Journal of Reproduction and Development, Vol. 48, No. 2, 2002 —Original— TRAIL-Decoy Receptor-1 Disappears in Granulosa Cells of Atretic Follicles in Porcine Ovaries Satoko WADA1), Noboru MANABE1), Naoko INOUE1), Mizuho NAKAYAMA1), Toshikatsu MATSUI1) and Hajime MIYAMOTO1) 1) Unit of Anatomy and Cell Biology, Department of Animal Sciences, Kyoto University, Kyoto 606-8502, Japan Abstract. To reveal the specific regulatory molecules that control granulosa cell apoptosis during follicular atresia, we immunohistochemically examined the localization of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its receptors in porcine ovaries. A marked reduction in the expression of decoy receptor-1 (DcR1), which has high affinity for TRAIL, was demonstrated in granulosa cells of atretic follicles, but no marked differences were seen in expression of TRAIL or other TRAIL-receptors (death receptor-4 or death receptor-5) in granulosa cells between healthy and atretic follicles. No positive staining for DcR2 was seen. We presum that TRAIL and its receptors are involved in induction of apoptosis in granulosa cells during atresia, and that DcR1 plays an inhibitory role in granulosa cell apoptosis. Key words: Apoptosis, TRAIL-decoy receptor, Follicular atresia, Granulosa cell, Pig (J. Reprod. Dev. 48: 167–173, 2002) n mammalian ovaries, more than 99% of the [15–19]. However, it has not been determined follicles undergo a degenerative change known whether the FasL-Fas system mediates apoptosis in as atresia at varying stages of follicle development pig ovaries. Previously, we revealed species- [1, 2]. A number of studies of follicular atresia have specific differences in the apoptotic process in revealed the morphological and biochemical granulosa cells and indicated that local mechanisms characteristics of atretic follicles [3, 4]. Recent of regulation of granulosa cell apoptosis may be findings have suggested that apoptosis, originally different among mammalian species [20]. described by Kerr et al. [5], is the mechanism As apoptotic stimuli and intracellular signal underlying ovarian follicular atresia [6, 7]. The transduction pathways involved in granulosa cell degeneration of atretic follicles in porcine ovaries apoptosis remain to be determined. We have been can be explained, at least in part, by apoptosis of studying which trigger molecules induce granulosa granulosa cells [8–11]. We previously confirmed cell apoptosis, and how intercellular apoptotic that granulosa cells undergo apoptosis but that no signals are transmitted in granulosa cells. A novel apoptosis occurs in cumulus cells during follicular cell death ligand, TNF-related apoptosis-inducing atresia in porcine ovaries [12–14]. In rodents, Fas ligand (TRAIL; also known as Apo-2 ligand), a ligand (FasL) and its receptor Fas/APO-1/CD95 cytotoxic cytokine homologous to FasL, was (Fas) may regulate granulosa cell apoptosis in identified in 1995 [21–23]. Apoptosis mediated by ovarian follicle atresia and luteal cell degeneration TRAIL is regulated by the expression of two death receptors, death receptor-4 (DR4; also known as Accepted for publication: December 8, 2001 Correspondence: N. Manabe TRAIL-R1) and death receptor-5 (DR-5; also known 168 WADA et al. as TRAIL-R2 or TRICK 2), and three unique decoy 3-aminopropyltriethoxysilane (Sigma Aldrich receptors, decoy receptor-1 (DcR1; also known as Chemicals, St. Louis, MO, USA). The sections were TRAIL-R3, LIT or TRID), decoy receptor-2 (DcR-2; fixed with precooled acetone for 5 min at – 80 C. also known as TRAIL-R4 or TRUNDD) and After washing with PBS (pH 7.4; Wako), the sec- osteoprotegerin (OPG), that inhibit apoptosis [24– tions were preincubated with diluted normal rabbit 36]. DcR1 and DcR2 have two extracellular serum (1/200 dilution with PBS; Wako) for 10 min cysteine-rich domains which show close homology at RT. Goat polyclonal antibodies against human to those of DR4 and DR5. TRAIL-induced TRAIL, DR4, DR5, DcR1 and DcR2 (Santa Cruz Bio- apoptosis was inhibited by cellular transfection technology, Santa Cruz, CA, USA) were diluted at with DcR1 or DcR2 [32]. Although the biological 1/150 with PBS containing 40 mg/ml BSA (PBS- functions of the TRAIL receptor system are largely BSA; Sigma), and then applied to the sections. The unknown, this system has been suggested as sections were incubated with each first antibody for contributing to the selective abolishment of 18 h at 4 C, washed with PBS containing 0.05% unnecessary cells under physiological conditions Tween 20 (PBS-Tw; Sigma), and then incubated [37]. with 25 µg/ml of FITC-conjugated rabbit anti-goat In the present study, to determine the phys- IgG (American Qualex, La Mirada, CA, USA) iological roles of TRAIL and its receptors on diluted with PBS-BSA containing 20 µg/ml of pro- granulosa cell apoptosis in porcine ovarian follicles, pidium iodide (PI; Sigma) for 90 min at RT for we immunohistochemically examined the changes nuclear staining. They were washed with PBS-Tw, in localization of TRAIL and its receptors in porcine mounted with glycerol, and then examined with a follicles during follicular atresia. confocal laser scanning microscope (FV3000, Olympus). Materials and Methods Statistical analysis All experiments involving follicle isolation were Immunohistochemistry for TRAIL and its receptors repeated with separate groups (six sows/group) Frozen sections of follicular sections of pig for independent observation. Wilcoxon’s signed- ovaries were prepared as described previously rank test for histological estimation was carried out [38–42]. Briefly, individual preovulatory antral using StatView IV on a Macintosh computer. follicles, 3–5 mm in diameter, were dissected from Differences at P<0.05 were considered significant. ovaries obtained from mature sows at a slaughter- house. Under a surgical dissecting microscope (SZ40, Olympus, Tokyo, Japan), the follicles were Results classified as morphologically healthy or atretic, and further subdivided into early and progressed TRAIL and its receptors, DR4, DR5, DcR1 and atretic follicles [40]. A part of the follicular fluid of DcR2, in frozen sections of healthy and atretic each follicle was collected by a 1-ml syringe. Fluid follicles of porcine ovaries were visualized by the from each follicle was separated by centrifugation immunofluorescence technique, and nuclear at 3,000 g for 10 min, and then estradiol-17β and morphology was visualized by PI staining. Positive progesterone levels in the follicular fluid were signals for TRAIL (Fig. 1A and C), DR4 (Fig. 1E and quantified using [125I]-RIA kits (Bio-Mèrieux, G) and DR5 (Fig. 2A and C) were observed in Marcy-l'Etolle, France). When the progesterone/ granulosa cells and the cells of theca internal and estradiol-17β ratio was less than 15, the follicle was external layers of both healthy (Fig. 1A, E and 2A) classified as healthy according to our previous find- and atretic follicles (Fig. 1C, G and 2C). Intense ings [9–14]. Then, each follicle was put on filter staining for TRAIL was seen in granulosa cells paper, mounted in OTC compound (Miles Lab., lining the antral cavity of healthy antral follicles Elkhart, IN, USA), and rapidly frozen in dry ice-iso- (Fig. 1A) and those separating the antral cavity pentane (Wako Pure Chemical, Osaka, Japan) from the basement membrane of atretic follicles mixture. Serial sections 5 µm thick were cut on a (Fig. 1C). Extremely strong immunoreactivity for cryostat (Jung CM1500; Leica, Heidelberger, Ger- DR4 was detected in granulosa cells of both healthy many), and mounted on glass slides precoated with and atretic follicles (Fig. 1E and G) and theca TRAIL RECEPTORS IN PIG GRANULOSA CELLS 169 Fig. 1. Localization of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and TRAIL-death receptor-4 (DR4) in the sections of healthy (A, B, E and F) and atretic (C, D, G and H) follicles of pig ovaries. The frozen sections were double-stained with anti-TRAIL (A and C) and -DR4 (E and G) antibodies and with propidium iodide (B, D, F and H). G, granulosa layer; BM, basement membrane; TI, theca interna layer. × 200. interna layers of atretic follicles (Fig. 1G). Intense D, F and H). Large oval nuclei were seen in staining for DR5 was detected in granulosa cells granulosa cells of healthy follicles (Fig. 1B and F, lining the antral cavity of healthy follicles (Fig. 2A) and 2B and F), but many small condensed nuclei, a and those suspended in the cavity of atretic follicles morphological hallmark of apoptotic cells, were (Fig. 2C), but weak signals were seen throughout seen in granulosa cells of atretic follicles (Fig. 1D the theca interna and externa layers in both follicles. and H, and 2D and H). Positive staining for DcR1 was demonstrated in granulosa cells and theca internal and external layers of healthy follicles (Fig. 2E). Intense positive Discussion immunostaining was seen in granulosa cells located on the inner surface of the follicular wall. In Over the last decade, several cell death ligands atretic follicles, however, no positive signal for (TNF-α, FasL, TRAIL etc.) and their receptors DcR1 was observed in granulosa cells, but scattered (TNFR1, Fas, DR4, DR5, DcR1, DcR2 etc.) have been weak signals were seen throughout the theca found [24–26, 43–45]. Recent studies have internal and external layers (Fig. 2G). No positive increased our understanding of selective apoptotic staining for DcR2 was shown (data not shown). cell death under both physiological and pathological Nuclear morphology was examined by PI staining conditions through the cell death ligand-receptor in the frozen sections (Fig. 1B, D, F and H, and 2B, interaction. Most follicles selectively undergo 170 WADA et al. Fig. 2. Localization of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-death receptor-5 (DR5) and TRAIL-decoy receptor -1 (DcR1) in sections of healthy (A, B, E and F) and atretic (C, D, G and H) follicles of porcine ovaries. The frozen sections were double-stained with anti-DR5 (A and C) and -DcR1 (E and G) antibodies and with propidium iodide (B, D, F and H).