The overall goals of the proposed research are to examine regulatory mechanisms that influence post-mitotic development of testicular germ cells. The major calcium binding protein in mammalian sperm is calspermin. We have cloned the CDNA encoding this protein. Analysis of the sequence and of the gene revealed that calspermin is derived from a transcription unit that also encodes a novel calcium-calmodulin dependent protein kinase. This cDNA has also been cloned and sequenced. Whereas the kinase mRNA is first detected in early primary spermatocytes and declines to a low level following the last meiotic division, calspermin mRNA is first expressed in pachytene primary spermatocytes and continues to increase as cell complete meiosis and undergo terminal differentiation. We have isolated genomic DNA that encodes the entire transcription unit and propose to characterize the structural organization of the gene. We will then utilize transfected mammalian cells, a testicular transcription system and transgenic mice to determine the molecular mechanisms that control cell specific mRNA expression. The hypothesis is that alternative transcriptional initiation followed by a requisite unique RNA processing event are involved in calspermin production. Expression vectors have been used to produce calspermin and the kinase and to identify key amino acids required for function and/or regulation of activity. A constitutive kinase has been used to develop mammalian cell lines that express the enzyme in a regulated fashion and to identify a transcriptional DNA element that is trans-activated by the active enzyme. We propose to characterize the mechanism by which the overexpression of the kinase results in a G2 cell cycle block and the transcription factor responsive to the kinase. Transgenic mice will be utilized to evaluate the consequences of altering the concentration of the kinase on spermatogenesis and sperm motility. Different promoters will be employed to change the temporal expression and/or amount of the proteins during germ cell development. We predict that the kinase will play a role in the onset, progression or completion of meiosis whereas calspermin will be involved in terminal differentiation of the spermatid or sperm motility.
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