Immunolocalization of the Oostatic and Prothoracicostatic Peptide, Neb-TMOF, in Adults of the Fleshfly,Neobellieria bullata

Abstract

The hexapeptide Neb-TMOF (H-NPTNLH-OH, trypsin modulating oostatic factor of the gray fleshfly,Neobellieria bullata)22Abbreviations used: BB, brilliant blue; BFB, Bromophenol blue; BSA, bovine serum albumine; CFA, complete Freund's adjuvant; ELISA, enzyme-linked immunosorbent assay; IE, immunogen enhancer; IFA, incomplete Freund's adjuvant; PAGE, polyacrylamide gel electrophoresis; PTSH, prothoracicostatic hormone; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline; TMOF, trypsin modulating oostatic factor.occurs in vitellogenic ovaries and is involved in negative feedback regulation of trypsin biosynthesis in the gut of late vitellogenic females. Polyclonal antisera were raised against the synthetic peptide and were used to identify and immunolocalize Neb-TMOF epitopes in different fleshfly tissues. Neb-TMOF-immunoreactive material first appears in the cortical layer of young vitellogenic oocytes and later spreads over the yolk granules. This suggests a pinocytosis with the three yolk polypeptides (vitellogenins). Controls treated with the preimmune sera or with anti-Neb-TMOF antiserum preadsorbed to Neb-TMOF peptide coupled to a solid phase support did not stain. There was no immunostaining in the central nervous system (brain and ventral nerve cord), the retrocerebral complex, the fat body, or the testes. Western blot analysis showed that the anti-Neb-TMOF antisera specifically recognize a putative hormone precursor polypeptide (Mr75 kDa) from vitellogenic ovaries. This protein is virtually absent from the hemolymph. It is not immunologically related to the three yolk polypeptides, since it is not recognized by yolk polypeptides antisera. In adult females the ovary appears to be the only site of synthesis of Neb-TMOF and of its precursor. Immunopositive staining is found in the apical areas of ovarian follicle cells, suggesting these cells as a site of hormone precursor biosynthesis. This is the first demonstration that a protein colocalized with yolk proteins might act as a precursor for a folliculostatic hormone.