Abstract Somatic cell nuclear transfer was used to produce live piglets from cultured fetal fibroblast cells. This was achieved by exposing donor cell nuclei to oocyte cytoplasm for approximately 3 h before activation by chemical means. Initially, an experiment was performed to optimize a cell fusion system that prevented concurrent activation in the majority of recipient cytoplasts. Cultured fibroblast cells were fused in medium with or without calcium into enucleated oocytes flushed from superovulated gilts. Cybrids fused in the presence of calcium cleaved at a significantly (P < 0.05) greater rate (69%, 37 out of 54) after 2 days of culture compared with those fused without calcium (10%, 7 out of 73), suggesting that calcium-free conditions are needed to avoid activation in the majority of recipient cytoplasts during fusion. In the second experiment, cybrids fused in calcium-free medium were activated approximately 3 h later with ionomycin, followed by incubation in 6-dimethylaminopurine to determine d...
Leptin is a product of the ob gene that is produced primarily by adipose tissue. Leptin and its receptors are found within the ovary, but it is unclear what function this hormone has in the ovary. Using immunohistochemistry, we determined that leptin is found in most cell types in the murine ovary, with the highest staining levels observed in the oocyte. Leptin receptor was also expressed in all of the main ovarian cell types, with the thecal cell layer exhibiting the highest staining levels. Leptin administration did not affect spontaneous or induced maturation of either isolated denuded oocytes or cumulus-oocyte complexes, but it did significantly increase the rate of meiotic resumption in preovulatory follicle-enclosed oocytes ( P < 0.01). Measurements of cAMP within oocytes cultured with leptin showed that this enhanced ability to resume meiosis does not occur via activation of phosphodiesterase 3B and subsequent cAMP reduction. These results provide evidence that leptin affects oocyte maturation when the oocyte is cultured within its normal follicular environment. It is suggested that leptin may induce the production of another factor, possibly from thecal cells, that directly or indirectly acts on the oocyte to initiate germinal vesicle breakdown in this species.
In cooperatively breeding groups of mammals, reproduction is usually restricted to a small number of individuals within the social group. Sexual development of mammals can be affected by social environment, but we know little regarding effects of the cooperative-breeding system on males. Cotton-top tamarin (Saguinus oedipus oedipus) offspring typically do not reproduce in their natal group, even though they may be physically mature. We examined neonatal and pubertal development in captive male cotton-top tamarins as an example of reproductive development within a cooperative-breeding system and to compare cotton-top tamarins with the general primate model. Puberty was characterized using both hormonal and physical measures. Data were collected on urinary levels of LH, testosterone (T), dihydrotestosterone (DHT), cortisol, and the ratio of DHT to T; testicular development; body weight; and breeding age. We determined that 1) pubertal LH secretion began at Week 37, 2) a surge of T secretion followed at Weeks 41–44, and 3) an increase in the metabolism of T to DHT may have occurred at an average age of 48.6 wk. Most of the rapid weight gain was completed by Week 24, before hormonal increases and rapid scrotal growth. We concluded that rapid pubertal testicular growth in captive cotton-top males was completed by an average 76 wk, but that completion of the individual pubertal spurt can occur between 56 and 122 wk. In a cooperative-breeding system, the opportunity for successful reproduction is dictated by the social environment, but we found no evidence that male offspring were developmentally suppressed in their natal social groups. Our findings suggest that puberty in male New World callitrichid primates occurs more quickly than puberty in Old World primates, even though both have similar patterns of development.
Dmrt1is a recently described gene that is specifically expressed in the gonads and is required for postnatal testis differentiation. Here, we describe the transcriptional mechanisms regulating theDmrt1proximal promoter in testicular Sertoli cells. A genomic clone containing exon 1 of the ratDmrt1gene and more than 9 kilobases of 5′ flanking sequence was isolated and characterized. Several prominent transcriptional start sites were identified, with the major site located 102 bases from the translational start. TheDmrt15′ flanking region from ?5000 to + 74 was transcriptionally active in primary Sertoli cells, and deletion analysis of this fragment identified 2 major regions needed for fullDmrt1promoter function. These regions were located between ?3200 and ?2000 base pairs (bp) and downstream of ?150 bp relative to the major transcriptional start site. DNase I footprint analysis of the region downstream of ?150 bp revealed 3 regions that are bound by proteins from Sertoli cell nuclear extracts. Site-directed mutagenesis of these regions identified 2 elements that activate theDmrt1promoter and 2 that repress it. The positive elements bind the transcription factors Sp1, Sp3, and Egr1, suggesting that these transcription factors play a critical role inDmrt1regulation in the testis.