Assessment of luteal rescue and desensitization of macaque corpus luteum brought about by human chorionic gonadotrophin and deglycosylated human chorionic gonadotrophin treatment

The objective of the current study was to investigate the mechanism by which the corpus luteum (CL) of the monkey undergoes desensitization to luteinizing hormone following exposure to increasing concentration of human chorionic gonadotrophin (hCG) as it occurs in pregnancy. Female bonnet monkeys were injected (im) increasing doses of hCG or dghCG beginning from day 6 or 12 of the luteal phase for either 10 or 4 or 2 days. The day of oestrogen surge was considered as day ‘0’ of luteal phase. Luteal cells obtained from CL of these animals were incubated with hCG (2 and 200 pg/ml) or dbcAMP (2.5,25 and 100 M) for 3h at 37°C and progesterone secreted was estimated. Corpora lutea of normal cycling monkeys on day 10/16/22 of the luteal phase were used as controls. In addition thein vivo response to CG and deglycosylated hCG (dghCG) was assessed by determining serum steroid profiles following their administration. hCG (from 15–90 IU) but not dghCG (15-90 IU) treatment in vivo significantly (P < 0.05) elevated serum progesterone and oestradiol levels. Serum progesterone, however, could not be maintained at a elevated level by continuous treatment with hCG (from day 6–15), the progesterone level declining beyond day 13 of luteal phase. Administering low doses of hCG (15-90 IU/day) from day 6–9 or high doses (600 IU/day) on days 8 and 9 of the luteal phase resulted in significant increase (about 10-fold over corresponding control P < 0.005) in the ability of luteal cells to synthesize progesterone (incubated controls) in vitro. The luteal cells of the treated animals responded to dbcAMP (P < 0.05) but not to hCC added in vitro. The in vitro response of luteal cells to added hCG was inhibited by 0,50 and 100% if the animals were injected with low (15-90 IU) or medium (100 IU) between day 6–9 of luteal phase and high (600 IU on day 8 and 9 of luteal phase) doses of dghCG respectively; such treatment had no effect on responsivity of the cells to dbcAMP. The luteal cell responsiveness to dbcAMP in vitro was also blocked if hCG was administered for 10 days beginning day 6 of the luteal phase. Though short term hCG treatment during late luteal phase (from days 12—15) had no effect on luteal function, 10 day treatment beginning day 12 of luteal phase resulted in regain ofin vitro responsiveness to both hCG (P < 0.05) and dbcAMP (P < 0.05) suggesting that luteal rescue can occur even at this late stage. In conclusion, desensitization of the CL to hCG appears to be governed by the dose/period for which it is exposed to hCG/dghCG. That desensitization is due to receptor occupancy is brought out by the fact that (i) this can be achieved by giving a larger dose of hCG over a 2 day period instead of a lower dose of the hormone for a longer (4 to 10 days) period and (ii) the effect can largely be reproduced by using dghCG instead of hCG to block the receptor sites. It appears that to achieve desensitization to dbcAMP also it is necessary to expose the luteal cell to relatively high dose of hCG for more than 4 days     

Significance of the maternal levels of serum alpha-fetoprotein, human chorionic gonadotropin and unconjugated estriol in various congenital developmental defects in pregnancy trimester II]

Maternal serum alpha-fetoprotein (MSAFP), human chorionic gonadotropin (hCG) and unconjugated oestriol (uE3) concentrations were measured in maternal serum samples from 21 pregnancies with neural-tube defects, 4 pregnancies with ventral wall defects (VWD) and 1662 unaffected pregnancies in women. These congenital malformations were confirmed by ultrasound scanning. The mean multiplate of the median (MoM) for AFP and uE3 was significantly different from the control values in cases of open NTD (AFP median MoM = 5.95, p < 0.001, uE3 median MoM = 0.2, p < 0.001), while hCG values did not differ from those of matched controls (hCG median MoM = 0.9). The biological basis of altered levels of uE3 in pregnancies with fetal NTDs is unclear.     

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.     

Synthesis of alpha subunit of human chorionic gonadotrophin by presumptive HeLa cells

Summary Several cell lines, originally thought to be derived from a human placenta at term but possibly HeLa-contaminated, have been studied. These cells secrete a protein indistinguishable immunochemically from the alpha subunit of chorionic gonadotropin but not the beta subunit of chorionic gonadotropin or placental lactogen. Complete chorionic gonadotropin was detected but amounted to less than 1% of the level of the alpha subunit. The cells also produce an alkaline phosphatase similar to placental alkaline phosphatase in immunochemical, gel-electrophoretic, and heat-denaturation properties. They induce tumor growth when inoculated into nude mice. These cells are aneuploid and have a model chromosome number of 66. The common HeLa karyologic markers, designated 1, 2, and 3, and A-type glucose-6-phosphate dehydrogenase are present in these cells. HeLa cells have not previously been shown to secrete theα subunit of hCG.     

Predictive value of plasma human chorionic gonadotrophin beta subunit in diagnosing ectopic pregnancy after in vitro fertilisation and embryo transfer.

A study was conducted aimed at establishing a range of plasma concentrations of the beta subunit of human chorionic gonadotrophin that might predict ectopic pregnancy after in vitro fertilisation and embryo transfer. From May 1984 to February 1986, 161 consecutive pregnancies at the Monash University in vitro fertilisation unit were analysed by determining plasma beta human chorionic gonadotrophin concentrations between two and 10 weeks after oocyte collection. Eighty eight ongoing singleton pregnancies, 25 multiple pregnancies, 27 first trimester spontaneous abortions, 12 anembryonic pregnancies, and nine ectopic pregnancies resulted from these conception cycles. When compared with values for ongoing singleton pregnancies two weeks after oocyte collection plasma beta human chorionic gonadotrophin concentrations in ectopic pregnancies were significantly lower (p less than 0.05; Wilcoxon rank sum test). Two weeks after oocyte collection all plasma beta human chorionic gonadotrophin concentrations in the set of ectopic pregnancies were below 30.6 IU/l, which corresponded to the lower quartile (25th percentile) of beta human chorionic gonadotrophin concentrations in ongoing singleton pregnancies. The beta human chorionic gonadotrophin concentration corresponding to the lower quartile of ongoing singleton pregnancies at each week of gestation was used to derive the predictive value of various statistics in detecting ectopic pregnancy in patients after in vitro fertilisation. The sensitivity, specificity, predictive value of a positive result, predictive value of a negative result, and efficiency of a single plasma beta human chorionic gonadotrophin concentration in predicting ectopic pregnancy were 100%, 68.1%, 16.7%, 100%, and 70%, respectively, two weeks after oocyte collection. These results suggest that a single determination of the plasma beta human chorionic gonadotrophin concentration beginning 14 days after oocyte collection is clinically useful in predicting the outcome of pregnancy achieved by in vitro fertilisation. Ectopic pregnancy after in vitro fertilisation is more likely when beta human chorionic gonadotrophin concentration is below the lower quartile of values in ongoing singleton pregnancies achieved by the technique.     

LH and hCG Action on the Same Receptor Results in Quantitatively and Qualitatively Different Intracellular Signalling

Human luteinizing hormone (hLH) and chorionic gonadotropin (hCG) act on the same receptor (LHCGR) but it is not known whether they elicit the same cellular and molecular response. This study compares for the first time the activation of cell-signalling pathways and gene expression in response to hLH and hCG. Using recombinant hLH and recombinant hCG we evaluated the kinetics of cAMP production in COS-7 and hGL5 cells permanently expressing LHCGR (COS-7/LHCGR, hGL5/LHCGR), as well as cAMP, ERK1/2, AKT activation and progesterone production in primary human granulosa cells (hGLC). The expression of selected target genes was measured in the presence or absence of ERK- or AKT-pathways inhibitors. In COS-7/LHCGR cells, hCG is 5-fold more potent than hLH (cAMP ED₅₀: 107.1±14.3 pM and 530.0±51.2 pM, respectively). hLH maximal effect was significantly faster (10 minutes by hLH; 1 hour by hCG). In hGLC continuous exposure to equipotent doses of gonadotropins up to 36 hours revealed that intracellular cAMP production is oscillating and significantly higher by hCG versus hLH. Conversely, phospho-ERK1/2 and -AKT activation was more potent and sustained by hLH versus hCG. ERK1/2 and AKT inhibition removed the inhibitory effect on NRG1 (neuregulin) expression by hLH but not by hCG; ERK1/2 inhibition significantly increased hLH- but not hCG-stimulated CYP19A1 (aromatase) expression. We conclude that: i) hCG is more potent on cAMP production, while hLH is more potent on ERK and AKT activation; ii) hGLC respond to equipotent, constant hLH or hCG stimulation with a fluctuating cAMP production and progressive progesterone secretion; and iii) the expression of hLH and hCG target genes partly involves the activation of different pathways depending on the ligand. Therefore, the LHCGR is able to differentiate the activity of hLH and hCG.     

Production of antibodies specific to human chorionic gonadotropin in mice immunized against its chemical analogs

Mice immunized against DS₅-hCG-Β and DS₆-hCG-Β, chemical analogs of Β-subunit of human choriogonadotropin (hCG-Β) in which 5 and 6 disulphide bonds respectively were reduced and alkylated, were found to produce antibodies specific to hCG without significant crossreactivity with human lutropin (hLH) as tested in a radioimmunoassay. In contrast, mice immunized against the native hCG-Β subunit produced hLH crossreacting antibodies. While the anti-DS5, DS6-hCG-Β serum was capable of selectively blocking the binding of [¹²⁵I]-hCG to rat testicular LH/hCG receptors, it failed to inhibit the binding of [₁₂₅I]-hLH to the same receptors. The radioimmunoassay for hCG using the mouse anti-DS₅, DS₆-hCG-Β serum was not as sensitive as that employing rabbit anti-DS₅, DS₆-hCG-Β serum. The minimal detection limit was 5 ng/ml for the mouse antibody as compared to 1 ng/ml for the rabbit antibody. Supported by U.S. Public Health Service Grant HD 08766 to OPB. An erratum to this article is available at .     

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.     

Polyamines control human chorionic gonadotropin production in the JEG-3 choriocarcinoma cell

The effect of inhibition of ornithine decarboxylase with difluoromethylornithine (DFMO) and the resultant lowering of polyamine levels upon human chorionic gonadotropin (hCG) production in JEG-3 choriocarcinoma cells was investigated. DFMO (10 mM) totally inhibited ornithine decarboxylase activity. In DFMO-treated cells, cellular spermidine concentrations fell to nondetectable levels (less than 1% of control values) within 24 h and spermine concentrations were reduced to 41.9% of controls over 6 days. DFMO caused a 70-80% inhibition of hCG production. Levels of mRNA for both the alpha and beta subunits of hCG were also inhibited relative to mRNA for tubulin. Exogenous putrescine normalized hCG production in a dose-dependent manner. Other diamines, including cadaverine, 1,3-diaminopropane, 1,6-diaminohexane, and 1,7-diaminoheptane, were ineffective in reestablishing hCG production in DFMO-treated cells. Dibutyryl cAMP (1 mM) stimulated hCG production and increased levels of mRNA for the alpha and beta subunit 5-40-fold in both DFMO-treated and control cells. Polyamines appear to have a fundamental role in hCG production in JEG-3 choriocarcinoma cells. However, dibutyryl cAMP can partially overcome or circumvent the requirement for polyamines in hCG biosynthesis.     

Dependence of uterine cyclooxygenase2 expression on luteinizing hormone signaling

Several previous studies have demonstrated that uterine Cox2 (also known as Ptgs2) is required for implantation. Luteinizing hormone (LH) released from anterior pituitary gland and human chorionic gonadotropin released from placenta (hCG) can upregulate the uterine Cox2 gene expression. The Lhcgr knockout (herein designated LHRKO) animals have implantation failure even after estradiol and progesterone therapy. These findings led us to investigate the dependence of uterine Cox2 gene expression on LH signaling in LHRKO animals. The results revealed that, while Cox1 (also known as Ptgs1) mRNA levels were similar, Cox2 mRNA levels were lower in uterus of null animals than in wild-type siblings. Treatment with hCG did not increase Cox2 mRNA levels in null endometrial stromal or myometrial smooth-muscle cells unless gene therapy was performed to introduce native LHCGR. The Cox1 mRNA levels, on the other hand, did not change regardless of the introduction of native or activated Lhcgr or hCG treatment. The Cox2 mRNA increase paralleled the cAMP raise, suggesting that LH uses the cAMP second messenger system. Treating the wild-type uterine cells with hCG resulted in a Cox2 but not Cox1 mRNA increase. This increase became exaggerated when additional native LHCGR were introduced by gene therapy. In conclusion, deletion and reinsertion of Lhcgr further support that uterine Cox2 gene expression is dependent on LH signaling.     

On the role of luteinizing hormone-releasing hormone in the in vitro synthesis of bioactive human chorionic gonadotropin in human pregnancies

The dynamics of luteinizing hormone-releasing hormone (LHRH) induced human chorionic gonadotropin (hCG) production were studied in isolated placental cells from normal and anencephalic midterm and term gestations. A spontaneous release of immunoreactive hCG was first detected after 24-36 h of preparation in term control cells. The addition of LHRH at a concentration ranging from 10(-9) to 10(-6) M induced a threefold increase in this output of hCG. Placental cell responsiveness to LHRH varied according to the number of days of cell cultures, with maximal response on days 1 and 6. Placental cells from normal pregnancies incubated with 1 X 10(-6) M LHRH showed a release of both immuno- and bio-assayable hCG, which was four- to six-fold higher at midgestation than at term (p less than 0.001). In contrast, placental cells from pregnancies with anencephalic fetuses showed, at both stages of gestation, an hCG production that was comparable to that observed with normal term placental cells. We conclude that LHRH at a concentration appropriate for its placental receptor binding affinity induces a production of bioactive hCG in humans. Furthermore, our data suggest that anencephaly changes the placental response of hCG to LHRH stimulation.     

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.     

Site-specific glycan analysis of human chorionic gonadotropin beta-subunit from malignancies and pregnancy by liquid chromatography--electrospray mass spectrometry

Glycosylation is an important posttranslational modificationin proteins, and aberrant glycosylation occurs in malignancies.Human chorionic gonadotropin (hCG) is a glycoprotein hormoneproduced in high concentrations during pregnancy. It is alsoexpressed as particular glycoforms by certain malignancies.These glycoforms, which are called "hyperglycosylated" hCG (hCGh),have been reported to contain more complex glycan moieties.We have analyzed tryptic glycopeptides of the ß-subunitof hCG of various origins by liquid chromatography (LC) connectedto an electrospray mass spectrometer. Site-specific glycan structureswere visualized by the use of differential expression analysissoftware. hCGß was purified from urine of two patientswith testicular cancer, one with choriocarcinoma, one with aninvasive mole, two pregnant women at early and late gestation,from a pharmaceutical preparation and culture medium of a choriocarcinomacell line. N-glycans at Asn-13 and Asn-30 as well as O-glycansat Ser-121, Ser-127, Ser-132, and Ser-138 were characterized.In all samples, the major type of N-glycan was a biantennarycomplex-type structure, but triantennary structures linked toAsn-30 as well as fucosylation of the Asn-13-bound glycan areincreased in cancer-derived hCGß. There were significantsite-specific differences in the O-glycans, with constant core-2glycans at Ser-121, core-1 glycans at Ser-138, and putativesites unoccupied by any glycan. Core-2 glycans at either Ser-127or Ser-132 were enriched in cancer. The glycans of free hCGßwere larger and had a higher fucose content of Asn-13-linkedoligosaccharides than intact hCG. This may facilitate the detectionof this malignancy-associated variant by a lectin assay. Analysisof hCGh affinity purified with antibody B152 confirmed thatthis antibody recognizes a core-2 glycan on Ser-132.     

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: