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J. A. Mitchell and B. R. Van Kainen

Summary. The effects of ethanol on uterine sensitivity to induction of decidualization and deciduoma growth were determined. Rats were ovariectomized, given an oestrogen-progesterone regimen to optimize induction and growth of deciduoma and randomly assigned to one of three ethanol treatment groups: (i) days 1–4 (pre-induction/period of sensitivity), (ii) days 5–9 (post-induction/period of growth), (iii) days 1–9 (periods of sensitivity and growth); or to a control group not treated with ethanol (pair-fed to treated groups). Ethanol (0, 1, 2, or 4 g kg−1) diluted in water was administered by stomach tube on the days prescribed. Decidualization was induced in one uterine horn by intraluminal injection of sodium phosphate buffer. Uterine sensitivity and decidual growth were assessed as cornu weight. Blood alcohol concentrations were measured by gas chromatography. Alcohol treatment reduced uterine sensitivity, but increased deciduoma growth. Blood alcohol concentrations rose to 133 mg% at 30 min, remained high for 90 min and declined to 82 mg% at 120 min. Thus, blood alcohol concentrations sufficient to induce mild intoxication in humans suppressed uterine sensitivity to decidualization and enhanced deciduoma growth in rats. As all ovarian steroid hormone support was exogenous, the effects of ethanol on deciduoma induction and growth were not due to alterations in the hypothalamic–pituitary–ovarian axis.

Keywords: decidualization; alcohol; implantation; rat

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J. A. Mitchell and R. E. Hammer

Summary. Nicotine (5·0 mg/kg) was injected (s.c.) twice daily on Day 1 or Days 1–4 or 1–5 of pregnancy. Cumulative doses of nicotine retarded embryo cell cleavage and substantially reduced embryo cell number (saline vs nicotine: 42·5 ± 1·7 vs 22·1 ± 1·9 nuclei/embryo, at 12:00 h on Day 5; P < 0·05). However, treatment for even 1 day (Day 1) significantly reduced cell number (saline vs nicotine: 42·5 ± 1·7 vs 30·5 ± 0·9, at 12:00 h day on Day 5; P < 0·01). Nicotine injection also resulted in a marked and prolonged reduction in oviduct blood flow (pretreatment vs 90 min after nicotine: 0·61 ± 0·06 vs 0·37 ± 0·10 ml/min·g−1; P < 0·005). The results indicate that, in the rat, even a brief exposure to nicotine, the chief alkaloid of tobacco, reduces oviducal blood flow and the rate of embryo cell proliferation. The embryo is therefore susceptible to the effects of nicotine before implantation.

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J. A. Mitchell and H.-W. Denker

Summary. Localization of uterine arylamidase activity varied between species: arylamidase was found primarily in the apical aspect of uterine epithelial cells in the rabbit, hamster and non-pregnant rat; only moderate staining was observed in these animals in the endometrial stroma. By contrast, arylamidase localization was primarily stromal in the guinea-pig at all stages studied while the luminal epithelium was devoid of reactivity. In all species, uterine enzyme activity increased before implantation but decreased in the vicinity of the blastocyst once implantation had begun. A generalized increase over the entire length of the uterus was seen during the preimplantation phase in the uterine epithelium of the rabbit and in the endometrial stroma of the guinea-pig. Increase in stromal activity appeared to indicate predecidual transformations which were embryo-dependent (i.e. localized to the implantation site) in the rat, or embryo-independent (i.e. occurring throughout the uterus) in the guinea-pig. A subsequent decrease in enzyme activity occurred in the vicinity of the implanting embryo irrespective of the cell type involved (epithelium in the rabbit, stroma/decidua in the rat and guinea-pig). Since arylamidases of the type studied here are integrated membrane proteins, the uniformity of changes observed in different species may reflect profound changes in membrane properties of endometrial cells as an element of the implantation reaction.

Keywords: uterus; arylamidase; aminopeptidase; implantation; epithelium; stroma

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K. L. Simpson, J. A. Keelan and M. D. Mitchell

Parturition is associated with changes in the production of inflammatory mediators by gestational tissues. An explant system was established to study the change in response of human amnion to various regulating factors during labour. Disks of tissue (6 mm) were excised from amnion membranes obtained either at term by Caesarian section before labour (n = 5–6) or after spontaneous vaginal delivery (n = 3–7). After 24 h equilibration in media, the tissues were treated with interleukin 1β (10 ng ml−1), tumour necrosis factor α (100 ng ml−1), lipopolysaccharide (5 μg ml−1) and dexamethasone (1 μmol l−1) or an appropriate vehicle control for 24 h (n = 3 wells per treatment). Media were harvested and interleukin 10, interleukin 6 and prostaglandin E2 concentrations were determined by immunoassay. In tissues taken both before and after the onset of labour, basal interleukin 10 production by amnion explants was near to the limit of detection. Basal production rates of PGE2 by amnion explants were significantly higher (P < 0.0012; Mann–Whitney U test) in tissues taken during labour than in tissues taken before the onset of labour, while interleukin 6 production was not significantly altered by labour. Production rates of interleukin 6 and prostaglandin E2 were significantly increased by interleukin 1β, tumour necrosis factor α and lipopolysaccharide in explants from tissues taken during and before labour, while the responsiveness of interleukin 10 production to these treatments was inconsistent. Dexamethasone had no effect on interleukin 6 production by amnion explants, but significantly inhibited prostaglandin E2 production, although this inhibition was approximately 30% lower in tissues obtained after the onset of labour. These results support the presence of inflammatory positive feedback cycles, coincident with a deficiency of an anti-inflammatory factor within gestational tissue, which may be involved in the progression or maintenance of labour.

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R. E. Hammer, H. Goldman and J. A. Mitchell

Summary. Subcutaneous injection of nicotine (0·5 or 5 mg/kg body wt) resulted in a marked and prolonged reduction in uterine blood flow and intrauterine oxygen tension in pseudopregnant rats. By 10 min after nicotine administration (5 mg/kg) uterine perfusion was reduced by 40%, remained suppressed for 90 min and returned to the pre-treatment level by 120 min. Rats receiving the 0·5 mg nicotine/kg also showed a marked reduction in uterine blood flow, although the response was slower in onset and longer in duration. Nicotine (5 mg/kg) also resulted in a sustained decrease in intrauterine oxygen tension from a control value of 48·9 ± 3·6 to 22·2 ± 2·6 mmHg at 45–60 min and 21·7 ± 1·5 mmHg at 60–90 min. The frequency and amplitude of fluctuations in intrauterine oxygen tension were still reduced by 90 min after treatment.

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J. Falconer, M. D. Mitchell, L. A. Mountford and J. S. Robinson

Summary. Plasma samples were obtained from 8 conscious rhesus monkeys at 3–4 day intervals throughout the menstrual cycle. Oxytocin concentrations were significantly higher in mid-cycle (Days 10–11) than at Days 4–5 (P < 0·01) and Days 28–29 (P < 0·02, Wilcoxon signed rank test). No significant correlations between oxytocin and oestradiol or progesterone were found.

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M. D. Mitchell, A. P. F. Flint, J. S. Robinson and G. D. Thorburn

Summary. In the fetal cotyledons and membranes, PGE production (determined in a continuous superfusion system) was significantly greater than that of PGF (P < 0·01 and P < 0·02 respectively). The fetal cotyledon also produced more PGE than the maternal cotyledon (P< 0·01) and more PGF after parturition than before (P < 0·01).

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M. J. N. C. Keirse, M. D. Mitchell and A. P. F. Flint

Summary. 15-Hydroxyprostaglandin dehydrogenase (PGDH) was measured in vitro in myometrium and maternal cotyledons from 6 sheep and in fetal cotyledons from 3 sheep before and after parturition. Maternal cotyledons contained more PGDH than fetal cotyledons or myometrium. At parturition, PGDH activity decreased in myometrium but increased in fetal and maternal placental tissue. The enzymatic activity observed in vitro did not result in a large, consistent release of prostaglandin F metabolites into the utero-ovarian vein in vivo, and such activity may have a strictly localized effect in ovine parturition.

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Miguel J Xavier, Lisa A Mitchell, Kristen E McEwan, Rodney J Scott and R John Aitken

The Big Blue λSelect-cII selection system has been employed along with whole-exome sequencing to examine the susceptibility of the male germ line to mutation in two challenging situations (i) exposure to a chemotherapeutic regime including bleomycin, etoposide and cis-platinum (BEP) and (ii) the ageing process. A 3-week exposure to BEP induced complete azoospermia associated with a loss of developing germ cells and extensive vacuolization of Sertoli cell cytoplasm. Following cessation of treatment, spermatozoa first appeared in the caput epididymis after 6 weeks and by 12 weeks motile spermatozoa could be recovered from the cauda, although the count (P < 0.001) and motility (P < 0.01) of these cells were significantly reduced and superoxide generation was significantly elevated (P < 0.001). Despite this increase in free radical generation, no evidence of chromatin instability was detected in these spermatozoa. Furthermore, embryos obtained from females mated at this 12-week time point showed no evidence of an increased mutational load. Similarly, progressive ageing of Big Blue mice had no impact on the quality of the spermatozoa, fertility or mutation frequency in the offspring despite a significant increase in the mutational load carried by somatic tissues such as the liver (P < 0.05). We conclude that the male germ line is highly resistant to mutation in keeping with the disposable soma hypothesis, which posits that genetic integrity in the germ cells will be maintained at the expense of the soma, in light of the former’s sentinel position in safeguarding the stability of the genome.

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Razan A Elkhatib, Marine Paci, Romain Boissier, Guy Longepied, Yasmina Auguste, Vincent Achard, Patrice Bourgeois, Nicolas Levy, Nicolas Branger, Michael J Mitchell and Catherine Metzler-Guillemain

During spermiogenesis the spermatid nucleus is elongated, and dramatically reduced in size with protamines replacing histones to produce a highly compacted chromatin. After fertilisation, this process is reversed in the oocyte to form the male pronucleus. Emerging evidence, including the coordinated loss of the nuclear lamina (NL) and the histones, supports the involvement of the NL in spermatid nuclear remodelling, but how the NL links to the chromatin is not known. In somatic cells, interactions between the NL and the chromatin have been demonstrated: LEM-domain proteins and LBR interact with the NL and respectively, the chromatin proteins BAF and HP1. We therefore sought to characterise the lamina-chromatin interface during spermiogenesis, by investigating the localisation of six LEM-domain proteins, two BAF proteins and LBR, in human spermatids and spermatozoa. Using RT-PCR, IF and western blotting, we show that six of the proteins tested are present in spermatids: LEMD1, LEMD2 (a short isoform), ANKLE2, LAP2β, BAF and BAF-L, and three absent: Emerin, LBR and LEMD3. The full-length LEMD2 isoform, required for nuclear integrity in somatic cells, is absent. In spermatids, no protein localised to the nuclear periphery, but five were nucleoplasmic, receding towards the posterior nuclear pole as spermatids matured. Our study therefore establishes that the lamina-chromatin interface in human spermatids is radically distinct from that defined in somatic cells. In ejaculated spermatozoa, we detected only BAF and BAF-L, suggesting that they might contribute to the shaping of the spermatozoon nucleus and, after fertilisation, its transition to the male pronucleus.