The G-protein coupled receptor GPR54 and its ligand, KiSS-1-derived peptide kisspeptin-54, appear to play an important role in the mechanism of puberty. This study measures the release of kisspeptin-54 in the stalk-median eminence (S-ME) during puberty and examines its potential role in the pubertal increase in LHRH-1 release in female rhesus monkeys. First, developmental changes in release of kisspeptin-54 and LHRH-1 were assessed in push-pull perfusate samples obtained from the S-ME of prepubertal, early pubertal, and midpubertal female rhesus monkeys. Whereas LHRH-1 levels in 10-min intervals had been measured previously for other experiments, kisspeptin-54 levels in 40-min pooled samples were newly measured by RIA. The results indicate that a significant increase in kisspeptin-54 release occurred in association with the pubertal increase in LHRH-1 release and that a nocturnal increase in kisspeptin-54 release was already observed in prepubertal monkeys and continued through the pubertal period. Second, we measured kisspeptin-54 release in the S-ME of midpubertal monkeys at 10-min intervals using a microdialysis method. Kisspeptin-54 release in the S-ME was clearly pulsatile with an interpulse interval of about 60 min, and approximately 75% of kisspeptin-54 pulses were correlated with LHRH-1 pulses. Finally, the effect of kisspeptin-10 on LHRH-1 release was examined with the microdialysis method. Kisspeptin-10 infusion through a microdialysis probe significantly stimulated LHRH-1 release in a dose-dependent manner. Collectively, the results are consistent with the hypothesis that kisspeptin plays a role in puberty.

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Trophoblast differentiation and early placental development are essential for the establishment of pregnancy, yet these critical events are not readily investigated in human pregnancy. We used embryoid bodies (EBs) prepared from human embryonic stem (hES) cells as an in vitro model of early human development. The levels of human chorionic gonadotropin (hCG), progesterone, and estradiol-17beta in medium from hES cell-derived EBs grown in suspension culture for 1 wk were higher than unconditioned culture medium or medium from undifferentiated hES cells or spontaneously differentiated hES cell colonies. EBs were explanted into Matrigel (MG) "rafts" and cultured for up to 53 d. During the first 7-10 d of three-dimensional growth in MG, small protrusions appeared on the outer surface of EBs, some of which subsequently extended into multicellular outgrowths. The secretion of hCG, progesterone, and estradiol-17beta began to increase on approximately d 20 of MG culture and remained dramatically elevated over the next 30 d. EBs maintained in suspension culture failed to demonstrate this elevation in hormone secretion. Suspension-cultured and MG-embedded EBs exhibited widespread expression of cytokeratins 7/8, demonstrating extensive epithelial differentiation as well as consistent hCG expression. We propose that hES cell-derived EBs may be a useful model for investigation of human trophoblast differentiation and placental morphogenesis.

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