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M. P. McLean and J. B. Miller

Summary. Rabbits were given 50 i.u. hCG, i.v., to initiate ovulation and pseudopregnancy (Day 0) and were treated, s.c., with or without a 1-cm Silastic oestradiol implant. Serum progesterone concentrations were measured at 4-day intervals and 3-hydroxy-3-methylglutaryl–coenzyme A (HMG–CoA) reductase activity was estimated by the conversion of HMG to mevalonate in microsomes from corpora lutea removed on Days 4, 8, 12, 16 and 20 of pseudopregnancy (4 rabbits/day). Total HMG–CoA reductase activity was significantly (P < 0·05) higher in control rabbits on Days 8 and 12 (5·29 ± 0·63 and 5·5 ± 0·28 nmol/min/mg protein, respectively) compared to oestradioltreated rabbits (2·57 ± 0·25 and 4·03 ± 0·23 nmol/min/mg protein, respectively). On Days 16 and 20, total HMG–CoA reductase activity was not different in control and oestradiol-treated animals. There was no difference in the levels of the active fraction of HMG–CoA reductase, which represented <20% of the total enzyme activity, in control and oestradiol-treated rabbits (<780 pmol/min/mg protein, Day 12). These results indicate that oestradiol does not alter the active form, but can reduce the total activity of HMG–CoA reductase in the rabbit corpus luteum without a decline in serum progesterone. Therefore, neither total nor active forms of HMG–CoA reductase are directly related to progesterone secretion. This suggests that other sources of cholesterol may contribute to progesterone production in the rabbit.

Keywords: HMG–CoA reductase; corpus luteum; progesterone synthesis; oestradiol action; lipoproteins

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A. D. JOHNSON, M. B. SIGMAN and W. J. MILLER

Summary.

Mature male rats and domestic fowl were injected subcutaneously with 109CdCl2; some rats were pre-treated 24 or 2 hr previously with Zn or Se, respectively. Testis, muscle and liver samples were taken at 5, 10, 20 or 40 min post-injection. Tissues were homogenized and centrifuged to obtain nuclear, heavy and light mitochondrial, microsomal, and the resultant 103,000 g supernate fractions. This supernate was partitioned by acetone which resulted in non-protein and protein-bound Cd fractions. In the liver nuclear fraction, there was only 10% of the total 109Cd compared to 50 and 30% in the muscle and testis, respectively. These trends were reversed in the 103,000 g supernate. Pre-treatment with Zn or Se reduced the 109Cd in this fraction in the testis and muscle. In these organs, there was more 109Cd in the mitochondrial fractions of the domestic fowl than in the rat. Pre-treatments decreased 109Cd in the testicular microsomes and in the protein of the 103,000 g supernate. After 5 min, there was essentially no free element in the testis of the domestic fowl. If subcellular location is indicative, the domestic fowl and the Zn and Se pre-treated rats appear to have very different mechanisms of defence against Cd.

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C. W. EMMENS, D. J. COLLINS, J. J. HOBBS, B. G. MILLER and W. H. OWEN

Summary.

A number of 4,4′-dihydroxybibenzyls has been prepared, with resolution of some isomers. Most are pro-oestrogens and all are anti-oestrogens of varying potency. Dextro- and laevo-α,α′-dimethyl-4,4′-dihydroxybibenzyl (d- and l-dma) have the same anti-oestrogenic and antifertility potencies in mice, but threo- and erythro-α-ethyl-α′-methyl-4,4′-dihydroxybibenzyl (threo- and erythro-mea) differ markedly in all properties examined. Erythro-mea, although a fairly potent pro-oestrogen, is highly anti-oestrogenic intravaginally and a potent antifertility agent with an med of less than 1 μg/day. dl-Hexoestrol (dl-dea) is also a potent anti-oestrogen and fairly potent antifertility agent, with an med about 10 μg/day.

This series of compounds contrasts with the stilbene-4,4′-diols in possessing members with exceptional antifertility potency, not readily explained by their oestrogenic activities.

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M. Winter, J. Pirmann, R. E. Falvo, B. D. Schanbacher and J. Miller

Summary. The effects of s.c. administration of oil, testosterone, 5α-dihydrotestosterone, 5α-androstane-3α,17β-diol and oestradiol-17β on plasma concentrations of LH and FSH were determined in 5 orchidectomized dogs. The dosages for the androgens and oestradiol-17β were 500 and 50 μg/kg body weight, respectively. Testosterone and oestradiol-17β significantly reduced plasma gonadotrophin concentrations, although the onset and duration of their suppressive effects differed. Dihydrotestosterone and oil had no effect on either gonadotrophin. Administration of androstanediol had no effect on plasma concentrations of LH but did cause a temporary and significant reduction in FSH. It is concluded that testosterone and oestradiol-17β are major regulators of gonadotrophin secretion in the male dog, but the 5α-reduction of testosterone seems to play only a minor role in this control.

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S. Damle, A. R. LaBarbera, J. B. Miller and M. Hunzicker-Dunn

Summary. Quartered CL from 7-day pseudopregnant rabbits were incubated at 37°c for 0–180 min in the presence of BSA, LH or adrenaline in Krebs–Ringer–bicarbonate buffer. Total progesterone at each time point was quantified in homogenates of tissue plus incubation media and expressed relative to CL protein. Progesterone increased linearly with time during the first 30 min of incubation in the presence of BSA. LH and adrenaline markedly accelerated progesterone accumulations relative to the BSA control. At 10 min, progesterone accumulation in the presence of LH and adrenaline were 2·4 and 5·9 times that in the absence of stimulators, respectively. Both hormones caused concentration-dependent increases in progesterone and the apparent ED50 was 0·75 μg/ml for LH and adrenaline. The CL obtained from ovaries of 7-day pseudopregnant rabbits are therefore capable of an acute steroidogenic synthetic response to LH as well as adrenaline.

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S. P. Brinsko, B. A. Ball, P. G. Miller, P. G. A. Thomas and J. E. Ellington

This study was designed to investigate the development of day 2 embryos obtained from young and aged mares, co-cultured with oviductal epithelial cells obtained from mares in each age group in a 2 × 2 crossover design. Young, fertile mares (n = 19; 2–7 years of age) and aged, subfertile, mares (n = 16; 17–24 years of age) were used as embryo and oviductal epithelial cell donors. Embryos (n = 37) were collected from the oviducts 2 days after ovulation and were paired (embryos obtained from young mares with embryos obtained from aged mares) so that eight pairs were co-cultured with young mare oviductal epithelial cells and eight pairs were co-cultured with aged mare oviductal epithelial cells. Five additional embryos obtained from young mares were co-cultured with oviductal epithelial cells from either young mares or aged mares but were not paired. Embryos were co-cultured for 7 days at 38.5°C in 5% CO2 or until morphological degeneration was detected. The proportions of paired embryos that reached the blastocyst stage were similar for embryos obtained from young mares and embryos obtained from aged mares after co-culture with oviductal epithelial cells from young mares (6 of 8 versus 5 of 8) or from aged mares (6 of 8 versus 5 of 8), respectively. Although the overall rate of development of embryos to blastocyst from both young mares and aged mares was similar, blastocysts developing from embryos obtained from aged mares were inferior to blastocysts obtained from young mares in terms of number of cell nuclei, quality score, and diameter at day 7. The results of this experiment indicate that the high rate of early embryonic loss in aged, subfertile mares may be due to inherent developmental defects in their embryos, but does not appear related to the ability of embryos from aged, subfertile mares to reach the blastocyst stage.

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S. M. Skinner, G. J. Killian, L. A. Miller and B. S. Dunbar

The effectiveness of zona pellucida antigens in immunizing white-tailed deer to reduce fertility was evaluated by analysing the constituent deer zona pellucida proteins and their immunogenicity. Does were immunized with porcine zona pellucida antigens. The antibodies were characterized using immunohistochemical and immunoblot analysis, in which zona pellucida proteins were separated by one-dimensional and two-dimensional PAGE. Deer anti-porcine zona pellucida antibodies were found to recognize all the major proteins of the porcine zona pellucida. These antibodies also recognized several proteins of deer zona pellucida, indicating that it is possible to break immune tolerance in the deer using such a protocol. The antibodies were also found to recognize peptides of 55 and 75 kDa that were produced by expressing cDNA clones containing antigens of major glycoproteins of rabbit zona pellucida. Furthermore, antibodies against rabbit zonae pellucidae recognized antigens in zonae of paraffin-embedded deer ovaries. Taken together, these experiments demonstrate the crossreactive nature of a number of zona pellucida epitopes found in deer and in several other species. They also illustrate the immunogenicity possible in such an immunization protocol, and provide valuable probes for the investigation of follicular development in this and other species.

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D. W. Miller, D. Blache, R. Boukhliq, J. D. Curlewis and G. B. Martin

Nutrition influences the reproductive axis via alteration of gonadotrophin secretion. However, a link between nutrition and the secretion of GnRH, which drives the axis, has yet to be established. The aim of the present study was to measure the change in the concentrations of metabolic substances in the cerebrospinal fluid of adult male sheep offered a diet designed to maintain constant gonadotrophin secretion (Group M; n = 6), or a diet known to increase gonadotrophin secretion (Group M + L; n = 6). On days 1, 3 and 10 of the dietary treatments, cerebrospinal fluid and jugular blood were sampled and analysed for metabolic fuels (glucose, amino acids and free fatty acids) and metabolic hormones (insulin, insulin-like growth factor I, GH, prolactin, cortisol and the thyroid hormones). On day 11 of the dietary treatment, LH pulse frequency and mean FSH concentrations in Group M + L had increased relative to Group M and to day 0. Plasma concentrations of prolactin and insulin on days 3 and 10, and glucose and insulin-like growth factor I on day 10, were higher in Group M + L than in Group M, but only cerebrospinal fluid concentrations of insulin, glucose and certain amino acids were affected by the dietary treatments on days 3 and 10. Cerebrospinal fluid, but not plasma, concentrations of aspartate, tyrosine, cystine, phenylalanine and arginine on day 3, and glutamine, γ-aminobutyric acid, threonine, alanine on days 3 and 10, were higher in Group M + L relative to Group M. On day 10, plasma and cerebrospinal fluid concentrations of arginine, phenylalanine, proline, tyrosine, methionine and phosphoserine, but only the plasma concentrations of linoleic acid, aspartate and serine, were higher in Group M + L than in Group M. Concentrations of triiodothyronine, thyroxine, and cortisol in plasma and cerebrospinal fluid were not affected. These results show that the nutritional stimulation of gonadotrophin secretion is accompanied primarily by fluctuations in plasma and cerebrospinal fluid concentrations of insulin and certain amino acids, which suggests that, when nutritional status is improved, insulin, amino acids and possibly glucose interact to modulate GnRH secretion.