The fibrous sheath (FS) is a cytoskeletal structure that encases the axoneme in the principal piece of the spermatozoon tail. In the rat, it is composed of several proteins of which a 75 kDa polypeptide (FS 75) is the most prominent. The objectives of this study were to clone and sequence this protein and to characterize its transcriptional and translational origins during spermatogenesis. We succeeded in isolating two overlapping cDNAs encoding half of the downstream segment of the FS 75 protein. Both clones were obtained by screening a rat testicular phagemid cDNA library with an anti-FS 75 polyclonal antibody. The upstream portion of the FS 75 mRNA containing the initiation codon for translation was obtained by using the Polymerase Chain Reaction (PCR) technique, a pair of specific primers and a lambdagt11 cDNA rat library. The amino acid sequence of the longest possible open reading frame of the rat FS 75 was found to be almost identical to two previously cloned major FS polypeptides of mouse spermatozoa. Sequence analysis of the rat FS 75 cDNA revealed two A&barbelow;-K&barbelow;inase A&barbelow;nchoring P&barbelow;rotein (AKAP) domains and several kinase phosphorylation sites supporting the idea that this protein plays a crucial role in the motility of spermatozoa. The presence of a potential N-mistoylation site suggests that this protein may bind covalently to the inner leaflet of the plasma membrane (PM) which may explain the close relationship between the FS and PM from early development in round spermatids (step 2 of spermiogenesis) to maturation in spermatozoa. Developmental Northern Blot analysis and in situ hybridization revealed that the FS 75 mRNA is mainly haploid expressed with an abundant level of mRNA in round spermatids. Maximum levels of the FS 75 polypeptide, determined by immunocytochemistry, correlated with a rapid decline in corresponding mRNA levels in step 14--16 spermatids. Since transcriptional termination occurs several steps earlier
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.20592 |
| Date | January 1998 |
| Creators | Moshonas, Demetra. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Anatomy and Cell Biology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001605529, proquestno: MQ44226, Theses scanned by UMI/ProQuest. |
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