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R. Wordinger, J. Nile and G. Stevens

Summary. Pregnant CD-1 mice were injected with diethylstilboestrol (10μg/kg body weight) in 0·1 ml maize oil, or maize oil alone, on Day 16 of gestation. Six experimental and 6 control female progeny were killed daily from birth until Day 7 and uterine tissues were examined by light microscopy. In-utero exposure to diethylstilboestrol resulted in hypertrophy of luminal epithelial cells and premature formation of uterine glands. The initial sign of uterine gland formation was invagination of the uterine surface epithelial cell layer into the underlying connective tissue stroma. A temporal difference occurred between control animals and those exposed to diethylstilboestrol: uterine gland formation first occurred in experimental progeny on Day 4, but not until Day 5 in control progeny. Uterine glands which extended deep into the connective tissue stroma to the myometrium were present in diethylstilboestrol-treated progeny by Day 7, but remained in the superficial endometrial connective tissue stroma in control animals. The results indicate that prenatal exposure of mice to diethylstilboestrol causes uterine epithelial cell hypertrophy at birth and the premature formation of uterine glands during the first week of neonatal uterine development.

Keywords: uterine glands; development; diethylstilboestrol; mouse

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CA Maltin, MI Delday, KD Sinclair, J Steven and AA Sneddon

The possibility that early fetal programming affects health or disease status in adult life has been considered in relation to tissues such as the cardiovascular system but not with respect to skeletal muscle. Since muscle mass and function are important for life, it is pertinent to ask whether events during the development of muscle in utero can affect the performance of the tissue in later life. This review discusses the factors that influence muscle performance, outlines the current understanding of myogenesis and examines how manipulations alter myogenic outcome after birth. The performance of muscle is determined by the number, type and size of the muscle fibres, these in turn being affected by a number of factors, and the evidence indicates that the proportions of types of muscle fibre have a heritable component. The formation of muscle occurs early in embryogenesis and it appears that the major impacts on myogenesis are associated with extremes of treatment or embryo manipulations. The impact of extremes of treatment or embryo manipulations on myogenesis is seen in the secondary fibres whereas primary fibres appear to be insensitive or protected. Overall, the opportunities for manipulation of myogenesis in utero to improve adult performance are limited.

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The ultrastructure of four roe-deer blastocysts at different stages of embryonic development were studied. During delayed implantation, the outer surface of the trophoblast possessed numerous microvilli and periodic invaginations or caveolae. There was a marked lack of organelles such as mitochondria or endoplasmic reticulum in the cytoplasm of the trophoblast cells, though many lipid droplets, granular inclusions and a lamina of fine fibrillae were present. Elongation of the blastocyst was associated with a decrease in the size and number of the microvilli, the disappearance of lipid droplets and granular inclusions, a reduction in the amount of fibrillar material and a dramatic increase in the development of mitochondria, granular endoplasmic reticulum, ribosomes and Golgi apparatus.

The histology of the ovaries and uterus was studied in thirty-one roe deer. No prominent changes occurred in the ovaries at any stage of development; all ovaries possessed active CL and showed signs of follicular growth and atresia. Changes in the degree of mitotic activity, epithelial cell height, endometrial vascularity and stromal oedema were observed in the uterus throughout the period of delayed implantation and during the phase of rapid embryonic growth. Elongation of the embryo was associated with a marked decline in the height of the glandular epithelium and an increase in endometrial vascularity.

The most important ultrastructural changes in the uteri of six roe deer were observed in the endometrial glands. Delayed implantation was associated with the accumulation of numerous supranuclear vesicles derived from the Golgi apparatus, while the resumption of embryonic growth was correlated with their sudden disappearance. When elongation had been completed, there was a sudden decrease in the cellular activity of these endometrial glands.

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P. J. Booth, D. A. Stevens, M. E. Collins and J. Brownlie

Large-scale in vitro bovine embryo production systems commonly use genital tracts obtained from an abattoir as a source of both cumulus–oocyte complexes and co-culture feeder cells. Tissues derived from this source may be contaminated with non-cytopathogenic bovine viral diarrhoea virus (BVDV) since, in several countries surveyed, approximately 1% of animals tested are persistently infected with this pathogen. Therefore, the use of such material in in vitro fertilization systems presents a potential risk for the transmission of BVDV to bovine embryos and via embryo transfer. This potential was investigated by obtaining oviduct epithelial cells and granulosa cells, which are commonly used as feeder cells, from cattle persistently infected with BVDV and examining them for the presence of BVD viral antigen (p80 non-structural protein and gp53 envelope glycoprotein) by indirect immunofluorescent histochemistry, and also viral RNA (encoding the p80 region) by in situ hybridization. In addition, titres of virus present in oviduct, ovary and blood were assayed by immunodetection on calf testis cell cultures. Luminal epithelial cells from the oviduct and primary cultures of granulosa cells and oviduct epithelial cells from such cattle were shown to contain both viral antigen and RNA. The susceptibility of both cell types to BVDV infection was further established by inoculating primary cell cultures of cells derived from cattle not infected with BVDV with a cloned isolate of non-cytopathogenic BVDV (Pe515). RNA encoding BVDV and the antigen were detected 12 h after inoculation. Viral titres present in oviduct, ovary and blood were between ≥102.2 and 107; ≥102.2 and 106.75; and 103.5 and 104.25 tissue culture infective doses, (TCID)50 g−1, respectively. Control tissues from cattle not infected with BVDV, tested in each of the preceding techniques, were negative. These data establish that ovary and oviduct of persistently infected animals harbour non-cytopathogenic BVDV and that granulosa cells and oviduct epithelial cells, which are used as co-culture cells during bovine embryonic development in vitro and which, in the case of granulosa cells, constitute the cumulus investment surrounding the oocyte, are a vehicle for the potential transmission of BVDV to developing embryos.

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Christina J McManus, Miroslav Valent, Steven L Hardy and Robert L Goodman

Seasonal anoestrus in the ewe results from enhanced oestrogen negative feedback. Recent data have implicated the ventromedial preoptic area (vmPOA) as an important site of oestrogen action. This study addressed whether NO acts within the vmPOA to inhibit LH during seasonal anoestrus. In Experiment 1, microimplants containing Nω-nitro-l-arginine methyl ester (l-NAME, NOS inhibitor), S-methyl thiocitrulline (SMTC, neural NOS (nNOS) inhibitor) or empty implants (control) were administered during mid-anoestrus to the vmPOA. l-NAME, but not SMTC, significantly increased LH pulse frequency. For Experiment 2, ewes in late anoestrus were administered 7-nitroindazole (7NI; nNOS inhibitor), l-NAME, SMTC, or empty implants. 7NI, but not l-NAME or SMTC, increased LH pulse frequency. In Experiment 3, the effects of microimplants and microinjections of l-NAME were compared in mid-anoestrus. Microinjections of l-NAME (300 nl at 10 μg/μl) increased LH pulse frequency, but microimplants did not. In late anoestrus, similar microinjections were ineffective. Taken together, the results of Experiments 1–3 suggested that NO inhibition may be stronger during the middle than at the end of seasonal anoestrus. To test this hypothesis, ewes in Experiment 4 received microinjection of l-NAME or vehicle thrice during the non-breeding season; none of the treatments increased LH pulse frequency. These results indicate that NO plays a role in the vmPOA in suppressing LH secretion during seasonal anoestrus because NOS inhibitors were consistently stimulatory when LH pulse frequency was low. However, the inconsistent and modest effects of these inhibitors suggest that NO actions in this area cannot completely account for the effects of inhibitory photoperiod.

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Christine A Swann, Steven J B Cooper and William G Breed

In mammals, before fertilization can occur, sperm have to bind to, and penetrate, the zona pellucida (ZP). In the laboratory mouse, which has been used as a model system for fertilization studies, sperm–ZP binding has been found to be mediated by a region at the carboxy terminal, encoded by exon 7 of the Zp3 gene. This region shows considerable interspecific sequence diversity with some evidence of adaptive evolution in mammals, suggesting that it may contribute to species-specific sperm–ZP binding. However, in a previous study of sequence diversity of ZP3 of three species of Australian murine rodents, we found an identical protein sequence of the region encoded by exon 7. Here, we expand this earlier study to determine the sequence diversity of this region in 68 out of the 130 species of Australasian murine rodents. Maximum likelihood analyses, using representatives of both New Guinean and Australian taxa, provide evidence of positive selection at three codons adjacent to, or within, the putative combining-site for sperm of ZP3, but this was not evident when the analysis was restricted to the Australian taxa. The latter group showed low levels of both intra- and inter-generic sequence divergences in the region encoded by exon 7 of Zp3, with little evidence that this region contributes to species specificity of sperm–ZP binding. These findings suggest that the selective forces acting on the Zp3 exon 7 region during the evolution of the Australasian murine rodents have been variable, and that positive selection has only occurred in a few lineages.

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Claire Geddes, K. E. Kendle, Carole W. Shanks and Margaret J. Steven

Summary. Inhibition of ovulation by RMI 12,936 was associated with suppression of the pro-oestrous peak of hypothalamic dopamine. The antiovulatory effect was not reversed by administration of oestrogen, was partly reversed by progesterone and was fully reversed by oestrogen and progesterone. Hypophysial sensitivity to LH-RH, known to be reduced by RMI 12,936, remained low when ovulation was restored by steroid treatment. Administration of oestrogen did not restore the pro-oestrous peak of hypothalamic dopamine and ovulation was not induced following administration of l-DOPA in RMI 12,936-treated animals. It was concluded that RMI 12,936 is antioestrogenic as well as antiprogestational, that oestrogen is necessary for induction of full hypothalamic-hypophysial responsiveness to pro-gesterone and that a hypothalamic dopaminergic pathway may have a non-essential role in the control of ovulation possibly associated with increasing hypophysial sensitivity to LH-RH.

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Jonathan W Boyd, Thomas J Lechuga, Charlotte A Ebner, Michael A Kirby and Steven M Yellon

The hypogastric nerve is a major pathway innervating the uterine cervix, yet its contribution to the processes of cervical ripening and parturition is not known. The main objective of this study was to determine the effect of hypogastric nerve transection on remodeling of the cervix and timing of birth. As an initial goal, processes associated with remodeling of the peripartum cervix were studied. The cervix was obtained from time-dated pregnant rats on days 15, 19, 21, and 21.5 of pregnancy, and post partum on the day of birth (day 22). The cervix was excised, post-fixed overnight, and sections stained to evaluate collagen content and structure or processed by immunohistochemistry to identify macrophages or nerve fibers. The census of macrophages and density of nerve fibers in the cervix peaked on day 21, the day before birth, and then declined post partum. These results replicate in time course and magnitude previous studies in mice. To address the main objective, the hypogastric nerve was bilaterally transected on day 15 post-breeding; sham-operated rats served as controls. Pups were born in both groups at normal term. Transection of the hypogastric nerves did not affect remodeling of collagen or the census of macrophages or the density of nerve fibers in the cervix. These findings support the contention that enhanced innervation and immigration of immune cells are associated with remodeling of the cervix and parturition, but that a neural pathway other than the hypogastric nerve may participate in the process of cervical ripening.

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Steven M Yellon, Lauren A Grisham, Genevieve M Rambau, Thomas J Lechuga and Michael A Kirby

The transneuronal tracer pseudorabies virus was used to test the hypothesis that connections from the cervix to the forebrain and hypothalamus are maintained with pregnancy. The virus was injected into the cervix of nonpregnant or pregnant mice, and, after 5 days, virus-labeled cells and fibers were found in specific forebrain regions and, most prominently, in portions of the hypothalamic paraventricular nucleus. With pregnancy, fewer neurons and fibers were evident in most brain regions compared to that in nonpregnant mice. In particular, little or no virus was found in the medial and ventral parvocellular subdivisions, anteroventral periventricular nucleus, or motor cortex in pregnant mice. By contrast, labeling of virus was sustained in the dorsal hypothalamus and suprachiasmatic nucleus in all groups. Based upon image analysis of digitized photomicrographs, the area with label in the rostral and medial parvocellular paraventricular nucleus and magnocellular subdivisions was significantly reduced in mice whose cervix was injected with virus during pregnancy than in nonpregnant mice. The findings indicate that connections from the cervix to brain regions that are involved in sensory input and integrative autonomic functions are reduced during pregnancy. The findings raise the possibility that remaining pathways from the cervix to the forebrain and hypothalamus may be important for control of pituitary neuroendocrine secretion, as well as for effector functions in the cervix as pregnancy nears term.

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David C Kersey, David E Wildt, Janine L Brown, Rebecca J Snyder, Yan Huang and Steven L Monfort

The luteal phase of the giant panda has been exclusively assessed by studying urinary hormone patterns in a very few individuals. To better understand hormonal dynamics of protracted progestagen excretion in this endangered species, we monitored hormonal metabolites in the fibrous faeces of multiple females in the USA and China. Giant pandas that were anoestrual during the breeding season excreted baseline progestagen throughout the year. In contrast, there were two distinctive periods when progestagen excretion increased in females that experienced behavioural oestrus, the first being modest, lasting for 61–122 days, and likely reflecting presumptive ovulation. This increase was far surpassed by a secondary rise in progestagen excretion associated with a rejuvenated luteal capacity or hormone production from an extra-gonadal source. The duration of this ‘secondary’ rise in progestagen excretion averaged ∼45 days and terminated in a decline to baseline coincident with parturition or the end of a non-parturient luteal interval. Data revealed that, even with a complex, biphasic progestagen profile, the longitudinal patterns produced by giant pandas were relatively consistent among animals and across years within individuals. However, progestagen excretion patterns throughout this period could not be used to discriminate among non-pregnant, pregnant or pseudopregnant states.