Protein tyrosine kinase expression in the porcine ovary.

Various growth factor receptors contain intrinsic tyrosine kinase activity, indicating that protein tyrosine kinases (PTK) play an important role in signal transduction pathways for cell proliferation and differentiation. To identify oocyte-derived factors which control follicle cells as well as oocyte-controlling factors produced by follicle cells, we examined the expression of genes which contain the PTK domain in the porcine ovary, using a polymerase chain reaction-based amplification technique with degenerate oligonucleotide primers that are specific to the PTK domain. Clones for the porcine homologues of platelet-derived growth factor receptor alpha (PDGFRalpha) and of insulin-like growth factor-I receptor (IGF-IR) were found during follicle growth both in oocytes and follicle cells. Clones for the porcine homologues of focal adhesion kinase (FAK), of c-kit and of fms-like tyrosine kinase (FLT)-3 were found only in oocytes. Moreover, after 24 h of in-vitro maturation of the cumulus-oocyte complexes, clones for the porcine homologues of FLT-1, of FLT-4, of Tie2 and of RYK in oocytes were observed. Immunohistochemical studies revealed the existence of PDGFRalpha, platelet-derived growth factor A (PDGFA), FAK and FLT3 in oocytes at various stages of folliculogenesis. These results suggest that fluctuations in the expression of these PTK genes may be involved in follicle growth and maturation.

Immunochemical characteristics of a novel cell death receptor and a decoy receptor on granulosa cells of porcine ovarian follicles.

Previously, we prepared an IgM monoclonal antibody(PFG-1) which specifically recognized a cell-membraneglycoprotein (PFG-1 antigen; 55 kD, pI 5.9),immunohistochemically reacted with granulosa cells ofhealthy follicles but not of atretic follicles, andinduced granulosa cell apoptosis. In the presentstudy, an IgM monoclonal antibody (PFG-3) capable ofinducing granulosa cell apoptosis and an IgGmonoclonal antibody (PFG-4) not capable of inducingapoptosis were produced against granulosa cellsprepared from healthy antral follicles of porcineovaries. Two-dimensional Western blotting analysisrevealed that PFG-3 specifically recognized twocell-membrane proteins (named PFG-3-1 andPFG-3-2/PFG-1 antigens; 42 kD, pI 5.2 and 55 kD, pI5.9, respectively) of healthy granulosa cells, andthat PFG-4 recognized the same two cell-membraneproteins. In atretic granulosa cells, PFG-3-2/PFG-1antigen disappeared. Immunochemical reactions of theseantibodies were only detected in follicular granulosacells but not any other ovarian tissues or organs.PFG-3 and PFG-4 immunohistochemically reacted withgranulosa cells of healthy and atretic follicles. Whenthe isolated granulosa cells prepared from healthyfollicles were cultured in medium containing PFG-3,the cells underwent apoptosis, and co-incubation withPFG-4 inhibited PFG-3-inducible apoptosis. Theseobservations suggested that PFG-3-2/PFG-1 antigen isa novel cell death receptor which is different fromthe apoptosis-mediating receptors (Fas/Apo-1/CD95 orTNF receptor), and that PFG-3-1 antigen may act as adecoy receptor and inhibit apoptotic signal transmission.

Monoclonal antibodies against pig ovarian follicular granulosa cells induce apoptotic cell death in cultured granulosa cells.

Two monoclonal antibodies capable of inducing granulosa cell apoptosis were produced against granulosa cells prepared from antral follicles of pig ovaries. The healthy follicles, 4-5 mm in diameter, were dissected from the ovaries of gilts, and then granulosa cells were isolated. BALB/c female mice were immunized with the isolated granulosa cells. Antibodies against the granulosa cells were detected by immunofluorescent staining using frozen ovarian sections. The isolated spleen cells prepared from immunized mice producing antibodies against the granulosa cells were fused with Sp2/O-Ag 14 mouse myeloma cells by standard hybridization techniques. Two hybridoma clones, PFG-1 and PFG-2, which produced specific IgM antibodies against granulosa cells were selected. Western blotting analysis revealed that PFG-1 and PFG-2 antibodies specifically recognized cell-membrane proteins with molecular weights of 55 and 70 kD and isoelectric points of 5.9 and 5.4, respectively. The monoclonal antibodies immunohistochemically reacted with granulosa cells of healthy follicles. When the isolated granulosa cells prepared from healthy follicles were cultured in medium containing 0.1 or 10 micrograms/m/PFG-1 or PFG-2 antibodies, respectively, the cells underwent apoptosis as determined by nuclear morphology, DNA electrophoresis and flow cytometric analysis. In conclusion, these two monoclonal antibodies against granulosa cells have cell-killing activity in cultured granulosa cells.