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M. M. Singh and W. D. Booth

Summary. Day-6 rabbit blastocysts were incubated with 3H-labelled neutral steroids. Pregnenolone metabolism was insignificant, but progesterone was extensively metabolized to 5β-pregnanedione (5β-pregnane-3,20-dione) and 5β-pregnanolone (5β-pregnan-3β-ol-20-one). Some dehydroepiandrosterone (DHA) was converted to 5-androstene-3β,17β-diol; androstenedione and testosterone were interconverted, and both 4-ene steroids were metabolized to 5α-reduced compounds. There was no evidence for oestrogen synthesis from progesterone or androgens. Exogenous cofactors had no significant effect on the course of steroid metabolism. Oestradiol and oestrone were determined in blastocysts, and high levels of oestrogen were found in blastocysts from superovulating rabbits. Injection of [3H]oestradiol into pregnant rabbits gave rise to radioactivity associated primarily with oestradiol in uterine tissue, uterine flushings and blastocysts.

It is concluded (1) that the capacity of the Day-6 rabbit blastocyst to metabolize progesterone and androgen may be utilized in vivo to protect the blastocyst from excess maternal progesterone, and to involve androgens in an anabolic role, and (2) that blastocyst oestrogen may be partly of maternal origin.

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M. M. Singh and C. E. Adam

Summary. Progesterone concentrations in peripheral plasma of 27 rabbits were determined by radioimmunoassay daily from Days 1 to 7. The rate of increase was significantly higher in pregnant than in pseudopregnant rabbits from Days 5 to 7 (P < 0·02), but not before Day 5. Transfer of Day-4 or -5 blastocysts to synchronous, pseudopregnant recipients resulted in a significant rise in progesterone levels in comparison with those in sham-operated controls (P < 0·01) or pseudopregnant does (P < 0·01).

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M. M. Singh, V. Wadhwa, N. Sethi and V. P. Kamboj

Summary. 'Tube-locked' morulae and blastocysts were recovered from the ampulla of the oviduct of centchroman-treated mice between Days 4 and 12 post coitum and transferred to the uteri of pseudopregnant female mice. Pregnancy and implantation rates were lower and the post-implantation resorption rate was higher in the treated than in the control group. There was little difference in the pregnancy or implantation rates between embryos recovered on Days 4 or 12 post coitum, but the resorption rate increased with increasing duration of embryos in the oviducts and was 100% for the Day-12 embryos. The resorption rate was similar even when these embryos were transferred to the sterile uterine horn of unilaterally pregnant mice. Centchroman did not produce any deleterious effect on embryos which survived until Day 19 of pregnancy in foster mothers. The average fetal weight was also comparable to those of control fetuses.

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M. M. Singh, S. Sreenivasulu and V. P. Kamboj

The duration of the anti-implantation action of a single oral post-coital dose (1.25 mg kg−1) of a triphenylethylene anti-oestrogen, centchroman, was determined in adult rats. The effects of centchroman were compared with those of tamoxifen. In rats undergoing delay, centchroman administered orally on day 7 post-coitum prevented the induction of implantation of delayed blastocysts by an implantation inducing dose (1 μg per rat, s.c.) of oestrone which was administered earlier than 120 h after centchroman treatment. In tamoxifen (0.2 mg kg−1, orally) pretreated rats, oestrone administered at 144 h or later induced implantation. In cyclic rats treated with centchroman at intervals of 168 h and mated with males of proven fertility, implantation was prevented only when the interval between centchroman treatment and nidatory oestrogen secretion was less than 120 h. None of the females conceived when treated regularly at intervals of 120 h during exposure to fertile males. Discontinuation of treatment resulted in the occurrence of normal implantations in rats that mated 48 h or later after the last dose of centchroman, since in these animals the interval between anti-oestrogen treatment and nidatory oestrogen secretion was greater than 120 h. These findings suggest that the duration of the anti-implantation action of a single oral antifertility dose of centchroman in rats is about 120 h. Recovery of normal blastocysts from rats treated continuously with this dose of centchroman at these intervals suggests lack of significant effect on follicular maturation, ovulation, fertilization, preimplantation development or mating behaviour. Tamoxifen appears to be slightly longer acting and the duration of its action was about 144 h.

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M. M. Singh, V. Bhalla, V. Wadhwa and V. P. Kamboj

Summary. A single oral administration of centchroman (1·25 mg/kg) to adult female rats within 24 h of mating induced slight acceleration in the rate of transport of embryos through the oviducts. The compound did not seem to produce any deleterious effect on preimplantation embryonic development since well organized and apparently normal embryos were collected from the genital tract up to Day 12 of pregnancy. The recovery rate of embryos from centchroman-treated rats was, however, significantly reduced after Day 4 of pregnancy. There was some stimulation in the rate of cleavage of embryos and morula to blastocyst transformation, but retardation in the shedding of the zona pellucida. The rate of blastocyst formation was not altered when 6–8-cell embryos collected from the oviducts of control rats were transferred to the uteri of control or centchroman-treated females. A delay in zona shedding was observed in the centchroman-treated recipients.

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Kusum Vermat, M. M. Misro, H. Singh, Sudha Mahajan, A. R. Ray and S. K. Guha

Summary. An alternate co-polymer of styrene and maleic anhydride was dissolved in dimethylsulphoxide and injected into the vas deferens of rats. The polymer was retained in the vas deferens and the morphological changes detected were confined to the mucosa. When the polymer was removed by flushing dimethylsulphoxide, the mucosal structure became normal within 2 weeks.

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S. M. Totey, G. Singh, M. Taneja, C. H. Pawshe and G. P. Talwar

Summary. Cumulus–oocyte complexes, 5596, were cultured for 24 h in either TCM-199 or Ham's F-10 with or without gonadotrophins and supplemented with either 20% buffalo oestrous serum (BES) or fetal calf serum (FCS). The maturation rates of oocytes cultured in TCM-199 or Ham's F-10 medium supplemented with 20% BES were 47·4 ± 17·8 and 44·8 ± 25·6, respectively. Addition of luteinizing hormone (LH) (5 μg ml−1) significantly improved the maturation rate in the Ham's F-10 medium supplemented with 20% BES (76·8 ± 18·3), but follicle-stimulating hormone (FSH) (0·5 μg ml−1) and oestradiol (1 μg ml−1) failed to synergize with LH (71·7 ± 19·5). In the TCM-199 system, LH failed to enhance the maturation rate but the addition of FSH and oestradiol significantly enhanced the proportion of mature oocytes (42·7 ± 1·4 and 81·7 ± 14·5, respectively; P < 0·05). Frozen–thawed spermatozoa prepared in Bracket and Oliphant (BO) medium and treated with 5 mmol caffeine l−1 ± 10 μg heparin showed a higher fertilization rate (29·8%) than those treated in Hepes–Talp and treated with 10 μg heparin ml−1 (19·6%). Fertilization rate was significantly improved when fresh ejaculated spermatozoa treated with 5 mmol caffeine l−1 and 10 pg heparin in BO medium (50%) was used. Rate of cleavage and development were also higher when in vitro fertilization was carried out with fresh ejaculated spermatozoa treated with caffeine and heparin (34·1 and 36·8%, respectively) than with frozen–thawed spermatozoa (27·0 and 22·0%, respectively).

Development rate was enhanced when fertilized ova were cultured in ligated rabbit oviduct (28·0%) than when co-cultured on oviductal cell monolayers (8·2%). The results indicate that oocytes cultured in medium supplemented with BES and gonadotrophins reveal high rates of maturation and development to the blastocyst stage after fertilization with fresh ejaculated spermatozoa.

Keywords: oocytes; maturation; fertilization; culture; buffalo

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F C F Dias, M I R Khan, M A Sirard, G P Adams and J Singh

Microarray analysis was used to compare the gene expression of granulosa cells from dominant follicles with that of those after superstimulatory treatment. Cows were allocated randomly to two groups (superstimulation and control, n=6/group). A new follicular wave was induced by ablation of follicles ≥5 mm in diameter, and a progesterone-releasing device controlled internal drug release (CIDR) was placed in the vagina. The superstimulation group was given eight doses of 25 mg FSH at 12-h intervals starting from the day of wave emergence (day 0), whereas the control group was not given FSH treatment. Both groups were given prostaglandin F2 α twice, 12 h apart, on day 3 and the CIDR was removed at the second injection; 25 mg porcine luteinizing hormone (pLH) was given 24 h after CIDR removal, and cows were ovariectomized 24 h later. Granulosa cells were collected for RNA extraction, amplification, and microarray hybridization. A total of 190 genes were downregulated and 280 genes were upregulated. To validate the microarray results, five genes were selected for real-time PCR (NTS, FOS, THBS1, FN1, and IGF2). Expression of four genes increased significantly in the three different animals tested (NTS, FOS, THBS1, and FN1). The upregulated genes are related to matrix remodeling (i.e. tissue proliferation), disturbance of angiogenesis, apoptosis, and oxidative stress response. We conclude that superstimulation treatment i) results in granulosa cells that lag behind in maturation and differentiation (most of the upregulated genes are markers of the follicular growth stage), ii) activates genes involved with the NFE2L2 oxidative stress response and endoplasmic reticulum stress response, and iii) disturbs angiogenesis.

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G. Singh, M. M. Singh, S. C. Maitra, W. Elger, V. Kalra, S. N. Upadhyay, S. R. Chowdhury and V. P. Kamboj

Summary. RU-38486 or ZK-98734 treatment (3 mg/day, s.c.) to intact or hysterectomized adult female rats on Days 5–7 post coitum induced changes characteristic of luteolysis. Ultrastructurally, the luteal cells exhibited an extensive vacuolization of the cytoplasm and perinuclear areas, degeneration of mitochondrial cristae, massive accumulation of lipid droplets, increase in number of lysosome like granules and heterochromatinization of the nucleus. In general, RU-38486 induced more marked degeneration of the luteal cells than did ZK-98734. There was also a significant decrease in peripheral plasma progesterone concentrations in treated rats. We suggest that these antiprogestagens act via inhibition of luteal function in addition to their antagonism at the uterine progesterone receptor level.

Keywords: antiprogestagens; ultrastructure; corpora lutea; progesterone; rat

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S G Hannesdóttir, X Han, T Lund, M Singh, R van der Zee, I M Roitt and P J Delves

Immunosterilization is an attractive alternative to surgical castration. Gonadotropin-releasing hormone (GnRH) controls the production of the gonadotropins thereby having an orchestrating effect on the reproductive hormone cascade and spermatogenesis. Induction of neutralizing antibody can abrogate the effect of the hormone. Current GnRH-based vaccines often require strong adjuvants and/or multiple injections of the vaccines to overcome variability in the response. Heat shock proteins (hsp) have been used as carrier molecules because of their powerful intrinsic ability to enhance an immune response to associated antigens. A GnRH-analogue, GnRH-d6-Lys, was conjugated to recombinant Mycobacterium tuberculosis hsp70. Male BALB/c mice were immunized i.p. with GnRH-hsp70 in the mild adjuvant Ribi or in incomplete Freund’s adjuvant (IFA). The initial immunizations were done on pre-pubertal 3-week-old mice, with boosts at 5 and 8 weeks of age. The mice were killed at 10 weeks of age and GnRH-specific antibodies and serum testosterone levels measured. All the immunized mice mounted GnRH-specific antibody responses, with no difference in the mice immunized with GnRH-hsp70/Ribi or with GnRH-hsp70/IFA. There was substantial atrophy of the urogenital complex and significantly (P < 0.0005) reduced levels of testosterone-dependent testicular relaxin-like factor mRNA expression. Mice immunized with GnRH-hsp70/Ribi showed substantially reduced (P < 0.001) serum testosterone levels. These results indicate that hsp70 may serve as a particularly advantageous carrier for GnRH-based vaccines.