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

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 influence 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 [125I]iododeoxyuridine. 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.     

Human chorionic villous macrophages as a fetal biological shield from maternal chorionic gonadotropin

In addition to its most well characterized biological role in the rescue and maintenance of corpus luteum function, human chorionic gonadotropin (hCG) also stimulates the onset of fetal gonadal steroidogenesis. However, excess hCG is teratogenic to fetal gonadal tissues, and therefore hCG must be tightly regulated. Although there is an anatomical barrier between the fetal vessels and maternal blood, other mechanisms may regulate hCG levels. In the present study, we investigated whether human chorionic villous macrophages degraded maternal hCG. Isolated human macrophages incorporated and degraded hCG in a time-dependent manner. Human placental villous macrophages and phorbol myristate acetate (PMA)-treated THP-1 cells expressed the gene encoding an exon 9-deleted form of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptor; expression of the full-length receptor was not determined. While both PMA-treated or untreated THP-1 cells could uptake hCG into their cytoplasms, hCG degradation and excretion of its byproducts only progressed in PMA-treated THP-1 cells. In conclusion, hCG internalization and degradation are different processes in macrophages that protect fetal gonadogenesis from excess hCG. The exon 9-deleted LH/CG receptor, but not the full-length receptor, is involved in the degradation of cytoplasmic hCG by organ-specific, dominant–negative interactions.     

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     

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.     

Structural studies on equine chorionic gonadotropin

The amino acid sequence of the subunit of equine chorionic gonadotropin (eCG, also pregnant mare serum gonadotropin, PMSG) has been determined. Overlapping peptides from tryptic and chymotrypic digests were isolated by a two-dimensional peptide mapping technique and sequenced by the Edman procedure. The proposed amino acid sequence of eCG is: (**Denotes carbohydrate attachment points.) This sequence differs significantly from that proposed by Rathnamet al. (1978) for equine follitropin subunit; in particular, their sequence lacked the first fourteen residues.For the subunit we have placed in sequence 104 amino acid residues by direct sequence determination and peptide overlap procedures; in addition, 37 residues have been placed provisionally by homology with the human chorionic gonadotropin (hCG) sequence and composition and/or sequence data for the peptides isolated in the present studies. Difficulties in the procurement of the hormone have stalled completion of the -subunit amino acid sequence determination. The data now available indicate that eCG -subunit is highly homologous to hCG subunit and the subunits of luteinizing hormone from the pituitary gland of the several species so far described. The proposed partial sequence of eCG is:     

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。     

Effect of desialylated human chorionic gonadotropin (hCG) on the bioactivity of rat Leydig cells

Human chorionic gonadotropin is a glycoprotein hormone that, like LH, stimulates steroidogenesis in gonadal cells. Using a desialylation process, 95 per cent of the sialic acid residues from an intact standard hCG molecule were eliminated and then the electrophoretic properties and the bioactivity of the desialylated hCG were determined. Using rat Leydig cells as a biological model, the binding affinity to LH receptors of Leydig cell membranes, steroidogenic activity and second messenger production were studied. The results indicate that the loss of sialic acid from the hCG molecule slightly increases the binding activity to LH receptors and results in steroidogenic activity with an increased ED₅₀. Cyclic AMP production was significantly reduced however and arachidonic acid release was not observed. Several possible mechanisms that could explain these results are discussed. © 1998 John Wiley & Sons, Ltd.     

Differentiation in human amniotic fluid cell cultures: Chorionic gonadotropin production

Summary Two of the distinguishable cell classes subcultured from human amniotic fluid were examined for their capability to produce human chorionic gonadotropin (hCG) as determined by radioimmunoassay. The class that predominates in most cultures used for prenatal genetic diagnosis, previously termed AF (for amniotic fluid), secretes hCG into the culture medium. Dermal fibroblasts do not, nor does another type of cultured cell from amniotic fluid, previously termed F because of a resemblance to fibroblasts. Primary AF cultures produce more hCG than do subcultures. Evidence that this hormone is intact hCG is provided by its immunoreactivity with antisera raised against the β-subunit and against the intact molecule of hCG. Furthermore, a dose-response curve for hormone in culture medium is parallel to that of highly purified intact hCG. It is postulated that AF cultures are derived from fetal membranes and retain properties of trophoblast. Research supported by Grand HD 11379 from the National Institutes of Health.     

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.     

Isolation of functional human trophoblast cells and their partial characterization in primary cell culture

Summary Human trophoblast isolation and cell culture procedures were examined to identify variables that enhance secretion of chorionic gonadotropin (HCG) in primary culture. Brief exposure of unminced first-trimester placental specimens to a solution of trypsin-EDTA-DNAse, and isolation of the dispersed cells after Ficoll-hypaque centrifugation yielded primary cultures that were high in HCG secretion and content of epithelial-like cells. The gradual decline in HCG level with time in monolayer culture in these presumptive trophoblast cells was retarded by treatment with theophylline and cyclic adenosine monophosphate. Exposure to methotrexate (MTX) did not increase HCG secretion in normal trophoblast cells, in contrast to a 5-fold stimulation by MTX in the JAR line of choriocarcinoma cells. Clusters of polygonal cells in primary culture progressively lost their capacity to secrete HCG and their epithelial-like morphology. However, they could be maintained as cell strains through approximately 15 passages over a period of 13 to 16 weeks.     

Effect of human chorionic gonadotropin on serum levels of progesterone and estrogens in the pregnant bonnet monkev (Macaca radiata)

Administration of human chorionic gonadotropin to pregnant bonnet monkeys(Macaca radiata) at 55–60 days and 130–140 days of pregnancy resulted in a significant increase in serum progesterone levels. This effect could be observed even in lutectomized monkeys. However, no significant change in the serum estrogen level was noticed. These results suggest that although no chorionic gonadotropin is detectable in the serum after 35 days of pregnancy, the foetoplacental steroidogenic system is still responsive to exogenous gonadotropic stimulation.     

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.     

Effects of meiosis-inhibiting agents and equine chorionic gonadotropin on nuclear maturation of canine oocytes

Experiments were conducted to determine the effects of meiosis-inhibiting-agents and gonadotropins on nuclear maturation of canine oocytes. The culture medium was TCM199 + 10 ng/ml epidermal growth factor supplemented with 25 microM beta-mercaptoethanol, 0.25 mM pyruvate, and 1.0 mM L-glutamine (Basal TCM). Initially, oocytes were cultured in Basal TCM alone or in Basal TCM + dibutylryl cyclic adenosine monophosphate (0.5, 1, 5, or 10 mM dbcAMP) for 24 hr. Dibutylryl cAMP inhibited resumption of meiosis in a dose-dependent manner; 60% of oocytes remained at the germinal vesicle (GV) stage after being cultured for 24 hr in 5 mM dbcAMP. The meiosis-inhibitory effect of dbcAMP appeared to be reversible, as the oocytes resumed meiosis and completed nuclear maturation after being cultured for an additional 48 hr in its absence. Oocytes were then cultured in Basal TCM alone or in Basal TCM + roscovitine (12.5, 25, or 50 microM) for 24 hr. Although approximately 60% of oocytes cultured in 25 microM roscovitine remained at the GV stage, this percentage was not significantly different from the 48% that also remained at the GV stage when cultured in its absence. Oocytes were cultured in Basal TCM + 25 microM roscovitine for 17 hr, exposed briefly to equine chorionic gonadotropin (eCG), and then cultured in Basal TCM for 48 hr. Short exposure of oocytes to eCG was beneficial, as it significantly increased the proportion of oocytes developing beyond germinal vesicle breakdown (P < 0.05) with approximately 20-30% of these were metaphase I (MI) oocytes. Study of the kinetics of nuclear maturation demonstrated that large numbers of oocytes remained at MI even after being cultured for 52 hr following brief exposure to eCG. This study showed that in vitro maturation of canine oocytes can be somewhat improved by short exposure of oocytes to eCG. However, further studies are still required to derive effective methods to mature canine oocytes in vitro.     

Label-free amperometric immunosensor for the detection of human serum chorionic gonadotropin based on nanoporous gold and graphene

A label-free amperometric immunosensor for fast and sensitive assay of human serum chorionic gonadotropin (hCG) is presented. hCG was immobilized on nanoporous gold (NPG) foils and using hydroquinone (HQ) redox species as indicator. The variation of amperometric response to the concentration of hCG, the target antigen, was evaluated by cyclic voltammetry in phosphate-buffered solution. Taking advantage of dual-amplification effects of the NPG foils and graphene sheets (GSs), the immunosensor exhibited a specific response to hCG in the range of 0.5–40.00 ng ml⁻¹ with a detection limit of 0.034 ng ml⁻¹ under optimal conditions. It was demonstrated that our proposed method possesses good accuracy, acceptable precision, and reproducibility. The NPG showed a better sensitizing effect and stability as immobilization matrices.     

Effect of media composition on the induction of chorionic gonadotropin by sodium butyrate in HeLa cells

Summary Production of the glycoprotein hormone α-subunit by HeLa cells and its induction by sodium butyrate are dependent on the choice of culture medium. Under identical growth conditions it was found that subunit synthesis in the presence of butyrate was highest in RPMI 1640, lowest in Medium 199 (M199), and intermediate in minimum essential medium (MEM) and Waymouth's MB 752/1. Cell growth was similar in all media examined and was retarded in the presence of butyrate. Alkaline phosphatase activity was also lower in M199 than in RPMI 1640, although, in general, the magnitude of this difference was less than that for the hormone subunit. Incorporation of [1-¹⁴C]butyrate by HeLa cells was simimar in both M199 and RPMI 1640, indicating that uptake and metabolism of the fatty acid were not significantly different under these conditions. In the presence of 3 mM butyrate, mixtures of RPMI 1640 and M199 gave intermediate levels of α-subunit and alkaline phosphatase compared to each medium alone. Intracellular levels of α-subunit as well as that of the culture medium were reduced in M199 compared to RPMI 1640 indicating that synthesis rather than secretion was altered. This work was supported by Grant CA 21534 from the National Institutes of Health, Bethesda, MD.     

Positive influence of tetracycline on human fetal kidney in serum-free organ culture

Summary Human fetal kidney explants can be maintained during 5 days in Leibovitz’s L15, a basic serum-free medium. Because culture conditions are minimal for growth and differentiation, DNA synthesis drastically decreases during the first 48 h, but stabilizes thereafter. The addition of insulin plus transferrin significantly restores this important cellular function in kidneys of fetuses younger than 16 wk. However, renal explants from older fetuses are more difficult to culture: they respond less to growth factors and are more prone to necrosis. The objective of this study was to verify the influence of tetracycline, an antibiotic with anti-collagenase potential, on cultured kidney explants aged 17 to 20 wk. The addition of 20μg/ml tetracycline did not influence DNA synthesis nor the effectiveness of insulin plus transferrin on cell proliferation. Nor did it change the activities of alkaline phosphatase and γ-glutamyltransferase, two enzymic markers of brush border differentiation. After 5 days in L15 alone, explants often showed necrosis and an important reduction in both weight and volume. Insulin plus transferrin significantly restored these parameters to control values observed at Day 0, but evidence of necrosis was still present. Tetracycline alone markedly reduced explant necrosis resulting in a significant increase in weight and volume. The effectiveness of insulin plus transferrin on explant morphometry was not improved when tetracycline was added as third factor. These results indicate that insulin plus transferrin restores explant mass through cell proliferation, whereas tetracycline does so possibly through a reduction in extracellular matrix degradation. The two effects are not additive in cultured mid-term fetal kidneys.