Treatment with eCG and hCG to induce onset of estrous cycles in ewes during the non-breeding season: Effects on follicular development and fertility.

Although combining of eCG and hCG administrations is known to enhance LH-like actions, there have been few studies where there was comparison of the effects of treatment of anestrous ewes with eCG and hCG and eCG alone. In Experiment 1, 18 ewes in seasonal anestrus were administered an intravaginal device (IVD) containing medroxyprogesterone acetate for 12 days, and at the time of IVD removal (D0), were allocated into the following groups (n = 6/group): no further treatment (control); 400 IU eCG (eCG); or 400 IU eCG and 200 IU hCG (eCG + hCG). There was greater ovarian follicular growth in the groups treated with gonadotropins, compared to the control, and there were greater progesterone concentrations in the eCG + hCG group on D9 (P < 0.05). In Experiment 2, 66 ewe lambs were assigned to the same treatment groups described for Experiment 1, and subsequently there was natural mating with rams. There was a greater rate of behavioral estrous manifestation in the eCG (88.5 %; 23/26) and eCG+hCG (85.2 %; 23/27), than control (30.8 %; 4/13; P < 0.05) group. Pregnancy rate was also greater in the eCG (34.6 %; 9/26) and eCG+hCG (18.6 %; 5/27) than control (0 %; 0/13; P < 0.05) group, whereas conception rate, considering only ewe lambs that were mated, was only greater in the eCG group. Although there were greater progesterone concentrations 9 days after treatment in the eCG+hCG group, there was no difference in follicular growth in anestrous ewes, nor was there an effect on estrous behavior manifestation and pregnancy rates in ewe lambs, compared to treatment with only eCG.

Effect of different chorionic gonadotropins on final growth of the dominant follicle in Bos indicus cows.

The aim of this study was to evaluate the effect of eCG or hCG on the final growth of the dominant follicle in Nelore (Bos indicus) cows submitted to fixed-time AI (FTAI). Eighty-four lactating cows with body condition score (BCS) of 2.9 (range 1-5) were used. At a random day of the estrous cycle (D0) cows received 2 mg estradiol benzoate and a reused intravaginal progesterone device (1.9 g). At D8, when the device was removed, 0.5 mg cloprostenol and 1 mg estradiol cypionate was given i.m., and cows were randomly assigned to receive on D8 one of the following treatments: Control (no treatment; n = 17), eCG (300 IU i.m.; n = 17), hCG 300 (300 IU i.m.; n = 18), hCG 200 IM (200 IU i.m.; n = 16) and hCG 200 SC (200 IU s.c.; n = 16). On the same day and 2 days later, cows were subjected to ovarian ultrasonography to evaluate the diameter of the largest follicle and to calculate follicular growth rate (D8 to D10). No differences were observed for the diameter of the largest follicle on D8 (P = 0.3) or D10 (P = 0.4) among treatments. However, the growth rate of the dominant follicle between D8 and D10 was greater for the groups eCG and hCG 300 and there were no differences between the other treatments (Control = 1.1 mm/day; eCG = 1.8 mm/day; hCG 300 = 1.8 mm/day; hCG 200 IM = 1.3 mm/day; hCG 200 SC = 1.3 mm/day; P = 0.02). In addition, more cows from the Group hCG 300 presented premature ovulation (44.4%) than cows from Control (5.8%), eCG (0%), or hCG 200 IM (12.5%), but did not differ from Group hCG 200 SC (18.7%). Regardless of treatment, the size of the largest follicle on D8 was different between cows that presented premature ovulation vs. cows that did not ovulate prematurely (11.3 mm vs. 9.9 mm, respectively; P = 0.01). Treatment with different hCG doses on D8 of a FTAI protocol failed to produce similar effects compared to eCG in terms of final follicular growth support and greater ovulatory follicle size. In addition, the groups hCG 300 and hCG 200 SC induced premature ovulation in a greater portion of cows. Thus, a single administration of hCG on D8 does not appear to be a reliable alternative to eCG treatment in FTAI protocols.

Efficacy of inclusion of equine chorionic gonadotrophin into a treatment protocol for anoestrous dairy cows.

AIM: To compare the performance of intravaginal devices containing 1.0 g (DIB) or 1.38 g (CIDR) progesterone and to determine the efficacy of inclusion of equine chorionic gonadotrophin (eCG) into progesterone-based anoestrous cow treatment protocols for New Zealand dairy cows. METHODS: Anoestrous cows (n = 1,906) from 12 herds were randomly assigned to four treatments: 100 µg gonadorelin (GnRH) at Day -10; 500 µg cloprostenol at Day -3; 100 µg GnRH at Day -1 and fixed time artificial insemination (FTAI) on Day 0 (gonadotrophin-prostaglandin-gonadotrophin [GPG] group, n = 475); GPG with CIDR device (1.38 g progesterone) inserted between Day -10 and Day -3 (CIDR group, n = 477); GPG with DIB device (1.0 g progesterone) inserted between Day -10 and Day -3 (DIB group, n = 477); and DIB with 400 IU eCG at Day -3 (DIB + eCG group, n = 477). Conception rates to FTAI and pregnancy at Day 28 were analysed using generalised estimating equations (GEE). Time to conception and time to return to oestrus were analysed using Kaplan-Meier survival analysis and Cox's proportional hazards regression. RESULTS: The proportion of cows conceiving to FTAI was 0.34 (95%CI = 0.29-0.37), 0.38 (95%CI = 0.34-0.43), 0.38 (95%CI = 0.33-0.42) and 0.41 (95%CI = 0.37-0.46) for GPG, CIDR, DIB and DIB + eCG groups, respectively. The proportion of cows pregnant by Day 28 was 0.55 (95%CI = 0.51-0.60), 0.57 (95%CI = 0.52-0.61), 0.56 (95%CI = 0.52-0.60) and 0.63 (95%CI = 0.59-0.67) for GPG, CIDR, DIB and DIB + eCG groups, respectively. There was an interaction between treatment and number of days calved (p < 0.05). Cows more than 60 days calved and treated with DIB + eCG had higher FTAI conception and 28-day pregnancy rates than cows treated with GPG (p < 0.001). Median interval to conception did not differ between treatments (p > 0.05). There were no differences between DIB and CIDR groups for any parameter (p > 0.05). The range of the relative risk distribution among herds comparing DIB + eCG to DIB groups was greater than that comparing CIDR to DIB groups for conception to FTAI and pregnancy at Day 28. CONCLUSIONS: The inclusion of eCG into progesterone-based anoestrous cow treatment protocols improves conception to FTAI and 28-day pregnancy rates in cows >60 days calved at treatment compared with a GPG protocol. There was no difference in clinical performance between DIB and CIDR devices. CLINICAL RELEVANCE: The combination of a low payload (1.0 g) progesterone releasing intravaginal device with eCG treatment at device removal within a GPG treatment is a clinically effective treatment for anoestrous in New Zealand dairy cows.

The classification, functions and clinical use of different isoforms of HCG.

HCG is composed of two subunits, HCGalpha and HCGbeta. During early pregnancy, HCG stimulates progesterone production in the corpus luteum, and injection of HCG is widely used to induce ovulation in assisted reproduction treatment (ART). Under experimental conditions, the free subunits have been shown to exert functions other than those of HCG, but the relevance of these remains to be determined. Intact HCG, free subunits and degraded forms of these occur in biological fluids, and determinations of these are important for diagnosis and monitoring of pregnancy, pregnancy-related disorders and several types of cancer. Development of optimal methods for the various forms has been hampered by lack of appropriate standards and expression of the concentrations of the various forms in units that are not comparable. Furthermore, the nomenclature for HCG assays is confusing and in some cases misleading. These problems can now be solved; a uniform nomenclature has been established, and new standards are available for HCG, its subunits HCGalpha and HCGbeta, the partially degraded or nicked forms of HCG and HCGbeta, and the beta-core fragment. This review describes the biochemical and biological background for the clinical use of determinations of various forms of HCG. The clinical use of HCG and studies on HCG vaccines are briefly reviewed.

Screening for Down syndrome at 14 weeks of pregnancy.

To investigate whether statistical parameters used in Down syndrome screening between 15 and 22 weeks of pregnancy can be used at 14 weeks, we assayed alpha-fetoprotein (AFP), unconjugated oestriol (uE3), total human chorionic gonadotrophin (hCG), free alpha-hCG, free beta-hCG, and inhibin-A in 16 pregnancies with Down syndrome in the 14th week of pregnancy and expressed values in multiples of the normal median. The median and standard deviation values for these 16 pregnancies were not materially different from those published for 15-22 weeks. It is reasonable, therefore, to offer Down syndrome screening using these markers starting at 14 completed weeks of pregnancy instead of 15 weeks. It needs to be recognized, however, that serum AFP measurement for neural tube defect screening is less effective at this time than between 16 and 18 weeks of pregnancy.

Potent thyrotropic activity of human chorionic gonadotropin variants in terms of 125I incorporation and de novo synthesized thyroid hormone release in human thyroid follicles.

Using a highly sensitive bioassay for TSH, in which human thyroid follicles incorporate 125I and release de novo synthesized thyroid hormone into the culture medium, the thyrotropic activities of various hCG preparations were studied. Under the culture conditions employed, bovine TSH (bTSH) was approximately 6- to 9-fold more active than human TSH (hTSH). Highly purified hCG prepared from urine of normal pregnant women (CR 127) had only a trivial thyrotropic activity equipotent to 0.00022 microU bTSH/U hCG or 0.0013 microU hTSH/U hCG (19.7 microU hTSH/mg hCG). Hybrid hCG (AB1ER) also elicited low thyrotropic activity (14.0 microU hTSH/mg), whereas crude hCG had moderate thyrotropic activity (0.041 hTSH microU/U hCG or 127 microU/mg protein). Deglycosylated hCG, a very weak LH/hCG receptor agonist, was the most potent agonist in thyroid follicles (588 microU hTSH/mg protein). hCGs purified from urine of patients with trophoblastic tumors had greater TSH-like activity (37-84 microU hTSH/mg protein) than purified hCG. Asialo-hCG purified from a patient with choriocarcinoma had very potent TSH-like activity (468 microU hTSH/mg). Submaximal doses of bTSH and hCG variants produced additive stimulation of thyroid function. Furthermore, the thyrotropic effect of hCG was inhibited by anti-TSH receptor antibody obtained from patients with myxedema. These in vitro findings suggest that although hCG is reported to exert potent cAMP-stimulating activity on rat thyroid-like cells (FRTL-5) and Chinese hamster ovary cells transfected with hTSH receptor complementary DNA (0.092-0.72 microU hTSH/U hCG), the thyrotropic activity induced by authentic hCG in human thyroid follicles is too weak to cause hyperthyroidism in normal pregnancy. However, hCG produced by some trophoblastic tumors, particularly asialo-hCG, has potent thyrotropic activity sufficient to cause clinically overt hyperthyroidism when produced excessively.

Peroxisome proliferators and retinoids affect JEG-3 choriocarcinoma cell function.

To examine the hypothesis that nutritional signals regulate trophoblast cell function, JEG-3 choriocarcinoma cells were treated with drugs that stimulate peroxisome proliferator-activated receptors (PPARs). These receptors are thought to mediate in part the effects of lipidic nutrients on gene expression. Because PPARs are modulated by interactions with retinoid-X receptors, we also examined the actions of the peroxisome proliferators in the presence of retinoids. Clofibric acid, a known peroxisome proliferator, suppressed JEG-3 cell growth in association with increases in the tumor suppressor p53 protein and its messenger RNA (mRNA). It reduced CG secretion and CG alpha and CG beta mRNAs in growing cells. However, clofibric acid did not induce peroxisome proliferation in the JEG-3 cells, as assessed by electron microscopy and immunostaining for catalase, a peroxisomal enzyme, or alter levels of mRNAs for peroxisomal proteins, sterol carrier protein-X/sterol carrier protein-2 and acyl-Coenzyme-A oxidase. The mitochondrial cholesterol side-chain cleavage enzyme, cytochrome P450scc, was modestly increased in some experiments. All-trans-retinoic acid and 9-cis-retinoic acid increased CG secretion and CG alpha and CG beta mRNAs, but clofibric acid blunted these stimulatory effects. WY 14,643, another peroxisome proliferator, also reduced CG gene expression without increasing mRNAs encoding peroxisomal proteins or altering P450scc mRNA. The mRNA for a human PPAR, NUC1, was demonstrated in JEG-3 cells, and NUC1 mRNA was shown to be upregulated by 8-bromo-cAMP. We conclude 1) that JEG-3 cells express a PPAR and are subject to regulation by PPAR stimulators; 2) that PPAR stimulation in JEG-3 cells does not promote peroxisome proliferation; and 3) that peroxisome proliferators and retinoids differentially regulate JEG-3 cell endocrine activities. We suggest from these findings that JEG-3 cells possess mechanisms to respond to nutrient cues.

Combination of the chorionic gonadotropin free beta-subunit with alpha.

The rate-limiting event for combination of hCG alpha- and beta-subunits in JAR choriocarcinoma cells is the rate of disulfide bond formation in the beta-subunit. This is accompanied by a conformational change that produces a combination-competent form of the beta-subunit. The combination reaction, however, is incomplete, and 50% of the synthesized beta molecules remain uncombined (free). In addition, 70% of biosynthetically labeled free beta is degraded in the cell. Possible explanations for incomplete dimer formation include 1) biochemical differences between the free and combined beta-subunits that limit combination of free beta, and 2) an inefficient combination reaction due to low intersubunit affinities or limiting concentrations of combination-competent subunits within the cell. To examine whether the biochemical differences between free and combined beta-subunits that we have previously observed affect the combined beta-subunits that we have previously observed affect the combination competence of free beta, free and dimer beta-subunits were purified from the culture medium and lysates of JAR cells and examined for their ability to combine with alpha purified from pregnancy urine in an in vitro combination assay. Secreted free and dimer beta obtained from culture medium combined to the same extent with urinary alpha. Although the combination efficiencies were lower for the intracellular forms, the free and dimer beta-subunits purified from cell lysates also combined to the same extent with urinary alpha. Thus, biochemical differences that exist between the beta forms do not prevent combination of free beta with alpha in an in vitro combination assay. To examine the second possibility, we speculated that if high concentrations of hCG subunits remained in the rough endoplasmic reticulum (ER) for extended periods of time, the extent of dimer formation would increase in the cell. To increase the residence time of hCG subunits in the ER, JAR cells were treated with carbonyl cyanide trifluoromethoxyphenylhydrazone, an agent that inhibits the translocation of hCG subunits from the ER to the Golgi. Treatment of cells with trifluoromethoxyphenylhydrazone in long and short term pulse-chase labeling studies did not result in an increase in the extent of dimer formation. Thus, the subunit combination reaction in JAR cells may be incomplete due to subtle conformational differences in the free beta-subunit; however, these differences do not inhibit the combination of the free beta-subunit in vitro.

Expression of glycoprotein hormone alpha-subunit by endocrine cells of the oxyntic mucosa is associated with hypergastrinemia.

Previous studies have shown that hyperplastic endocrine cells of the oxyntic mucosa in patients with atrophic gastritis may express immunoreactivity for the alpha-subunit of human chorionic gonadotropin (alpha-HCG, common to all glycoprotein hormones). Since this endocrine proliferation is regarded as dependent on the trophic effect of the concomitant hypergastrinemia, the relation between immunohistochemical expression of alpha-HCG by oxyntic endocrine cells and serum levels of gastrin were investigated. The study was performed on endoscopic gastric biopsies of the oxyntic mucosa from 49 patients subdivided into the following groups: A) with histologically normal mucosa and normogastrinemia (22 cases), B) with atrophic gastritis and normogastrinemia (12 cases), C) with normal mucosa and hypergastrinemia (Zollinger-Ellison syndrome, retained antrum) (7 cases) and D) with atrophic gastritis and hypergastrinemia (with or without pernicious anemia) (8 cases). The alpha-HCG immunoreactive cells were found in all hypergastrinemic patients (groups C and D), regardless of the concomitant pathological condition of the mucosa. These cells accounted for 7.8% to 44.7% of the number of Grimelius argyrophil cells in consecutive serial sections. In contrast, alpha-HCG-containing cells were exceptional or absent in most normogastrinemic patients. Their number was sizable in only two cases of group A and three cases of group B, where it ranged from 2.5% to 14.8% of the number of argyrophil cells. It was concluded that expression of alpha-HCG is another feature of oxyntic endocrine cells associated with hypergastrinemia in addition to those previously recognized such as development of hyperplasia and/or carcinoid tumors.

Similarity of the clearance rates of free alpha-subunit and alpha-subunit dissociated from intact human chorionic gonadotropin, despite differences in sialic acid contents.

It is well known that the MCR of proteins can be influenced by their carbohydrate structure; i.e. the presence of terminal galactose on proteins results in uptake by hepatic receptors for galactose-terminated glycoproteins. Thus, a protein with its galactose residues covered or removed exhibits a far longer half life in plasma than one with its galactose residues exposed. The free alpha-subunit of human CG (hCG) has been shown to have a different carbohydrate composition than does the alpha-subunit dissociated from the intact hormone. In our laboratory, analysis of alpha-subunits isolated from pregnancy urine indicated that the alpha-subunit dissociated from hCG (hCG alpha) contains primarily monosialylated oligosaccharides, whereas the free alpha-subunit contains more than one sialic acid per oligosaccharide. This difference in the degree of sialylation prompted us to examine the clearance rates of these two subunits. Accordingly, free alpha and hCG alpha were purified by affinity chromatography and labeled with 125I. The labeled subunits were injected iv into rats and serum samples were removed at various time intervals over a 2-h period. The amount of [125I]alpha-subunit remaining in the serum was determined by immunoprecipitation using an antiserum to hCG alpha. The disappearance curves for the two subunits were indistinguishable and could be analyzed by a biexponential model. The t 1/2 of the faster component was 5 min, while the t 1/2 of the slower component was 74 min. In order to determine whether or not terminal galactose was present on either the hCG alpha or the free alpha-subunit, the labeled molecules were subjected to lectin column chromatography using Ricin or peanut lectin linked to agarose. Both of these lectins bind galactose but have different specificities with respect to the penultimate sugar. Both subunit preparations contained only minor amounts of material which could bind or either lectin. However, after desialylation, both hCG alpha and free alpha bound extensively to Ricin, indicating the presence of penultimate galactose residues in both. We conclude that terminal galactose residues are not present on the oligosaccharides of either hCG alpha or free alpha-subunits, and that the difference observed in the sialic acid contents of the two subunits does not affect their rates of clearance.

Absence of detectable chorionic gonadotropin subunit messenger ribonucleic acids in the rat placenta throughout gestation.

We hypothesized that if a CG similar in structure to other glycoprotein hormones was present in the rat placenta, then mRNAs encoding its subunits would be detectable. To investigate the possible presence of a CG in the rat, we attempted to detect mRNAs encoding the alpha- and CG beta-like subunits in the placentae of timed pregnant rats by sensitive blot hybridization analyses using cloned cDNAs encoding alpha- and beta-subunits of LH derived from rat pituitary mRNA. No subunit-specific mRNAs in placentae of pregnant rats at 4-21 days gestation were detected at either high or low stringencies of hybridization. However, under similar conditions, subunit-specific mRNAs were readily observed in total pituitary RNA of normal as well as ovariectomized rats. Moreover, hybridization with a cDNA for alpha-tubulin, a major component of the cytoskeleton, yielded easily detectable bands in rat placental RNA. In addition, hybridization analysis, under low stringency conditions, of restriction enzyme digests of rat genomic DNA with a rat LH beta-cDNA, which would detect LH beta subunit-like genes, suggests the presence of a single gene that, in fact, encodes the rat LH beta-subunit. We conclude that mRNAs encoding for proteins structurally homologous to rat LH subunits are absent in the rat placenta and that only a single LH beta-like gene is present in the rat. The luteotropic activity associated with the rat placenta must be related to a gonadotropin-like hormone whose structure is dissimilar from that of CG.

Effects of crude and purified human chorionic gonadotropin on lymphocyte response.

Human chorionic gonadotropin (hCG) preparations with different biological activities were tested for their inhibitory effects on mitogenic of allogenic induced lymphocyte response. Various crude hormone batches inhibited the lymphocyte reaction in a dose-dependent manner. However, we found a varying suppression of lymphocyte response not correlated to the biological activity (2,660-4,300 IU/mg) of crude hormone. Fractions with very low gonadotropic activity (much less than 500 IU/mg) showed a 100-fold inhibition of lymphocyte reaction. Conversely, the enrichment of highly purified hCG with strong biological activity (greater than 10,000 IU/mg) had no inhibitory effect on mitogenic or allogenic induced lymphocyte transformation. Isoelectrofocussing and immunoelectrophoretic investigations indicated that the inhibition is probably caused by nondialyzable sialoglycoproteins. It is therefore very doubtful whether hCG plays an important part in maternal tolerance of the fetal allograft.