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

Efficient \u3cem\u3eIn Vitro\u3c/em\u3e Regeneration System From Seed Derived Callus of Perennial Ryegrass (\u3cem\u3eLolium Perenne\u3c/em\u3e) And Annual Ryegrass (\u3cem\u3eLolium Multiflorum\u3c/em\u3e)

The commercially important ryegrasses in cool temperate climates throughout the world are annual ryegrass (Lolium multiflorum L.) and perennial ryegrass (Lolium perenne L). Improvements through conventional breeding have been slow as they are usually heterozygous and highly self-infertile. Hence, there is a need to use modern biotechnological tools to the development of improved rye grass cultivars for incorporating value added traits. Successful transformation of rye grasses has been done using suspension cells, which is time consuming and laborious (Spangenberg et al., 1995, 1998). We report here a rapid and highly efficient in vitro plant regeneration system from seed derived callus in annual and perennial rye grasses

A Novel Genotype Independent Protocol for In Vitro Plant Regeneration from Mature Seed Derived Callus of Tall Fescue (\u3ci\u3eFestuca Arundinacea\u3c/i\u3e Schreb.)

Tall fescues (Festuca arundinacea Schreb.) are cool season forage and turf grasses of significant agricultural importance in different grassland countries. Genetic improvement of tall fescues by conventional selection procedures is slow, since these are predominantly, cross-pollinated, hexaploid and generally infertile (Jauhar, 1993). Genetic Engineering approaches for incorporation of agronomically useful traits may contribute to the development of improved tall fescue cultivars (Spangenberg et al., 1998). However for any genetic engineering studies, it is essential to develop a genotype-independent, reproducible and efficient in vitro plant regeneration protocol. In the present study, we analyzed the effects of different sterilization procedures for in vitro seed germination and studied the effects of different concentrations and combinations of 2,4-D and BAP on callus induction, growth and regeneration potential of two cultivars of tall fescue

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

DETERMINATION OF OCTANOL-WATER PARTITION COEFFICIENT OF NOVEL COUMARIN BASED ANTICANCER COMPOUNDS BY REVERSED-PHASE ULTRA-FAST LIQUID CHROMATOGRAPHY

Objective: The present study aims at the development of a reversed phase ultra-fast liquid chromatography (RP-UFLC) method for measurement of the lipophilicity (log P) between n-octanol and water for the newly synthesized coumarin derivatives in our laboratory.Methods: The synthesized compounds were dissolved in methanol and analyzed using XTerra RP18 column as the stationary phase and a mixture of methanol (0.25% v/v octanol) and buffer as the mobile phase with isocratic elution.Results: In this study we concentrated on the relationship between a reversed-phase ultra-fast liquid chromatography (RP-UFLC) retention parameters and log P of our synthesized compounds. Furthermore, a good correlation and very close values were obtained between the experimentally determined log P values and values obtained from Chemdraw.Conclusion: The developed method was found to be insensitive to any of the impurities present and moreover it requires very little sample for analysis