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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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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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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.
