Extraction of equine chorionic gonadotrophin from pregnant mare plasma by direct adsorption on chromatographic media

Equine chorionic gonadotrophin (eCG) is a hormone of practical value in veterinary medicine and animal production. Here we report a novel preparation procedure based on its direct adsorption onto anionic-exchange resins in a batch-wise mode. The active plasma is previously conditioned to reduce pH and ionic strength to required levels. After the adsorption stage, a 90% recovery of the initial eCG is achieved, with a concentration factor of about 50 and an enrichment factor around 500, with high preservation of biological activity. Further purification is carried out by cation-exchange column chromatography. The recovery for the whole process is higher than 70%, and the final potency of the preparation is close to 4000 IU/mg. The process is well suited for its application to the industrial scale.     

Purification and characterization of guinea-pig chorionic gonadotrophin

A human chorionic gonadotrophin-like protein (GF-1, 1.0 g) from the placentae of 50 guinea-pigs killed at Day 26 of gestation was purified by pH and ammonium salt fractionation followed by column chromatography on DEAE-Sephadex and filtration on Sephadex G-100. Relative to the Second International hCG standard (MRC 61/6) GF-1 had an immunological potency of 21 000 i.u./mg as measured in a specific hCG-beta radioimmunoassay and, using the ovarian ascorbic acid depletion assay, an apparent biological potency of 24 064 i.u./mg. Isoelectric focusing yielded 6 bands between pH 4.4 and 5.7 and the material comprised two non-covalently linked subunits. The Stokes' radii were 3.40 nm for the native preparation, and 2.38 nm and 3.15 nm for GF-1-alpha and GF-1-beta subunits respectively. The guinea-pig placenta therefore produces a chorionic gonadotrophin which on purification has physicochemical, biological and immunological properties similar to those of hCG.     

Purification and properties of baboon chorionic gonadotrophin

Baboon CG from the urine and placenta (Days 33-45 of gestation) was purified by ammonium acetate/alcohol extraction followed by ionic exchange, gel filtration and affinity chromatography. The CG activity was monitored using a double-antibody radioimmunoassay utilizing anti-baboon CG and hCG both as the labelled ligand and standard. Biological activity was measured by the rat luteal cell radioreceptor assay. The purified preparation exhibited heterogeneity in terms of its behaviour during ionic exchange chromatography and isoelectric focussing. Like hCG, baboon CG was made up of two non-covalently linked subunits: the Stokes' radii were 36.5, 29.0 and 22.0 X 10(-10) m for native CG, the beta subunit and the alpha subunit. The material focussed between pH 4.2 and 5.5. Relative to the second international hCG standard the biological potency of the purified urinary baboon CG was 4058 i.u. whilst the immunological activity was 4364 i.u. It is concluded that baboon and human CG are very similar with respect to their physicochemical properties and that baboon CG can be purified by the methods that have been developed for hCG.     

A unifying concept of chorionic gonadotrophin production in malignancy

Elevated blood levels of immunoreactive human chorionic gonadotrophin (HCG) have been reported in many patients with non-trophoblastic tumours, but also in various non-malignant conditions. Even normal tissues other than placenta have been shown to produce HCG, such as gonads, gastrointestinal tract, liver, and pituitary. Since HCG is produced, albeit at a low level, by a variety of normal tissues, there is no need to invoke the gene derepression theory to account for 'ectopic' HCG production. However, tumours associated with excess of biologically active HCG as evidenced by endocrinological abnormalities, such as precocious puberty or gynaecomastia, are very rare. We have reviewed the world literature and found 44 such tumours in the lung, adrenal gland, liver, gastrointestinal tract, and genitourinary tract (excluding the gonads). The analysis of their histological pattern shows that they typically contain syncytial giant cells or frankly choriocarcinomatous elements. In this respect they are like germ-cell tumours associated with excess HCG production. The precursor of the HCG-containing cells in 'somatic' tumours is unknown but their functional and morphological similarity to the trophoblast revives the old concept of pathophysiological correspondence between some malignant tumours and invasive trophoblast.     

Effect of equine chorionic gonadotropin on weaning-to-first service interval and litter size of female swine

We evaluated the effect of PMSG on the weaning-to-first service interval, total litter size and born alive litter size in swine. Four doses of PMSG (0, 500, 750 and 1,000 IU) were administered intramuscularly after weaning to sows at 3 different farms, grouped by parities (1, 2 and 3 or higher) and 2 distinct time periods. The associations among main effects and response variables were assessed by analysis of variance. Polynomial orthogonal terms were used to adjust the estimates of weaning-to-first service interval, total litter size and born alive litter size for the interaction effect of parity and PMSG treatment. The weaning-to-first service interval did not differ across periods and farms (P>0.05), although the interval was shorter (P<0.05) for Parity 3+ sows (4.97 d) than for Parity 1 sows (5.29 d), with no other differences in intervals observed across parities (P>0.05). Time period did not influence litter size (P>0.05), but there were differences in litter size across farms (P<0.05). Both litter size traits were lower for Parity 1 sows than for higher parity sows (P<0.05), but there were no differences in litter size between Parity 2 and 3+ sows (P>0.05). Litter size increased with PMSG dose in both Parities 1 and 2 (P<0.05), but not in Parity 3+ (P>0.05). A significant quadratic effect (P<0.05) of PMSG treatment in weaning-to-first service interval was observed for both Parity 1 and 2 sows, with the shortest intervals occurring with the 750 IU dose for Parity 1 sows. Administration of PMSG after weaning was associated with a shortened weaning-to-first service interval in Parity 1 sows and increased litter size in Parity 1 and 2 sows.     

Magnetic relaxation switch detection of human chorionic gonadotrophin

Functionalized nanoparticle contrast agents, also known as magnetic relaxation switches (MRS), were prepared to detect protein A and the beta subunit of human chorionic gonadotrophin (hCG-beta). Antibodies were attached to cross-linked iron oxide (CLIO) nanoparticles using standard peptide chemistry. Protein A was used as a simple model analyte, as it is naturally multivalent and can bind multiple CLIO-IgG simultaneously. The addition of PA to CLIO-IgG resulted in transverse relaxation time (T2) shortening compared to a blank control as seen by NMR relaxometry measurements. Analyte-induced aggregation was confirmed by light scattering particle size analysis. A two-particle system was designed to measure hCG-beta, as it is not multivalent and requires conjugation of a matched pair of monoclonal antibodies to CLIO (referred to as C95 and C97). Measurement of hCG-beta is important, as elevated serum levels are associated with malignancies including testicular and ovarian cancers. The addition of hCG-beta to C95 and C97 resulted in T2 shortening with a linear dynamic concentration range of 0.1 to 1 molecules of analyte per nanoparticle. Similar data were obtained for the hCG dimer. Observations with higher stoichiometric ratios of analyte to nanoparticle and increased nanoparticle valency were also made. This method can potentially be adapted to detect other biomarkers in solution.     

Variations in the properties of equine chorionic gonadotropin

The objectives of this paper are to review the chemical and biological properties of equine chorionic gonadotropin (eCG, PMSG) isolated from the serum. Comparisons are made with eCG isolated from endometrial cups, trophoblast cell culture medium, and low titer serum. The results show that eCG can vary, depending on the source, in both chemical and biological (LH and FSH activity) properties.     

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:     

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