An immunological approach to study gonadotropin action at the cellular and molecular level

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Two simple and rapid methods to detect monoclonal antibodies with identical epitope specificities in a large population of monoclonal antibodies

Two novel and simple methods have been developed for detecting monoclonal antibodies (MAbs) with identical epitope specificities from a large population of MAbs against human chorionic gonadotropin (hCG). The first method was based on the observation that following radioiodination many molecules of an antigen are altered in such a manner that they cannot be recognized by MAbs. The proportion of radiolabelled antigen (Bmax) able to bind to a MAb was characteristic of that MAb and MAbs having identical specificities showed identical Bmax values. Using this principle it was possible to identify MAbs having identical epitope specificities within a large population of MAbs against hCG. In the second method one test MAb was immobilized on a plastic surface through an immunochemical bridge. This MAb was then incubated with 125I-hCG previously complexed with a second MAb. Such a complex could bind to the solid phase MAb only if the two MAbs were not identical. The results obtained with both methods were concordant. With such methods it is possible to identify MAbs with identical epitope specificities immediately after the initial screening of the fusion. These methods do not require subcloning, ascites production, or the purification and iodination of large number of MAbs

Effect of estrogen deprivation on the reproductive physiology of male and female primates

The availability of CGS 16949A, CGS 20267 and CGP 47645, a series of aromatase inhibitors (AIs) having high specific activity and specificity, made possible this study wherein the need for estrogen (E) for regulating (a) follicular maturation/ovulation, luteal function and pregnancy establishment, and (b) testicular function of the bonnet monkey (Macaca radiata) has been examined. Generally these compounds, used in the range of 500 μg to 2.5 mg/day did not inhibit follicular maturation although they did reduce E levels. Although low doses had no effect on ovulation it appears that relatively high doses of CGS 20267 and CGP 47645 could be inhibiting it. Three oral doses of letrozole (CGS 20267, each dose of 2 mg) during the follicular phase resulted in the formation of multiple follicles in cycling females, and these could be ovulated by exogenous hCG (1000 IU) treatment. Although administration of AI during the early luteal phase had no effect on progesterone (P) production, it prevented pregnancy establishment. Whereas AI administration in the female had no significant effect on luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels (except at high drug dosages), it significantly increased serum testosterone (T) levels in the male. Sustained high levels of T (30-50 ng/ml) could be maintained for 100 days by administering 2.5 mg of CGP 47465 orally once in 5 days. Blockade of E synthesis in the male led to the disruption of testicular germ cell transformation, which in turn resulted in a significant reduction in sperm production. These studies with aromatase inhibitors in the monkey suggest that these compounds have a potential for use as fertility regulating agents in both the male and female primate

Conformation of the α-subunit of glycoprotein hormones: a study using polyclonal and monoclonal antibodies

The conformation of the common α-subunit of human glycoprotein hormones, luteinizing hormone (hLH), follicle-stimulating hormone (hFSH), thyroid-stimulating hormone (hTSH) and chorionic gonadotropin (hCG) was probed using a highly specific polyclonal antiserum against the α-subunit of hCG and several monoclonal antibodies (MAbs) produced against hCG which recognized the α-subunit in free and combined form. The α-subunit was found to be conformationally altered (compared to its conformation in the isolated state) when it was in combination with various β-subunits as indicated by shifts in the displacement curves of binding of [125I]hCG α to the polyclonal antiserum. The extent of the change was dependent on the β-subunit present with minimum change being observed with hLHβ, intermediate with hCGβ and maximum change with hFSH and TSH β-subunits. However, the affinity constants of this antiserum for all four hormones were nearly similar. Further, it was also found that binding of any one of the glycoprotein hormones to this antibody could be completely inhibited by any other hormone suggesting that the conformation of the α-subunit in all the four hormones is probably very similar. This was further investigated using five hCG MAbs capable of recognizing the α-subunit, but with different epitope specificities. All these MAbs could recognize all the four hormones suggesting the presence of the epitopes in these proteins. These epitopes were conformation specific since the MAbs did not bind reduced and carboxymethylated α-subunit. Displacement analysis using [125I]hCG as the tracer showed that two epitopes have nearly the same conformation in all the four hormones, while two were partially modified depending on the β-subunit present. Based on these results, it is concluded that the α-subunit of glycoprotein hormones has nearly the same conformation, though subtle differences do exist

Conformation of the α\alpha-subunit of glycoprotein hormones: a study using polyclonal and monoclonal antibodies

The conformation of the common $\alpha$-subunit of human glycoprotein hormones, luteinizing hormone (hLH), follicle-stimulating hormone (hFSH), thyroid-stimulating hormone (hTSH) and chorionic gonadotropin (hCG) was probed using a highly specific polyclonal antiserum against the $\alpha$-subunit of hCG and several monoclonal antibodies (MAbs) produced against hCG which recognized the $\alpha$-subunit in free and combined form. The $\alpha$-subunit was found to be conformationally altered (compared to its conformation in the isolated state) when it was in combination with various $\beta$-subunits as indicated by shifts in the displacement curves of binding of $[^{125}I]$hCG $\alpha$ to the polyclonal antiserum. The extent of the change was dependent on the $\beta$-subunit present with minimum change being observed with hLH$\beta$, intermediate with hCG$\beta$ and maximum change with hFSH and TSH $\beta$-subunits. However, the affinity constants of this antiserum for all four hormones were nearly similar. Further, it was also found that binding of any one of the glycoprotein hormones to this antibody could be completely inhibited by any other hormone suggesting that the conformation of the $\alpha$-subunit in all the four hormones is probably very similar. This was further investigated using five hCG MAbs capable of recognizing the $\alpha$-subunit, but with different epitope specificities. All these MAbs could recognize all the four hormones suggesting the presence of the epitopes in these proteins. These epitopes were conformation specific since the MAbs did not bind reduced and carboxymethylated $\alpha$-subunit. Displacement analysis using $[^{125}I]$hCG as the tracer showed that two epitopes have nearly the same conformation in all the four hormones, while two were partially modified depending on the $\beta$-subunit present. Based on these results, it is concluded that the $\alpha$-subunit of glycoprotein hormones has nearly the same conformation, though subtle differences do exist

Alterations in Sperm Characteristics of Follicle-Stimulating Hormone (FSH)-Immunized Men Are Similar to Those of FSH-Deprived Infertile Male Bonnet Monkeys

The quality of sperm ejaculated by bonnet monkeys and normal, healthy proven fertile volunteer men, both actively immunized with ovine follicle-stimulating hormone (oFSH), was examined at different times of study for chromatin packaging and acrosomal glycoprotein concentration by flow cytometry. Susceptibility of sperm nuclear DNA to dithiothreitol (DTT)-induced decondensation, as measured by ethidium bromide binding, was markedly high compared with values at day 0 in men and monkeys during periods when FSH antibody titer was high. Sperm chromatin structure assay yields $\alpha t$ values, which is another index of chromatin packaging. Higher $\alpha t$ values, signifying poor packaging, occurred in both species following immunization with heterologous pituitary FSH. The binding of fluorosceinated pisum sativum agglutinin (PSA-FITC) to acrosome of sperm of monkeys and men was significantly low, compared with values at day 0 (control) during periods when cross-reactive FSH antibody titer was high and endogenous FSH was not detectable. Blockade of FSH function in monkeys by active immunization with a recombinant oFSH receptor protein corresponding to a naturally occurring messenger RNA (mRNA) also resulted in production of sperm with similar defects in chromatin packaging and reduced acrosomal glycoprotein concentration. Thus, it appears that in monkeys and men, lack of FSH signaling results in production of sperm that exhibit defective chromatin packaging and reduction in acrosomal glycoprotein content. These characteristics are similar to that exhibited by sperm of some class of infertile men. Interestingly, these alterations in sperm quality occur well ahead of decreased sperm counts in the ejaculate