Effects of luteinizing hormone and human chorionic gonadotropin on corpus luteum cells in a spheroid cell culture system

The human corpus luteum (CL) is a highly vascularized, temporarily active endocrine gland and consists mainly of granulosa cells (GCs), theca cells (TCs), and endothelial cells (ECs). Its cyclic growth and development takes place under the influence of gonadotropic hormones. If pregnancy does occur, human chorionic gonadotropin (hCG) takes over the function of luteinizing hormone (LH) and, in contrast to LH, extends the functional life span of the CL. In this study, we investigated the effects of hCG and LH in a spheroidal cell culture model of CL development. Our data indicate that GCs secrete factors under the control of hCG that increase sprout formation of EC-spheroids. We demonstrate that the most prominent of these factors is VEGF-A. Furthermore, we found that both LH and hCG decrease sprout formation of GC-spheroids. After forming EC-GC coculture spheroids and consequently bringing GCs and ECs in close contact, sprouting increased under the influence of hCG, however not under LH. These experiments provide evidence for an hCG dependent functional switch in the GCs after coming in contact with ECs. Moreover, it demonstrates the considerably different effects of hCG and LH on GCs although their signaling is transmitted via the same receptor.     

Serum chorionic gonadotropin levels determined by radioreceptorassay and early diagnosis of pregnancy in the cynomolgus monkey (Macaca fascicularis)

The radioreceptorassay developed to determine serum luteinizing hormone level in the cynomolgus monkey was evaluated for its usefulness in early pregnancy diagnosis by the detection of serum chorionic gonadotropin (CG). Blood samples were collected at weekly intervals from the 1st to the 5th week after conception to determine changes in circulating levels of CG. In the pregnancy cases, serum CG levels increased to above 50 μg/ml in almost all animals. By the determination of CG 3 weeks after conception, 86% of all pregnant cases exhibited a positive response. Cases that were negative 3 weeks after conception were followed by a repeated test in the next week. According to this test schedule, 95% of the pregnant cases were detected by 4 weeks after conception, and 5% were undetected as negative responses because their CG levels were low.     

Effects of culture conditions on the synthesis of human chorionic gonadotropin by placental organ cultures

Summary Culture conditions for maintaining first trimester human placenta in organ culture, which enhance the secretion of human chorionic gonadotropin (hCG), are described. Nutrient medium, oxygen tension and Gelfoam support matrix infuence the synthesis of hCG by these cultures. Placental tissue remained viable for the duration of experiments (12 days) as judged by the incorporation of tritiated thymidine into DNA and the lack of release of incorporated [¹²⁵Iiododeoxyuridine. Optimal conditions for hCG synthesis in placental organ culture included an atmosphere of 95% air and 5% CO₂ (approximately 20% O₂), CMRL 1066 medium containing fetal human or bovine serum, insulin, hydrocortisone and retinal acetate. Multiple pieces of placenta could be cultured in the same dish with an additive effect on hCG secretion. The functional responsiveness of these placental cultures was demonstrated by modulation of hCG synthesis with theophylline and 3′5′ dibutyryl cyclic AMP. Presented in part at the meeting of the American Association for Cancer Research, April 1978. This work is being submitted in partial fulfillment of the Ph.D. requirements in the Department of Biology, Catholic University of America.     

A model system for the biochemical study of luteinizing hormone/chorionic gonadotropin receptor synthesis

A model system for the biochemical study of LH/CG receptor synthesis has been developed. Culture conditions for porcine granulosa cells were adapted that maximized the selective induction of LH/CG receptors by cAMP-inducing stimuli with an elimination of background LH/CG receptor appearance. It was found that the addition of FSH (1.5 μg/ml) or cholera toxin (10 ng/ml) 1 day after plating resulted in optimal induction of the LH/CG receptor (20–60 pg [¹²⁵I]CG bound/μg DNA 72 h after addition) with virtually no LH/CG receptor appearance in the absence of added stimuli. Later additions of FSH or cholera toxin required insulin (1.0 μg/ml) which alone caused background LH/CG receptor appearance in the absence of any additional stimuli. Furthermore, insulin increased the general rate of cellular protein synthesis, whereas FSH or cholera toxin each decreased it. Thus, the use of FSH or cholera toxin, without insulin, may enable one to detect the synthesis of the LH/CG receptor by metabolic labeling techniques where background protein synthesis has been lowered.     

Role of gangliosides in gonadotropin and cholera enterotoxin stimulated steroidogenesis in isolated rat ovarian cells

Ovarian cells isolated from 26 day old rats responded to hCG (10 ng/ml) and cholera enterotoxin (100 ng/ml) $\text{in vitro}$ with a forty-five to fifty-fold increase in progesterone production. Both cholera enterotoxin and hCG-stimulated progesterone response was accompanied by a lag period. The duration of the lag period in the production of the progesterone depended on the concentration of gonadotropin or cholera enterotoxin, and with maximally stimulating dose it was 20–30 minutes. Addition of highly purified mixed gangliosides to the incubation medium abolished the stimulatory effect of cholera enterotoxin on progesterone response. In contrast, under identical experimental conditions, ganglioside addition produced no effect on progesterone response elicited by hCG or LH. Similarly mixed gangliosides did not prevent the specific binding of [¹²⁵I]hCG to the ovarian cells or to the membranes isolated from the ovary. In addition preincubation of [¹²⁵I]hCG with ganglioside did not alter the subsequent binding of the hormone to the ovarian cell surface receptor. These findings suggest that gangliosides are not involved in the hormone receptor interactions and subsequent receptor mediated physiological response.     

The effect of clomiphene citrate and human chorionic gonadotropin on the expression of CatSper1, CatSper2, LHCGR,and SF1 genes,as well as the structural changes in testicular tissue of adult rats

The purpose of this study was to investigate the effect of clomiphene citrate and human chorionic gonadotropin (HCG) on the structural changes, as well as the evaluation of the expression of cation channel sperm‐associated protein 1 (CatSper1), cation channel sperm‐associated protein 2 (CatSper2), luteinizing hormone/choriogonadotropin receptor (LHCGR), and steroidogenic factor 1 (SF1) genes in testicular tissue of rats. All rats divided into five groups as follows; G1 as the control group that received normal saline, G2 received olive oil, G3 received 100 IU/kg HCG, G4 received 5 mg/kg clomiphene citrate, and G5 received 5 mg/kg clomiphene citrate and 100 IU/kg HCG. At the end of the experiment period, Day 56, blood samples were taken and the serum was isolated. Then, histomorphometric analysis, hormonal assess, and real‐time polymerase chain reaction to measure the expression of CatSper1, CatSper2, LHCGR, and SF1 genes were performed. The results showed that the concentrations of testosterone, follicle‐stimulating hormone, and luteinizing hormone were decreased in the G4 group, whereas these parameters were increased in the G3 group. A comparison of the sperm quality indicated a significant reduction in the quality of sperm cells in the G4 group compared with other groups. The quality of sperm was significantly enhanced in the G3 and G5 groups in comparison with the G1 group. Also, our findings demonstrated that the expression of CatSper1, CatSper2, LHCGR, and SF1 genes were significantly elevated in the G3 group when compared with other experimental groups. According to the obtained results, it seems that clomiphene citrate reduces the process of spermatogenesis and the detrimental impacts of this compound would be neutralized by the administration of HCG.     

A test of maternal human chorionic gonadotropin during pregnancy as an adaptive filter of human gestations

The risk of abnormalities and morbidity among live births increases with advanced maternal age. Explanations for this elevated morbidity invoke several maternal mechanisms. The relaxed filter stringency (RFS) hypothesis asserts that mothers, nearing the end of their reproductive lifespan, reduce the stringency of a screen of offspring quality in utero based on life-history traits of parity and interbirth interval (IBI). A separate line of research implicates human chorionic gonadotropin (hCG) during pregnancy as a signal of offspring quality. We test the RFS hypothesis directly by examining whether the difference in gestational hCG across consecutive live births varies positively with the mother''s number of previous live births but inversely with her most recent IBI. We applied multivariable regression methods to a unique dataset of gestational hCG for over 500 000 live births from 2002 to 2007. The difference in gestational hCG across mothers'' consecutive live births varies positively with both mothers'' parity and IBI. These associations remain similar among older mothers (35+ years). Findings support the RFS hypothesis for the parity expectation but not for the IBI expectation. Further evidence for the RFS hypothesis among contemporary human gestations would have to invoke screening mechanisms other than hCG.     

Chemical and biological properties of the subunits of pregnant mare serum gonadotropin

The two subunits (α and β) of pregnant mare serum gonadotropin have been dissociated and partially characterized. Recombination of the biologically inactive subunits results in the restoration of both the follicle stimulating and leuteinizing activities of pregnant mare serum gonadotropin. In addition, the α subunit of pregnant mare serum gonadotropin can be combined with the β subunit of either ovine luteinizing hormone, human chorionic gonadotropin, or follicle stimulating hormone with generation of the specific activity expected of the β subunit.     

The effect of luteinizing hormone releasing hormone and anti-luteinizing hormone releasing hormone antibodies on chorionic gonadotropin and progesterone secretion by human placental villiin vitro

Gonadotropin releasing hormone has been located and found to be secreted by the human placenta in culture. Addition of the releasing hormone upto 1μg concentration in the placental cultures brings about stimulation of chorionic gonadotropin and progesterone secretion. Higher amounts of the decapeptide has an inhibitory influence on both the gonadotropin and the steroid production. The action of the releasing hormone on the placenta could be blocked by the anti-luteinizing hormone releasing hormone monoclonal antibodies indicating a possible site of action of the antibodies for control of fertility     

Gonadal receptors I. Evidence for irreversibility in the binding of human chorionic gonadotropin and human luteinizing hormone

The binding of human chorionic gonadotropin and human luteinizing hormone to particulate receptors of rat testes has generally been assumed to follow an equilibrium model similar to that proposed for many enzyme systems. Our work shows that equilibrium dissociation constant (K_d) and number of hormone binding sites (B_max) are highly sensitive to changes in hormone and/ or receptor concentration and to treatment received by tissue or receptor preparation prior to the assay. The results of binding assays obtained using receptor preparation pretreated with hormone (labeled as well as unlabeled) indicated that the binding reaction between hormone and receptor was irreversible and that pretreatment of the tissue with hormone greatly alters the number of high affinity gonadotropin binding sites in the testicular homogenate. Data from studies involving increasing receptor concentrations revealed that increasing the mass of particulate receptors in the binding assays leads to higher K_d as well as B_max values. These findings are incompatible with a binding model based upon occupancy of receptor sites and the state of equilibrium implied. The incompatibilities are analyzed and an alternate model advanced (Bhalla, V.K., Trowbridge, C.G., Chen, C.J.H., Lindeman, J.G. and Rojas, F.J. (1979) Biochim. Biophys. Acta 584, 436–453).     

Characterization of a lipid-associated gonadotropin receptor from the luteinized rat ovary

Gonadotropin receptors which bind luteinizing hormone (lutropin) and human chorionic gonadotropin (hCG) in the ovaries of immature female rats showed a 30-fold increase after treatment of animals with pregnant mare serum gonadotropin (PMSG) and hCG. This marked induction of lutrophin/hCG receptors in the rat ovary was not accompanied by a change in binding affinity for labeled hCG. Such luteinized ovaries have been found consistently to contain a small proportion of soluble receptor sites, which comprised about 5% of the total receptor population. The soluble receptor sites were present in the floating lipid fraction of the 360 000 × g supernatant of homogenate prepared from luteinized ovaries, and could not be detected in similar fractions prepared from interstitial cells or homogenates of the normal rat testis.The physico-chemical properties of the spontaneously soluble ovarian receptors were similar to those derived for detergent-solubilized receptors prepared by extraction of particulate ovarian binding fractions with Triton X-100. The affinity constant to the soluble ovarian receptor sites for [¹²⁵I]hCG was 0.70 · 10¹⁰ M^?1, and that of the receptors solubilized by Triton X-100 was 0.72 · 10¹⁰ M^?1. The sedimentation pattern of the soluble receptors during sucrose density gradient centrifugation showed extensive aggregation into rapidly sedimenting forms. However, centrifugation of the cytosol receptor in the presence of Triton X-100 gave a single 6.5 S component, corresponding to the solubilized receptors previously characterized in detergent extracts of the rat ovary and testis.The pesence of a spontaneously soluble lutropin/hCG receptor in ovarian cytosol fractions suggests that rapid synthesis and assembly of receptors in ovaries of PMSG-hCG-treated rats is accompanied by increased production of cytoplasmic receptor precursors; alternatively, this receptor population may represent a fraction that has been internalized or processed as during receptor turnover in the cell membrane.     

Detection of human chorionic gonadotrophin hormone using a label-free epoxysilane-modified capacitive immunosensor

A new and label-free capacitive immunosensor based on antibody-functionalized epoxysilane on a glassy carbon electrode has been developed for quantitative detection of human chorionic gonadotropin (hCG). Monitoring the changes in the capacitance signals of antibodies before and after the binding of the antigen provides the basis for an immunoassay. The performance and factors influencing the immunosensor were also studied. Under the optimized conditions, the developed immunosensor quantitatively detected serum hCG in the range of 18–450 mIU/ml with a detection limit of 5.0 mIU/ml (at 3δ). Thirty-five patients’ sera were assayed by the proposed immunosensor, and the results agreed with those given by the commercial radioimmunoassay test kit, with correlation coefficient of 0.998. Further research about the intrinsic electroactivity of antibodies and their target molecules would surely provide new and sensitive screening assays as well as extensive data regarding their interaction mechanisms.     

Brief report: Synthesis of ribonucleic acid in oocytes collected from squirrel monkeys and humans following chorionic gonadotropin administration

The time between administration of human chorionic gonadotropin (hCG) and follicle aspiration was analyzed for alterations in [³H]uridine incorporation [as an indicator of relative synthesis of ribonucleic acid (RNA)] of oocytes from squirrel monkeys and humans. There was a significant decline in both the uptake and incorporation of [³H]uridine in squirrel monkey oocytes by 36 hr following hCG administration to the animals, as compared with 16 hr after hCG. Similarly, RNA synthesis diminished in oocytes collected 35 hr after hCG in humans, as compared with 12 hr after hCG. This reduction in RNA synthesis of maturing oocytes is similar to that of other mammalian species. This provides evidence for an increased interval between hCG administration and follicle aspiration in order to recover mature oocytes for in vitro fertilization studies.     

Theoretical prediction of antigenic sites in the Β-subunits of human choriogonadotropin and luteinizing hormone

Using hydrophilicity and recognition values of amino acids, the antigenic sites of theΒ-subunits of human choriogonadotropin and luteinizing hormone were computed from their amino acid sequences. Six antigenic sites were calculated for human choriogonadotropinΒ-subunits: residues 3–8, 17–22, 59–65,100–106,110–116 and 134–139. For luteinizing hormoneΒ-chain three antigenic sites were calculated: residues 17–22,59–65, and 100–106; all these three sites of luteinizing hormoneΒ being identical to the corresponding sites in human choriogonadotropinΒ. There was no antigenic site in luteinizing hormone that was also not found in human choriogonadotropin. On the other hand, there were unique determinants in human choriogonadotropin that were not found in luteinizing hormone; these determinants were residues 3–8, 110–116 and 134–139     

Cloning of partial putative gonadotropin hormone receptor sequence from fish

A search for the presence of mariner-like elements in the Labeo rohita genome by polymerase chain reaction led to the amplification of a partial DNA sequence coding for a putative transmembrane domain of gonadotropin hormone receptor. The amplified DNA sequence shows a high degree of homology to the available turkey and human luteinizing and follicle stimulating hormone receptor coding sequences. This is the first report on cloning such sequences of piscine origin.     

磁性分离酶联免疫技术检测人类绒毛膜促性腺激素及其临床意义分析

应用磁性分离酶联免疫检测技术（ＭＡＬＡ）对２１０名正常非妊娠妇女的血清进行ＨＣＧ定量测定,平均值３．５４９ｍＩＵ／ｍｌ血清。另外,对１０名不全流产患者、１５名葡萄胎患者及５名早孕误诊患者血清进行ＨＣＧ定量测定,均作出准确诊断。ＨＣＧ定量测定在临床诊断上有重要意义。该法具有灵敏、快速、准确、无污染等特点。是目前临床生殖内分泌激素定量测定的好方法。     

Use of a radioreceptorassay (RRA) for human luteinizing hormone/chorionic gonadotropin (hLH/CG) for detection of early pregnancy and estimation of time of ovulation in macaques

A radioreceptorassay (RRA) for macaque luteinizing hormone (LH)/chorionic gonadotropin (CG) was adapted from the clinical RRA for human LH/CG, Biocept-G?, for the purposes of detection of pregnancy prior to day 20 of gestation and for estimation of the time of ovulation in macaques. The 90-min assay procedure was simple, accurate, and reliable. Seventy-five rhesus monkeys (Macaca mulatta) and 20 crab-eating monkeys (Macaca fascicularis) were tested for the presence of CG in the serum on estimated days 17–20 of pregnancy. Of a total of 160 tests, four false negative and 0 false positive tests were obtained, for an accuracy of 97.5%. The preovulatory LH peak was detected in 19 rhesus monkeys by semiquantitative RRA of LH/CG. Ovulation was confirmed in these 19 animals by the presence of a fresh corpus luteum at laparotomy 2–10 days after ovulation, collection of an embryo, pregnancy, or subsequent cycle history. The short, simple assay procedure and the low inter-and intraassay coefficients of variation (7.3 and 3.7%, respectively) allow use of this assay in an economical, predictive, as well as retrospective, capacity for estimation of the time of ovulation in rhesus monkeys. The sensitivity, reliability, species nonspecificity, simplicity, and rapidity of performance of this RRA for LH/CG are features which add up to a useful new management tool for breeding macaques for research purposes.