Relative ability of ovine follicle stimulating hormone and its β-subunit to generate antibodies having bioneutralization potential in nonhuman primates

The relative ability of ovine follicle stimulating hormone and its β-subunit, two potential candidates for male contraceptive vaccine, to generate antibodies in monkeys capable of bioneutralizing follicle stimulating hormone was assessed usingin vitro model systems. Antiserum against native ovine follicle stimulating hormone was found to be highly specific to the intact form with no cross-reactivity with either of the two subunits while the antiserum against β-subunit of follicle stimulating hormone could bind to the β-subunit in its free form as well as when it is combined with α-subunit to form the intact hormone. Both antisera could block the binding of the hormone to the receptor if the hormone was preincubated with the antibody. However, the follicle stimulating hormone β-antisera could only inhibit the binding of the hormone partially (33% inhibition) if the antibody and receptor were mixed prior to the addition of the hormone, while antisera to the native follicle stimulating hormone could block the binding completely (100% inhibition) in the same experiment. Similarly antisera to the native follicle stimulating hormone was significantly effective in blocking (100%) response to follicle stimulating hormone but not the β-subunit antisera (0%) as checked using anin vitro granulosa cell system. Thus the probability of obtaining antibodies of greater bioneutralization potential is much higher if intact hormone is used as an antigen rather than its β-subunit as a vaccine

Involvement of luteinizing hormone in the implantation process of the rat

The use of specific anti-FSH and anti-LH substances has shown that LH is the only pituitary gonadotrophin involved in the implantation process. Using different dosages of LH antiserum at different time intervals, it has been possible to arrive at a minimum effective dose (0.05 ml) which, when given on the 4th day at 10.00 hours, results in inhibition of implantation on the 8th day. We have shown that, at this dose, the antiserum is mainly inhibiting the oestrogen surge. It is proposed that an LH surge precedes an oestrogen surge on Day 4 of pregnancy

Evaluation of relative roles of LH and FSH in regulation of differentiation of Leydig cells using an ethane 1,2-dimethylsulfonate-treated adult rat model

The relative role of LH and FSH in regulation of differentiation of Leydig cells was assessed using an ethane 1,2-dimethylsulfonate (EDS)-treated rat model in which endogenous LH or FSH was neutralized from day 3 to day 22 following EDS treatment. Serum testosterone and the in vitro response of the purified Leydig cells to human chorionic gonadotropin (hCG) was monitored. In addition RNA was isolated from the Leydig cells to monitor the steady-state mRNA levels by RT-PCR for 17α-hydroxylase, side chain cleavage enzyme, steroidogenic acute regulatory protein (StAR), LH receptor, estrogen receptor (ER-α) and cyclophilin (internal control). Serum testosterone was undetected and the isolated Leydig cells secreted negligible amount of testosterone on stimulation with hCG in the group of rats that were treated with LH antiserum following EDS treatment. RT-PCR analysis revealed the absence of message for cholesterol side chain cleavage enzyme and 17α-hydroxylase although ER-alpha and LH receptor mRNA could be detected, indicating the presence of undifferentiated precursor Leydig cells. In contrast, the effects following deprival of endogenous FSH were not as drastic as seen following LH neutralization. Deprival of endogenous FSH in EDS-treated rats led to a significant decrease in serum testosterone and in vitro response to hCG by the Leydig cells. Also, there was a significant decrease in the steady-state mRNA levels of 17α-hydroxylase, cholesterol side chain cleavage enzyme, LH receptor and StAR as assessed by a semiquantitative RT-PCR. These results establish that while LH is obligatory for the functional differentiation of Leydig cells, repopulation of precursor Leydig cells is independent of LH, and also unequivocally establish an important role for FSH in regulation of Leydig cell function

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

VALIDATION OF STABILITY INDICATING ULTRA-FAST LIQUID CHROMATOGRAPHY METHOD FOR SIMULTANEOUS ESTIMATION OF ATENOLOL and NIFEDIPINE IN BOTH BULK AND PHARMACEUTICAL DOSAGE FORMS

Objective: The study depicts improvement of ensuing validation of a stability indicating technique for the simultaneous estimation of Atenolol and Nifedipine using Ultra-fast liquid chromatographic method (UFLC).Methods: The analysis is performed on Phenomenex Kinetex C18, (150 × 4.6 mm, 5μm) column using methanol and 0.1%ortho-phosphoric acids (75:25 v/v) as mobile phase with a flow rate of 1.3 ml/min. The eluents were checked with PDA detector at 237 nm.Results: In this optimized conditions Atenolol and Nifedipine elutes at a retention time of 2.79 and 4.50 min respectively individually the considered optimized condition is having linearity in the range from 10 to 50µg/ml of Atenolol and 4-20µg/ml of Nifedipine. The method was validated by following the ICH guidelines and their combination drug yield was exposed to acid and base stress, thermal stress, photolytic stress, hydrolytic stress, and oxidative stress conditions. All samples were studied by the given optimized method. In this Calibration curves were linear over studies ranges with correlation coefficient found between the ranges of 0.99 to 1.00.Conclusion: The proposed method was found to be accurate, precise, and specific and suitable for determination of both the drugs

Immunobiology of a synthetic luteinizing hormone receptor peptide 21-41

Immunization of adult male rabbits with a synthetic luteinizing hormone-receptor peptide (LH-RP; representing amino-acids 21-41 of the extracellular domain of the rat LH receptor) resulted in production of high-titer antibodies capable of interacting with particulate and cell-based LH receptors. The antibody produced was able to inhibit binding of 125I-labeled human chorionic gonadotropin (hCG) to a particulate sheep luteal LH receptor preparation by 40%-50%. Maximal inhibitory activity was correlated with high antibody titer. Immunocytometry revealed that the antibody could directly bind to cells having LH receptors, such as rat granulosa and Leydig cells. The antibodies recognized a 77-kilodalton membrane protein in Western blots of mouse testicular extracts. Interaction of endogenous Leydig cell LH receptor with the LH-RP antibody resulted in both hormone agonist and antagonistic activities. The hormone-mimicking activity (increase in serum testosterone over control) was confined only to the early phase of immunization when the antibody titer was low. Blockade of LH receptor during the later part of immunization resulted in a significant reduction in serum testosterone over controls and inhibition of spermatogenesis. DNA flow cytometry showed that a specific and significant inhibition of meiosis (transformation of primary spermatocytes to round and elongated spermatids P < .01) and spermiogenesis (transformation of round spermatids to elongated spermatids P < .0001) occurred following blockade of LH function

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 alphat values, which is another index of chromatin packaging. Higher alphat 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

Characteristics and Source Apportionment of Black Carbon (BC) in a Suburban Area of Klang Valley, Malaysia

Black carbon (BC) is of concern due to its contribution to poor air quality and its adverse effects human health. We carried out the first real-time monitoring of BC in Malaysia using an AE33 Aethalometer. Measurements were conducted between 1 January and 31 May 2020 in a university area in a suburban location of the Klang Valley. The measurement period coincided with the implementation of a movement control order (MCO) in response to COVID-19. The mean concentration of BC before the MCO was 2.34 µg/m3 which decreased by 38% to 1.45 µg/m3 during the MCO. The BC is dominated by fossil-fuel sources (mean proportion BCff = 79%). During the MCO, the BCff concentration decreased by more than the BCbb concentration derived from biomass burning. BC and BCff show very strong diurnal cycles, which also show some weekday–weekend differences, with maxima during the night and just before noon, and minima in the afternoon. These patterns indicate strong influences on concentrations from both traffic emissions and boundary layer depth. BC was strongly correlated with NO2 (R = 0.71), another marker of traffic emission, but less strongly with PM2.5 (R = 0.52). The BC absorption Ångström exponent (AAE) ranged between 1.1 and 1.6. We observed pronounced diurnal cycles of lower AAE in daytime, corresponding to BCff contributions from traffic. Average AAE also showed a pronounced increase during the MCO. Our data provides a new reference for BC in suburban Malaysia for the public and policy-makers, and a baseline for future measurements