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M. Wilkinson

Summary. LHRH-stimulated LH and FSH secretion was studied in hemipituitaries, in vitro, obtained from several dystrophic mouse mutants (male: 129/ReJ-dy; 129B6F1/J-dy; C57BL/6J-dy and C57BL/6J-dy2J; female: 129B6F1/J-dy) and a dystrophic hamster mutant (male and female CHF-147). Without exception, pituitary tissue from dystrophic animals released significantly more FSH than did tissue obtained from controls. LH secretion was more variable; in the male mice release was inhibited, whereas in the male dystrophic hamsters secretion was elevated above normal. The female mouse mutant pituitary released more LH whereas in the female hamster LH secretion was normal.

The reduction in body weight of the mutants studied could have contributed to the observations of impaired anterior pituitary function.

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H. Badawi and M. Wilkinson

Summary. Daily subcutaneous injections of 100 μg melatonin given to prepubertal female rats housed in 14L:10D or 12L:12D failed to delay puberty as evidenced by the age at which vaginal opening occurred; neither the Sprague–Dawley nor the Wistar strain rats were responsive to melatonin treatment. Reproductive organ weights (ovaries and uteri) at vaginal opening were unaffected by such treatment. Administration of melatonin through the drinking water in doses of 100, 500 or 1000 μg/day did not alter the timing of puberty or the reproductive organ weights in rats of the Sprague–Dawley or Long–Evans strains (housed in 12L:12D). Our experimental methods are identical to a previous report and we have no explanation for our failure to reproduce the earlier results.

Keywords: melatonin; puberty; vaginal opening; rat

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M. Wilkinson and W. H. Moger

Summary. Pituitaries from immature male and female rats (5, 13, 21, 27 and 30 days of age) were removed at 08:00 or 16:00 h and stimulated with GnRH in vitro. In female rats more LH was released from pituitaries taken at 16:00 h than at 08:00 h on Days 21, 27 and 30, but not on Days 5 and 13. There were no differences in male rats in responsiveness of pituitaries taken at these two times. The ability of GnRH to induce release of more LH in the late afternoon may help to synchronize hormone output to coincide with first ovulation.

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E. C. Todoroff, M. Wilkinson and A. Bonen

Summary. Ovariectomized rats were treated with oestradiol benzoate and progesterone or GnRH. Prolonged exercise (running 4 days per week for 6 weeks) markedly potentiated the oestrogen/progesterone-induced release of LH and FSH, but the pituitary response to an injection of GnRH was unaffected. In contrast, at 24 h after a single exercise bout there was no apparent effect on steroid and GnRH stimulated LH and FSH responses although an acute exercise session given on the day of the LH surge inhibited steroid-induced LH release in some rats. We conclude that strenuous, prolonged exercise-training in the ovariectomized rat seems to modify the ability of the hypothalamus to release GnRH. The results were not attributable to a single bout of exercise since the gonadotrophin responses immediately or 24 h after such exercise did not parallel the results observed in the trained rats.

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S. Garagna, M. Zuccotti, J. B. Searle, C. A. Redi and P. J. Wilkinson

Summary. Twenty-two adult male common shrews were collected from 5 sites in the vicinity of Oxford (UK) close to the zone of hybridization between two karyotypic races. The shrews were subdivided into 3 karyotypic categories: homozygotes, simple Robertsonian heterozygotes (which form one or more trivalents at prophase I of meiosis) and complex Robertsonian heterozygotes (which form a quadrivalent). The ratio of primary spermatocytes to round spermatids was determined from transverse sections of seminiferous tubules, to provide an indication of germ cell death. In no individual was there severe germ cells loss. Homozygotes had the highest mean spermatocyte: spermatid ratio and complex heterozygotes the lowest, but there was substantial individual variation and the differences were not significant. Complex heterozygotes also had a higher proportion of defective seminiferous tubules and lower testis weights than did other categories and it is reasonable to propose that, as a population, complex heterozygotes have reduced fitness relative to other categories on the basis of spermatogenic performance. However, there is no evidence from studies of spermatogenesis that simple Robertsonian heterozygotes are less fit than homozygotes.

Keywords: common shrew; Robertsonian rearrangements; spermatogenesis; Robertsonian heterozygotes

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Jessica A McCoy, Benjamin B Parrott, Thomas R Rainwater, Phillip M Wilkinson and Louis J Guillette Jr

Despite the widespread occurrence of environmental sex determination (ESD) among vertebrates, our knowledge of the temporal dynamics by which environmental factors act on this process remains limited. In many reptiles, incubation temperature determines sex during a discrete developmental window just prior to and coincident with the differentiation of the gonads. Yet, there is substantial variation in sex ratios among different clutches of eggs incubated at identical temperatures during this period. Here, we test the hypothesis that temperatures experienced prior to the reported thermosensitive period for alligators (Alligator mississippiensis) can impact how the sex determination system responds to thermal cues later in development. Temperature shift experiments on eggs collected from the field within 24 h of oviposition were employed to decouple various maternal influences from thermal effects, and results demonstrate a previously undefined window of thermosensitivity occurring by stage 15 of embryonic development, six stages earlier than previously reported. We also examine the intrasexual expression of several male- and female-biased genes and show that while male-biased genes display no intrasexual differences, ovarian CYP19A1 (aromatase) transcript abundance differs by approximately twofold depending on thermal exposures experienced at early stages of embryonic development. These findings expand our understanding of the ESD in the alligator and provide the rationale for reevaluation of the temporal dynamics of sex determination in other crocodilians.

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R. A. L. Batt, Deborah M. Everard, Glenda Gillies, M. Wilkinson, Catherine A. Wilson and T. A. Yeo

Summary. Features of the reproductive axis in the genetically hypogonadal, obese mouse (genotype, ob/ob) were examined at 5–8 months of age and compared with those of wild-type litter mates. Hypothalamic concentrations of dopamine and 5-hydroxytryptamine were normal. Those of 5-hydroxyindoleacetic acid, noradrenaline and LH-RH were raised. LH-RH was biologically active. Pituitary concentration of LH was normal, but that of FSH was raised. Serum concentrations of LH and FSH, compared with those of wild-type animals, were normal and low, respectively. Gonad and accessory sex organ weights were reduced. These findings suggest that the release of FSH but not LH is defective in the ob/ob mouse.

Preliminary in-vitro experiments indicated that the pituitary gland responded normally or even supernormally towards LH-RH in its release of LH. The defect in the reproductive axis of the obese mouse may be due to inadequate release of LH-RH although an insensitivity of the pituitary gland towards LH-RH in its release of FSH cannot be excluded.

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Benjamin B Parrott, John A Bowden, Satomi Kohno, Jessica A Cloy-McCoy, Matthew D Hale, Jacqueline T Bangma, Thomas R Rainwater, Phillip M Wilkinson, John R Kucklick and Louis J Guillette Jr

Epigenetic modifications are key mediators of the interactions between the environment and an organism's genome. DNA methylation represents the best-studied epigenetic modification to date and is known to play key roles in regulating transcriptional activity and promoting chromosome stability. Our laboratory has previously demonstrated the utility of the American alligator (Alligator mississippiensis) as a sentinel species to investigate the persistent effects of environmental contaminant exposure on reproductive health. Here, we incorporate a liquid chromatography–tandem mass spectrometry method to directly measure the total (global) proportion of 5-methyl-2′-deoxycytidine (5mdC) in ovarian and whole blood DNA from alligators. Global DNA methylation in ovaries was significantly elevated in comparison with that of whole blood. However, DNA methylation appeared similar in juvenile alligators reared under controlled laboratory conditions but originating from three sites with dissimilar environmental qualities, indicating an absence of detectable site-of-origin effects on persistent levels of global 5mdC content. Analyses of tissues across individuals revealed a surprising lack of correlation between global methylation levels in blood and ovary. In addition, global DNA methylation in blood samples from juvenile alligators was elevated compared with those from adults, suggesting that age, as observed in mammals, may negatively influence global DNA methylation levels in alligators. To our knowledge, this is the first study examining global levels of DNA methylation in the American alligator and provides a reference point for future studies examining the interplay of epigenetics and environmental factors in a long-lived sentinel species.