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L. J. Wilton, V. S. Marshall, E. C. Piercy, and H. D. M. Moore

Oocytes aspirated from preovulatory (i.e. ≥ 2 mm) follicles of marmoset monkeys were graded for maturity according to the degree of cumulus expansion, grade I being most mature and grade IV least mature. After preincubation for 2–5, 9–11 or 21–29 h, 82% of oocytes could be fertilized using epididymal spermatozoa and only 2.3% were polyspermic. Fertilization rate was lowest (60%) in grade IV oocytes and all oocytes preincubated for 2–5 h (53%). Fertilization rate increased to 92% in oocytes preincubated for 21–29 h. Embryos developed in vitro to a mean of eight cells. Embryo development was unaffected by oocyte maturity but correlated with preincubation time. Oocytes preincubated for 2–5 h developed into embryos with significantly fewer cells than those preincubated for 9–11 or 21–29 h (P < 0.001). Fifty-six per cent of embryos showed delayed cleavage and these had fewer cells than non-delayed embryos (P < 0.001). When oocytes were preincubated for 2–5 h, development of all resulting embryos was delayed. However, only 17 and 58% of embryos developing from oocytes preincubated for 9–11 and 21–29 h, respectively, were delayed and this was independent of oocyte maturity.

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D. E. Wildt, M. Bush, S. J. O'Brien, N. D. Murray, A. Taylor, and J. A. Marshall Graves

Summary. Spermic electroejaculates (range in motile sperm/ejaculate, 0·50–122·9 × 106; mean ± s.e.m., 38·6 ± 4·9) were recovered from 47 of 48 adult koalas captured from 3 wild populations in Australia. Semen was characterized by (i) a high density of globular bodies, which prevented the estimation of sperm motility without dilution; (ii) a brownish colour; and (iii) an acidic pH. Spermatozoa were categorized on the basis of 10 head forms, most cells being a curved or hooked shape. The koala populations differed in sperm concentration and motility ratings, but not in testes size, testosterone production or proportions of spermatozoa with various head shapes. These data confirm that free-living koalas normally produce spermatozoa with a high incidence of structural heterogeneity almost solely confined to the head region; and demonstrate the utility and safety of conventional gamete and endocrine studies, approaches which will be useful for determining the impact of genetic isolation and venereal disease on species fertility.

Keywords: koala; marsupial; semen; electroejaculation; spermatozoa; testis; testosterone

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A. López Bernal, J. Bellinger, J. M. Marshall, S. Phaneuf, G. N. Europe-Finner, G. Asbóth, and D. H. Barlow

The expression of heterotrimeric (αβγ subunits) GTP-binding regulatory proteins (G proteins) and the activation of G protein-linked receptors in human granulosa cells were investigated. The cells were obtained from stimulated follicles in women undergoing in vitro fertilization and were cultured in serum-supplemented medium. Immunoblotting with specific antibodies showed that granulosa cell membranes express αs, αi3 αi1,2, αq,11 and β subunits. Three antibodies against αo failed to detect this protein. The cells responded to hCG and to prostaglandin E2 with a dose-dependent increase in cAMP formation, confirming the functional activation of Gαs. The α2 adrenoceptor agonist, clonidine, inhibited hCG-stimulated cAMP formation and this effect was blocked with pertussis toxin, thus involving a Gi-type protein, most likely Gαi2. Oxytocin provoked an increase in formation of inositol phosphates and intracellular calcium concentration, which was partly pertussis toxin resistant, providing evidence of Gαq,11 activation. However, a significant component of the response to oxytocin could be blocked by pertussis toxin, indicating Gi-mediated phospholipase C activation (by either αi or βγ subunits). These data demonstrate the presence of G proteins in granulosa cells and suggest a complex regulation of hormonal signalling. The concentration of cAMP in these cells depended on the balance of Gαs:Gαi activation, whereas activation of the inositol phospholipid pathway and rises in intracellular calcium involved both Gq,11 and Gi pathways.

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Courtney Barber, Yann Yap, Natalie J Hannan, Euan M Wallace, and Sarah A Marshall

Preeclampsia is a multisystem hypertensive disorder of pregnancy that remains one of the leading causes of maternal and perinatal morbidity and mortality worldwide. The widespread maternal endothelial dysfunction that underlies preeclampsia is thought to arise from excessive placental production of various factors combined with enhanced oxidative stress. While previous studies have reported elevated activin A in women diagnosed with preeclampsia, whether activin A can cause vascular dysfunction has not yet been thoroughly investigated. Here, we demonstrated that different subtypes of activin A receptors were localised to the endothelial and smooth muscle cells of mouse and human aortae. Then, the aorta of healthy female C57Bl6J mice (n = 8) were incubated for 24 h in various concentrations of recombinant activin A to mimic early pregnancy (5 ng/mL), late pregnancy (20 ng/mL) and preeclampsia (50 ng/mL). Vascular reactivity as assessed by wire myography revealed that only the preeclamptic level of activin A impaired agonist-mediated endothelium-dependent relaxation by reducing the vasodilator prostanoid contribution to relaxation. However, agonist-mediated endothelium-independent mechanisms were unaffected. Further investigations carried out on human aortic endothelial cells suggested that the impairment of aorta relaxation could also be driven by increased endothelial cell permeability, and decreased cell viability, adherence and proliferation. This is the first direct evidence to show that activin A can induce endothelial dysfunction in whole blood vessels, suggesting that at high circulating levels it may contribute to the widespread endothelial dysfunction in women with preeclampsia.