Monoclonal Antibodies against Pig Ovarian Follicular Granulosa Cells Induce Apoptotic Cell Death in Cultured Granulosa Cells

Abstract

ABSTRACT. 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-Ag14 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 µg/ml 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.-KEY WORDS: apoptosis, follicular granulosa cell, monoclonal antibody, swine ovary.

TRAIL-Decoy Receptor-1 Disappears in Granulosa Cells of Atretic Follicles in Porcine Ovaries.

“…We considere that TRAIL and its receptors play critical roles in induction and regulation of apoptosis in granulosa cells during follicular atresia of porcine ovaries, and that DcR1 may have a protective effect against the induction of apoptosis by TRAIL. Previous studies by other authors [15][16][17][18][19] as well as the findings of both the present and our previous studies [38][39][40] indicate that cell death ligands and receptors play critical roles in selective apoptosis of folicular cells during atresia. Our previous experiments using production of monoclonal IgM and IgG antibodies against unique cell-membrane proteins that exist only in granulosa cells of porcine ovaries indicated that other unknown receptor(s) may be involved in granulosa cell apoptosis [38][39][40].…”

“…Frozen sections of follicular sections of pig ovaries were prepared as described previously [38][39][40][41][42]. Briefly, individual preovulatory antral follicles, 3-5 mm in diameter, were dissected from ovaries obtained from mature sows at a slaughterhouse.…”

Species-specific Differences in Apoptotic Cell Localization in Granulosa and Theca Interna Cells during Follicular Atresia in Porcine and Bovine Ovaries.

“…However, it has not been determined which trigger molecules induce granulosa cell apoptosis, or how intercellular apoptotic signals are transmitted in the granulosa cells of atretic follicles [13]. To identify the trigger molecules for granulosa cell apoptosis and to define the molecular mechanism of apoptotic signal transmission pathways in the granulosa cells, we previously produced a specific monoclonal antibody that recognizes a novel cell death receptor, named PFG-1, expressed only on porcine granulosa cells and induces apoptosis in cultured granulosa cells [19][20][21]. PFG-1 is a transmembrane glycoprotein and belongs to the tumor necrosis factor/nerve growth factor receptor family, but is different from the Fas/APO-1/CD95 (Fas).…”

Apoptosis Occurs in Granulosa Cells but not Cumulus Cells in the Atretic Graafian Follicles in Multiparous Pig Ovaries.

“…The nature of the trigger molecule responsible for induction of granulosa cell apoptosis has not yet been determined, nor how the intercellular signals of apoptosis are transmitted in granulosa cells. Previously, we produced two specific monoclonal antibodies that react with cell-surface proteins of healthy granulosa cells [26]. When isolated granulosa cells prepared from healthy follicles of young gilts were co-cultured with these antibodies, apoptosis occurred in the cultured granulosa cells [26].…”