steroidogenesis was also stimulated by a synthetic fragment of the beta-subunit of human chorionic gonadotropin conforming to the peptide sequence residues 39--71, whereas peptide sequence residues 39--56 and three C-terminal fragments (residues 115--145, 111--145 and 101--145) failed to cause steroidogenesis. These studies suggest the presence in the beta-subunit of human chorionic gonadotropin of determinants recognized by the tissue receptors, a part of these determinants residing between amino acid residues 57--71.     

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.