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Simon I R Lane, Heng-Yu Chang, Phoebe C Jennings and Keith T Jones

Previous studies have established that when maturing mouse oocytes are continuously incubated with the Aurora inhibitor ZM447439, meiotic maturation is blocked. In this study, we observe that by altering the time of addition of the inhibitor, oocyte maturation can actually be accelerated by 1 h as measured by the timing of polar body extrusion. ZM447439 also had the ability to overcome a spindle assembly checkpoint (SAC) arrest caused by nocodazole and so rescue polar body extrusion. Consistent with the ability of the SAC to inhibit cyclin B1 degradation by blocking activation of the anaphase-promoting complex, we could also observe a rescue in cyclin B1 degradation when ZM447439 was added to nocodazole-treated oocytes. The acceleration of the first meiotic division by ZM447439, which has not been achieved previously, and its effects on the SAC are all consistent with the proposed mitotic role of Aurora B in activating the SAC. We hypothesize that Aurora kinase activity controls the SAC in meiosis I, despite differences to the mitotic cell cycle division in spindle architecture brought about by the meiotic mono-orientation of sister kinetochores.

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H. J. Stewart, D. S. C. Jones, J. C. Pascall, R. M. Popkin and A. P. F. Flint

Page Introduction 2 Structures of genes and mRNA molecules 2 Structure of genes 2 Sequences of promoters and enhancers 3 Signals for terminating RNA synthesis 5 Structure of mRNA molecules 5 Structure of genes coding for reproductive polypeptide hormones and hypothalamic releasing factors 7 Structure of gonadotrophin genes 7 Structure of placental lactogen, prolactin and growth hormone genes 10 Structure of the LHRH gene 12 Inhibin, activin and Müllerian duct-inhibiting hormone 13 Localization of gene products by in-situ hybridization 16 Pre- and post-translational processing 18 Pretranslational processing: differential splicing of RNA 18 Post-translational processing 18 Steroidogenic enzymes and steroid sulphatase 20 Steroid hormone receptors 22 Steroid receptor primary structure: amino acid sequences derived from cloned DNA 23 Steroid hormone receptors and oncogenesis 26 Mechanism of binding of receptors to DNA—the finger hypothesis 27 DNA sequences recognized by steroid receptors in eukaryotic cells 28 Fertilization and early development 30 Expression of

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R. E. Zimmerman, R. S. Nevin, D. J. Allen, C. D. Jones, M. E. Goettel and P. J. Burck

Summary. Acrosin and acrosomal hyaluronidase were inhibited by tetradecyl sodium sulphate (TDSS) in vitro at concentrations of < 10−4 M. TDSS prevented the removal in vitro of the cumulus oophorus by testicular hyaluronidase and the zona pellucida by acrosin. TDSS had a contraceptive effect in rabbits when administered intravaginally before coitus or released at levels of 1–3 μg/day from intrauterine silicone rubber devices.

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C L Tower, S Lui, N R Charlesworth, S D Smith, J D Aplin and R L Jones

Angiotensin II (Ang II) is locally generated in the placenta and regulates syncytial transport, vascular contractility and trophoblast invasion. It acts through two receptor subtypes, AGTR1 and AGTR2 (AT1 and AT2), which typically mediate antagonising actions. The objectives of this study are to characterise the cellular distribution of AGTR1 and AGTR2 at the maternal–fetal interface and explore the effects on cytotrophoblast turnover. Low levels of AGTR2 mRNA were detected in first trimester placental homogenates using real-time PCR. Immunohistochemistry using polyclonal antibodies against AGTR1 and AGTR2 detected the receptors in first trimester placenta, decidua basalis and villous tip outgrowths in culture. Serial staining with cytokeratin-7 was used to identify extravillous trophoblasts (EVTs). AGTR1 was found in the syncytiotrophoblast microvillous membrane, in a subpopulation of villous cytotrophoblasts, and in Hofbauer cells. AGTR1 was strongly upregulated in cytotrophoblasts in cell columns and villous tip outgrowths, but was absent in interstitial and endovascular EVTs within the decidua. AGTR2 immunostaining was present in Hofbauer cells and villous cytotrophoblasts, but was absent from syncytiotrophoblast. Faint staining was detected in cell column cytotrophoblasts and villous outgrowths, but not in EVTs within the decidua. Both receptors were detected in placental homogenates by western blotting. Ang II significantly increased proliferation of cytotrophoblasts in both villous explants and villous tip outgrowths, but did not affect apoptosis. Blockade of AGTR1 and AGTR2 together abrogated this effect. This study shows specific expression patterns for AGTR1 and AGTR2 in distinct trophoblast populations at the maternal–fetal interface and suggests that Ang II plays a role in placental development and generation of EVTs.

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G. R. Jones, A. G. Sacco, M. G. Subramanian, M. Kruger, S. Zhang, E. C. Yurewicz and K. S. Moghissi

Summary. Female rabbits (n = 36, 6 per group) were immunized with: (i) solubilized isolated porcine zona pellucida (SIZP), which contains ZP1, 82 kDa; ZP3α, 55 kDa; and ZP3β, 55 kDa; (ii) a purified preparation of ZP3α and ZP3β (ZP3); (iii) purified endo-β-galactosidase digested glycoproteins ZP3α-(EBGD) and (iv) ZP3β-(EBGD) (each about 30% deglycosylated); (v) chemically deglycosylated core proteins ZP3α-(DG) and (vi) ZP3β-DG (each > 92% deglycosylated). Rabbits injected with saline (n = 6) or Freund's adjuvant (n = 6) served as controls. Rabbits were bled weekly to monitor titres. Every six weeks two animals from each group (n = 16) were selected for unilateral oophorectomy followed by histological examination. Sections were scored for numbers of primary, secondary and tertiary follicles. Anti-ZP3 titres developed in all treatment groups and correlated with carbohydrate content (peak per cent [125I]labelled ZP3 binding by radioimmunoassay: SIZP 71·9 ± 1·2, ZP3 70·0 ± 2·5, ZP3α-EBGD 60·9 ± 5·3, ZP3β-EBGD 56·4 ± 5·0, ZP3α-DG 56·4 ± 4·0, ZP3β-DG 53·5 ± 4·3) (means ± sem). Animals immunized with SIZP, ZP3 and ZP3β-EBGD showed a statistically significant reduction in the number of primary, secondary and tertiary follicles compared with controls (P < 0·01, manova), whereas animals immunized with ZP3α-EBGD, ZP3α-DG and ZP3β-DG did not (P > 0·05, manova). These results demonstrate that immunization with purified ZP3α macromolecules (ZP3α-EBGD, ZP3α-DG) or ZP3β-DG does not produce histopathological changes in ovaries. Such deglycosylated ZP macromolecules represent potential target antigens for immunocontraceptive development.

Keywords: zona pellucida; deglycosylation; follicular development; ovarian histology; immunocontraception, rabbit

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H N Jones, C J Ashworth, K R Page and H J McArdle

Trans-placental transport of amino acids is vital for the developing fetus. Using the BeWo cell line as a placental model, we investigated the effect of restricting amino acid availability on amino acid transport system type A. BeWo cells were cultured either in amino acid-depleted (without non-essential amino acids) or control media for 1, 3, 5 or 6 h. System A function was analysed using α(methyl-amino)isobutyric acid (MeAIB) transcellular transport studies. Transporter (sodium coupled neutral amino acid transporter (SNAT1/2)) expression was analysed at mRNA and protein level by Northern and Western blotting respectively. Localisation was carried out using immunocytochemistry. MeAIB transcellular transport was significantly (P < 0.05) increased by incubation of the cells in amino acid-depleted medium for 1 h, and longer incubation times caused further increases in the rate of transfer. However, the initial response was not accompanied by an increase in SNAT2 mRNA; this occurred only after 3 h and further increased for the rest of the 6-h incubation. Similarly, it took several hours for a significant increase in SNAT2 protein expression. In contrast, relocalisation of existing SNAT2 transporters occurred within 30 min of amino acid restriction and continued throughout the 6-h incubation. When the cells were incubated in medium with even lower amino acid levels (without non-essential plus 0.5 × essential amino acids), SNAT2 mRNA levels showed further significant (P < 0.0001) up-regulation. However, incubation of cells in depleted medium for 6 h caused a significant (P = 0.014) decrease in the expression of SNAT1 mRNA. System L type amino acid transporter 2 (LAT2) expression was not changed by amino acid restriction, indicating that the responses seen in the system A transporters were not a general cell response. These data have shown that placental cells adapt in vitro to nutritional stress and have identified the physiological, biochemical and genomic mechanisms involved.