ABSTRACT
PHTF1 is an 84-86-kDa membrane protein found in the endoplasmic reticulum of male germ cells in rodents. There are no evident signs of PHTF1 in the spermatozoa released into the lumen of the seminiferous tubules but PHTF1 is present in the epididymal epithelium. Characterization of the epididymal Phtf1 messenger by Northern blot and reverse transcription-PCR identified a 3-kilobase transcript in the epididymis, similar to that previously reported in the testis. The transcript is present in the proximal part of the epididymis and it appears when the rats reach 4 wk of age. Through immunofluorescence analysis, PHTF1 was localized in the principal cells of the initial segment and the caput epididymis. Colocalization with different markers indicated PHTF1 is in the endoplasmic reticulum saccules applied to the trans face of the Golgi system. Western blot analyses revealed a shorter form of the protein--about 56-kDa versus the 84-kDa form found in the testis. Using the canine epididymal cell line CIM 20, transfected by N- and C-terminal myc-tagged PHTF1, we demonstrated that the 56-kDa epididymal form could result from proteolytical processing.
Subject(s)
Epididymis/physiology , Gene Expression Regulation, Developmental , Homeodomain Proteins/metabolism , Transcription Factors/metabolism , Animals , Animals, Newborn , Blotting, Western , Cells, Cultured , Dogs , Epididymis/cytology , Epithelial Cells/immunology , Epithelial Cells/metabolism , Gene Expression Regulation , Homeodomain Proteins/genetics , Homeodomain Proteins/immunology , Male , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Isoforms/metabolism , Protein Processing, Post-Translational , Rats , Rats, Sprague-Dawley , Receptors, Prostaglandin E/genetics , Receptors, Prostaglandin E, EP2 Subtype , Testis/physiology , Transcription Factors/genetics , Transcription Factors/immunologyABSTRACT
The Phtf1 gene encodes a membrane protein abundantly expressed in male germinal cells. Using a two-hybrid screening procedure we have identified FEM1B, an ortholog of the C. elegans feminization factor 1 (FEM-1), as a binding partner for PHTF1. We studied FEM1B expression in the rodent testis and found that Fem1b mRNA is present at high levels during meiosis and after, during spermiogenesis, in a similar manner to Phtf1 mRNA. Accordingly, Western blot and immunofluorescence revealed the presence of PHTF1 and FEM1B in the same cell types, and by coimmunoprecipitation we demonstrated the association between these proteins. We characterized some aspects of this interaction and showed that the ANK domain of FEM1B is necessary for the interaction with the amino extremity of PHTF1. Next, we found that FEM1B can bind several intracellular organelles and demonstrated that PHTF1 would recruit FEM1B to the endoplasmic reticulum membrane. Previous in vitro experiments had suggested that the human FEM1B was involved in apoptosis. After comparing expression profiles of FEM1B and PHTF1 with apoptotic events occurring in the normal seminiferous tubules, we suggest that neither FEM1B nor PHTF1 are directly implicated in apoptosis in this tissue.
Subject(s)
Apoptosis/physiology , Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Homeodomain Proteins/metabolism , Spermatozoa/cytology , Spermatozoa/metabolism , Transcription Factors/metabolism , Animals , Carrier Proteins/genetics , Cell Cycle Proteins/genetics , Cell Membrane/metabolism , Cells, Cultured , Endoplasmic Reticulum/metabolism , Homeodomain Proteins/genetics , Male , Mice , Mice, Inbred Strains , RNA, Messenger/analysis , Rats , Rats, Wistar , Seminiferous Tubules/cytology , Seminiferous Tubules/metabolism , Spermatogenesis , Transcription Factors/genetics , Transfection , Ubiquitin-Protein Ligase ComplexesABSTRACT
Phtf1 is a gene evolutionarily conserved from Drosophila to human that is abundantly expressed in testis. In adult rat, transcripts were abundant in germinal meiotic and postmeiotic cells. Phtf1-specific antibodies revealed weak activity in a juxtanuclear region of early pachytene spermatocytes. Labeling progressively extended to the entire cytoplasm of step 2-3 spermatids, became intense from step 4, and persisted until the end of spermiogenesis, when it was eliminated in the residual bodies. Phtf1 displayed the properties of an integral membrane protein. In transfected cells and haploid cells of rat seminiferous epithelium, it colocalized with ER markers (calnexin and calmegin, respectively). By using both ER and Golgi markers (TGN-38, p58), we were able to show that, in pachytene spermatocytes and in Golgi phase spermatids, phtf1 labeled a region neighboring the cis-Golgi that probably corresponded to the peripheral Golgi region. Phtf1 staining was not related to beta-COP, AP1, or AP2 aptamers, indicating that it was not transported between Golgi saccules or between the Golgi complex and plasma membrane. However, aptamer labeling showed that chlatrin vesicles could be engaged in a new traffic route, raising the possibility of a meiotic proacrosomal vesicle origin. Colocalization between phtf1 and calmegin decreased during the acrosomal phase. During the maturation phase, phtf1 was able to identify different ER domains, as described previously for the peripheral Golgi region. Phtf1 provides a potential new marker for Golgi modifications as well as for many of the obscure transformations undergone by the endoplasmic reticulum. It could help to elucidate the morphogenic events connected with the transformation of spermatogenic cells.
