Bovine cumulus expansion and corona-oocyte disconnection during culture in vitro.

Cumulus-oocyte complexes were aspirated from small antral follicles (3-5 mm in diameter) and divided into 2 groups: complexes in which a dark rim of corona cells were visible around the zona pellucida (group 1); and those in which the corona displayed the same density as the rest of the cumulus cell mass (group 2). Cumulus complexes of both groups were evaluated by acetoorcein staining, scanning electron microscopy (SEM) and 3H-uridine uptake during culture in vitro. Germinal vesicle breakdown was initiated at 7 h of culture in group 1 while more than half of the oocytes in group 2 already displayed germinal vesicle breakdown at the time of aspiration. However, whereas more than 80% of the oocytes in group 1 had reached metaphase II at 24 h of culture, almost half of the oocytes in group 2 were arrested in metaphase I after this time interval. As revealed by SEM the complexes of group 2 showed signs of expansion such as elongation of cumulus cells and presence of extracellular matrix already at the time of aspiration. In group 1 these features were noticed at 7-9 h of culture. A high level of metabolic coupling between corona cells and oocyte was maintained up to 9 h of culture in group 1 followed by a decrease to a constant low level at 13 h. In group 2 a high degree of coupling was maintained up to 7 h followed by a gradual decrease to a constant low level at 13 h. It is concluded that cumulus-oocyte complexes with a dark rim of corona cells as judged by stereomicroscopy mature at a higher rate and maintain unexpanded characteristics and efficient corona-oocyte coupling longer than complexes with even density of the cumulus mass. Consequently, the presence of a dark rim of corona cells may be used as a criterion for selection of oocytes for in vitro embryo production.     

Phosphodiesterase inhibitor 3-isobutyl-methyl-xanthine stimulates reproduction in rabbit females

The aim of our study was to examine the influence of 3-isobutyl-1-methyl-xanthine (IBMX), an inhibitor of cyclic adenosine monophosphate and cyclic guanosine monophosphate phosphodiesterases, on the reproductive efficiency of gonadotropin-stimulated rabbits (Oryctolagus cuniculus, Leporidae, Lagomorpha). The ovarian cycle and ovulation of control rabbits was induced by pregnant mare serum gonadotropin (PMSG) followed by administration of human chorionic gonadotropin (hCG; first series of experiments) or gonadotropin-releasing hormone (GnRH; second series of experiments). Experimental animals received PMSG and hCG together with IBMX (at 5 or 25 μg/animal) or GnRH together with IBMX (at 50 or 500 μg/animal). After ovulation and mating, in the first series of experiments, animals were killed; the pronuclear-stage eggs were flushed from the oviducts and cultured up to blastocyst cell stage. Numbers of ovarian corpora lutea, ovulated oocytes, and oocyte-derived embryos reaching blastocyst stage were determined. In the second series of experiments, all the animals were kept until parturition, when the pregnancy and birth rate, litter size, and number, viability, and body weight of pups were recorded. IBMX injections at doses of 5 or 25 μg/animal significantly increased the number of ovulations/corpora lutea, harvested zygotes, and embryos derived from these zygotes. Administration of IBMX at doses of 500 μg/animal or 50 μg/animal to nulliparous young animals (4.5 mo of age) significantly increased their pregnancy rate and birth rate, litter size, and litter weight. In multiparous old animals (2 yr of age), IBMX at a dose of 50 μg/animal, but not 500 μg/animal, significantly increased their pregnancy rate and litter size, but not the birth rate, number of pups per female, or litter weight. These data demonstrate that (1) IBMX can enhance the stimulatory effect of GnRH/gonadotropins on rabbit ovulation, oocyte maturation, embryo yield and development, pregnancy and birth rate, and number, viability, and body weight of pups; (2) nulliparous young females (4.5 mo of age) are more sensitive to IBMX treatments than the multiparous old animals (2 yr of age); and (3) cyclic nucleotides-dependent intracellular mechanisms are involved in control of rabbit reproductive functions and IBMX, an activator of these mechanisms, can be a stimulator of reproduction and fertility.     

A mutation in serca underlies motility dysfunction in accordion zebrafish

Zebrafish acquire the ability for fast swimming early in development. The motility mutant accordion (acc) undergoes exaggerated and prolonged contractions on both sides of the body, interfering with the acquisition of patterned swimming responses. Our whole cell recordings from muscle indicate that the defect is not manifested in neuromuscular transmission. However, imaging of skeletal muscle of larval acc reveals greatly prolonged calcium transients and associated contractions in response to depolarization. Positional cloning of acc identified a serca mutation as the cause of the acc phenotype. SERCA is a sarcoplasmic reticulum transmembrane protein in skeletal muscle that mediates calcium re-uptake from the myoplasm. The mutation in SERCA, a serine to phenylalanine substitution, is likely to result in compromised protein function that accounts for the observed phenotype. Indeed, direct evidence that mutant SERCA causes the motility dysfunction was provided by the finding that wild type fish injected with an antisense morpholino directed against serca, exhibited accordion-like contractions and impaired swimming. We conclude that the motility dysfunction in embryonic and larval accordion zebrafish stems directly from defective calcium transport in skeletal muscle rather than defective CNS drive.     

L1 (LINE-1) retrotransposable elements provide a "fossil" record of the phylogenetic history of murid rodents

The single most difficult problem in phylogenetic analysis is decidingwhether a shared taxonomic character is due to common ancestry or one thatappeared independently due to convergence, parallelism, or reversion to anancestral state. Mammalian L1 retrotransposons undergo periodicamplifications in which multiple copies of the elements are interspersed inthe genome. Because these elements apparently are transmitted only byinheritance and are retained in the genome, a shared L1 amplification eventcan only be an inherited ancestral character. We propose that L1amplification events can be an excellent tool for analyzing mammalianevolution and demonstrate here how we addressed several refractory problemsin rodent systematics using L1 DNA as a taxonomic character.     

Effects of chronic food restriction and treatments with leptin or ghrelin on different reproductive parameters of male rats

The existence of a close relationship between energy status and reproductive function is well-documented, especially in females, but its underlying mechanisms remain to be fully unfolded. This study aimed to examine the effects of restriction of daily calorie intake, as well as chronic treatments with the metabolic hormones leptin and ghrelin, on the secretion of different reproductive hormones, namely pituitary gonadotropins and prolactin, as well as testosterone, in male rats. Restriction (50%) in daily food intake for 20 days significantly reduced body weight as well as plasma PRL and T levels, without affecting basal LH and FSH concentrations and testicular weight. Chronic administration of leptin to rats fed ad libitum increased plasma PRL levels and decreased circulating T, while it did not alter other hormonal parameters under analysis. In contrast, in rats subjected to 50% calorie restriction, leptin administration increased plasma T levels and reduced testis weight. Conversely, ghrelin failed to induce major hormonal changes but tended to increase testicular weight in fed animals, while repeated ghrelin injections in food-restricted males dramatically decreased plasma LH and T concentrations and reduced testis weight. In sum, we document herein the isolated and combined effects of metabolic stress (50% food restriction) and leptin or ghrelin treatments on several reproductive hormones in adult male rats. Overall, our results further stress the impact and complex way of action of different metabolic cues, such as energy status and key hormones, in reproductive function also in the male.