Craniofacial abnormalities resulting from targeted disruption of the murine Sim2 gene.

Sim2 is a member of the basic helix-loop-helix PAS transcription factor gene family and is evolutionarily related to the Drosophila single-minded gene, a key regulator of central nervous system midline development. In an effort to determine the biological roles of Sim2 in mammalian development, we disrupted the murine Sim2 gene through gene targeting. Mice homozygous for the disrupted allele (Sim2 -/-) exhibit a cleft of the secondary palate and malformations of the tongue and pterygoid processes of the sphenoid bone. These craniofacial malformations are the most probable cause of aerophagia (air swallowing with subsequent accumulation of air in the gastrointestinal tract) and postnatal death exhibited by Sim2 -/- mice. The developing palates of the Sim2 -/- mice are hypocellular, and at embryonic day 14.5 contain excess extracellular matrix component hyaluronan (HA) compared with heterozygotes and homozygous wild-type littermates. HA plays an important role in the regulation and mechanics of palate development. Its premature accumulation in Sim2 -/- animal palates suggests a regulatory role for Sim2 in HA synthesis and in the establishment of craniofacial architecture.

Prolactin receptor gene expression and immunolocalization of the prolactin receptor in human luteinized granulosa cells.

Prolactin is mainly known for its role in breast development and lactation, but has been also implicated in other physiological functions such as immunoregulation and ovarian steroid production. Although prolactin and prolactin receptor (PRL-R) transcripts have been previously identified in the human ovary, the spatial localization of the receptor is unknown. To investigate the presence of PRL-R within the follicular apparatus, human luteinized granulosa cells were obtained at the time of follicular aspiration from women undergoing ovarian stimulation for IVF. RNA extracted from these cells was subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primers for the PRL-R gene. In addition, paraffin sections of isolated granulosa cells and sections of premenopausal human ovaries were immunostained with a mouse anti-human PRL-R monoclonal antibody. PRL-R were immunolocalized to the cell membrane of isolated luteinized granulosa cells and PRL-R transcripts were detected in the extracted RNA. No detectable staining was noted in secondary and early antral follicles in archived paraffin sections. These findings confirm the presence of PRL-R in human luteinized granulosa cells and suggest a localized role for PRL within the mature follicle. The absence of PRL-R in the early follicle suggests that the effects of prolactin are exerted around the time of ovulation.

Derivation of pluripotent stem cells from cultured human primordial germ cells.

Human pluripotent stem cells would be invaluable for in vitro studies of aspects of human embryogenesis. With the goal of establishing pluripotent stem cell lines, gonadal ridges and mesenteries containing primordial germ cells (PGCs, 5-9 weeks postfertilization) were cultured on mouse STO fibroblast feeder layers in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor, and forskolin. Initially, single PGCs in culture were visualized by alkaline phosphatase activity staining. Over a period of 7-21 days, PGCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells termed embryonic stem and embryonic germ (EG) cells. Throughout the culture period most cells within the colonies continued to be alkaline phosphatase-positive and tested positive against a panel of five immunological markers (SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) that have been used routinely to characterize embryonic stem and EG cells. The cultured cells have been continuously passaged and found to be karyotypically normal and stable. Both XX and XY cell cultures have been obtained. Immunohistochemical analysis of embryoid bodies collected from these cultures revealed a wide variety of differentiated cell types, including derivatives of all three embryonic germ layers. Based on their origin and demonstrated properties, these human PGC-derived cultures meet the criteria for pluripotent stem cells and most closely resemble EG cells.