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Ovary |
, TNF Receptor (TNFR) 2, and TNFR-Associated Factor 2 in Granulosa Cells During Atresia in Pig Ovaries1
a Unit of Anatomy and Cell Biology, Department of Animal Sciences, Kyoto University, Kyoto 606-8502, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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can induce both cell death and cell proliferation and exerts its effects by binding to either TNF receptor (TNFR) 1 or 2. When TNF-bound TNFR2 interacts with TNFR-associated factor 2 (TRAF2), expression of survival/antiapoptotic genes is up-regulated. In the present study we determined the changes in localization of TNF and TRAF2 and their mRNAs and the expression of TNFR2 in granulosa cells during follicular atresia in pig ovaries. In healthy follicles, intense signals for TNF and TRAF2 and their mRNAs were demonstrated in the outer zone of the granulosa layer, where many proliferating cells and no apoptotic cells were observed. In atretic follicles, decreased or trace staining for TRAF2 and its mRNA and decreased expression of TNFR2 were observed in the granulosa layer, where many apoptotic cells were seen. These findings suggested that TNF acts as a survival factor in granulosa cells during follicular atresia in pig ovaries.
apoptosis, follicle, granulosa cell, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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can induce both cell death and cell proliferation and exerts its effects by binding to TNF receptors (TNFRs) 1 and 2 [1, 2]. TNF induces apoptosis in a variety of tumor cells, and the death signal pathway has been suggested to be as follows [3–6]: 1) TNF binds to the extracellular domain of TNFR1, which contains an intracellular death domain (DD); 2) the intracellular DD of the receptor interacts with the DD of the adaptor protein (TNFR-associated DD protein; TRADD); 3) the DD of TRADD binds with the DD of another adaptor protein (Fas-associated DD protein; FADD); 4) FADD activates initiator caspase (procaspase-8); and 5) the caspase cascade is activated for intracellular transduction of the apoptotic signal (named the TNFR1-TRADD-FADD-caspase-8 signaling axis). In contrast, when TNF acts as a survival/antiapoptotic factor [3–6], 1) TNF binds to the extracellular domain of TNFR1, 2) the intracellular DD of the receptor interacts with the DD of TRADD, 3) the DD of TRADD binds with the DD of the receptor interacting protein (RIP), 4) RIP interacts with TNFR-associated factor-2 (TRAF-2), and 5) TRAF2 mediates the physical interaction of the TNFR1 signaling complex with the nuclear factor (NF)-
B-inducing inhibitor of B kinase (IKK) and the inhibitor of apoptosis proteins (cIAP) 1 (named the TNFR1-TRADD-RIP-TRAF2 signaling axis), and consequently, expression of survival/antiapoptotic genes is up-regulated. Thus, TRADD-RIP are key proteins at the point of divergence of cell death and cell proliferation in the TNFR1 signaling process, and TRAF2 is a good indicator of TNF-dependent cell proliferation.
Although TRAF2 cannot bind directly to TNFR1 as described above, it can interact directly with TNFR2, which is a non-DD-containing TNF receptor. TNFR2 can induce gene transcription for cell survival, growth, and differentiation, and therefore TNFR2 is involved in the antiapoptotic effect of TNF [2, 6]. Briefly, 1) TNF binds to the extracellular domain of TNFR2, 2) activated TNFR2 interacts with TRAF2, 3) NF-B is activated, and 4) consequently, expression of survival genes is up-regulated and apoptosis induced by TNF is prevented [3–6]. Thus, TRAF2 expression is considered to be a good indicator of TNF-dependent cell proliferation in both TNFR1 and TNFR2 signaling cascades [7].
Recently, Prange-Kiel et al. [8] showed that TNF induced both cell proliferation and cell death in primary cultured granulosa cells prepared from large antral follicles of pig ovaries. However, there has been no in vivo confirmed evidence of whether TNF acts as an apoptotic factor or as a survival/antiapoptotic factor in porcine ovarian follicles. In the present study, to determine the physiological roles of TNF and its receptor system in granulosa cell apoptosis, a key phenomenon in follicle selection during follicular atresia, we histochemically analyzed the changes in localization of TNF and TRAF2, a key adaptor protein, in granulosa cells during follicular atresia in pig ovaries. We examined the changes in localization and levels of expression of TNF and TRAF2 mRNAs by in situ hybridization, by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, or both. Moreover, the changes in expression of TNFR2 in granulosa cells during follicular atresia were examined by Western blot analysis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For immunohistochemical staining and TUNEL staining, the ovaries obtained from mature sows at a local slaughterhouse were immersed in 20% phosphate-buffered formalin (pH 7.4; Wako Pure Chemicals, Osaka, Japan), dehydrated through a graded ethanol series, and embedded in Histosec (Merck, Darmstadt, Germany). Serial sections 3 µm thick were cut on a microtome and mounted on glass slides precoated with 3-aminopropyltrimethoxysilane (Sigma Aldrich Chemicals, St. Louis, MO).
For in situ hybridization, small tissues were placed on filter paper, mounted in OCT compound (Miles Laboratory, Elkhart, IN), and then rapidly frozen in a dry ice-isopentane mixture. Serial sections 6 µm thick were cut on a cryostat (CM1500; Leica, Heidelberg, Germany) and mounted on glass slides precoated with 3-aminopropyltriethoxysilane (Sigma).
For immunohistochemical staining for TNF, TRAF2, and proliferating cell nuclear antigen (PCNA), serial paraffin sections were deparaffined, rehydrated, washed in distilled water, and immersed in 0.3% hydrogen peroxide in methanol for 30 min at room temperature (22–25°C). After washing with 0.01 M PBS pH 7.4, sections were preincubated with normal goat or horse serum (1:100 dilution with PBS containing 5% BSA:PBS-BSA) for 20 min at room temperature, rinsed with PBS-BSA, and then incubated with each primary antibody for 18 h at 4°C. Primary antibodies were as follows: rabbit anti-TNF polyclonal antibody (1:1000 dilution with PBS-BSA; Genzyme, Cambridge, MA), rabbit anti-TRAF2 polyclonal antibody (1:100 dilution with PBS-BSA; Santa Cruz Biotechnology, Santa Cruz, CA), and mouse anti-PCNA monoclonal antibody (1:1000 dilution with PBS-BSA; Sigma). Subsequently, the sections were washed with PBS-BSA and incubated with biotinylated secondary antibody (goat anti-rabbit immunoglobulin G [IgG] antibody for TNF and TRAF2, and horse anti-mouse IgG antibody for PCNA) for 30 min at room temperature. Immunoreactivity was visualized using a VectaStain avidin-biotin-peroxidase kit (Vector Laboratories, Burlingame, CA) for TNF and PCNA using a catalyzed signal amplification kit (DAKO, Carpinteria, CA) for TRAF2 according to the respective manufacturer's protocol. Sections were counterstained with methyl green, washed, dehydrated, mounted with Eukitt (Kindler, Freiburg, Germany), and then examined with a microscope (BX51; Olympus, Tokyo, Japan). In each experimental run, adjacent sections incubated without primary antibody or without any antibodies were prepared as negative controls.
As described previously [9–12], to visualize apoptotic cells, adjacent sections from each specimen were stained by the TUNEL method using an ApopTag kit (Intergen, New York, NY) according to the manufacturer's protocol. The following positive and negative controls were included in each experimental run. As negative controls, sections were incubated by omitting either terminal deoxynucleotidyl transferase or antidigoxigenin (anti-DIG) antibody. As a positive control, sections were treated with DNase I (1 µg/ml; Boehringer-Mannheim, Indianapolis, IN), 140 mM sodium cacodylate, 4 mM MgCl2, and 0.1 mM dithiothreitol in 30 mM Tris-HCl pH 7.2 for 10 min at room temperature. Paraffin sections prepared from young adult rat testis were used as physiological positive controls.
In situ hybridization for each TNF and TRAF2 mRNA was performed according to the method described in our previous report [13]. Briefly, frozen sections were fixed with 4% paraformaldehyde in PBS (PFA-PBS), treated with 0.2 N HCl, and digested with proteinase-K (1 µg/ml; Sigma). They were postfixed with 4% PFA-PBS, rinsed with PBS, prehybridized with hybridization solution (50% deionized formamide in 10 mM Tris-HCl pH 7.4, 1 mM EDTA, 600 mM NaCl, 1x Denhardt solution, 10 mg/ml yeast tRNA, 10 mg/ml salmon testicular DNA, and 5% dextran sulfate; Sigma) for 2 h at room temperature, and then hybridized with each DIG-labeled oligo-DNA probe for mRNA (1 µg/ml diluted with hybridization solution) for 18 h at 37°C. Sequences of DNA probes for TNF, TRAF2, and 28S rRNA (positive control) were 5'-GGATC ATCTT CTCGA ACCCC GAGTG ACAAG-3', 5'-GTTCA GGACG CAGAC AATGT TCT-3', and 5'-TGCTA CTACC ACCAA GATCT GCACC TGCGG CGGC-3', respectively. The sections were washed with 2x saline-sodium citrate (SSC) for 2 h, with 0.5x SSC for 2 h, and with 0.2x SSC for 1 h at 37°C, and rinsed with 100 mM Tris-HCl pH 7.5 containing 150 mM NaCl (THS), and then immersed in blocking solution (Boehringer) for 1 h at room temperature. They were incubated with alkaline phosphatase (AP)-conjugated sheep anti-DIG antibody (Boehringer; diluted 1:500 with the blocking solution) for 18 h at 4°C. After washing with THS, sections were equilibrated with 100 mM Tris-HCl pH 9.5, 100 mM NaCl, and 100 mM MgCl2 (THSM), and then incubated with THSM containing 0.4 mM nitroblue tetrazolium chloride, 0.4 mM 5-bromo-4-chloro-3-indolyl-phosphate-4-toluidine salt and 1 mM levamisole (DAKO) for 3 h at 4°C. They were washed with PBS, mounted with Histofine (Nichirei, Tokyo, Japan), and examined with a microscope. As negative controls, serial sections were incubated with sense probe, without antisense probe, or without anti-DIG antibody.
Western Blot Analysis and RT-PCR Analysis
As reported previously [13], individual preovulatory antral follicles 3–5 mm in diameter were dissected from the ovaries. With a surgical dissecting microscope (SZ40, Olympus), follicles were classified as morphologically healthy or atretic, and further subdivided into early and progressed atretic follicles (25 follicles/each group). A sample of follicular fluid from each follicle was collected using a 1-ml syringe, separated by centrifugation at 3000 x g for 10 min at 4°C, and then estradiol-17ß and progesterone levels were measured using [125I] radioimmunoassay kits (Bio-Mérieux, Marcy-l'Etolle, France) to confirm follicle classification. Follicles with a progesterone:estradiol-17ß ratio of less than 15 were classified as healthy according to our previous findings [9, 10]. Then, each follicle was opened using fine forceps, and oocyte-cumulus complexes were removed. The granulosa cells were collected, washed in PBS by centrifugation, and used for Western blot and RT-PCR analyses.
For Western blot analysis, the protein fraction (20 µg/lane) prepared from each sample as previously reported [9] was separated by 7.5% SDS-PAGE, and then transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ). The membranes were stained with 0.2% Ponceau S solution (Serva Electrophoresis, Heidelberg, Germany), immersed in blocking solution (0.1 M Tris HCl pH 7.6, 5% skim milk, 0.05 M NaCl, 0.001% Tween-20; Sigma), and incubated with rabbit anti-TNFR2 polyclonal antibody (Upstate Biotechnology, Lake Placid, NY; 1:200 dilution with 0.1 M Tris-HCl) for 18 h at 4°C. After washing, they were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Jackson Immunoresearch Laboratories Inc., West Grove, PA) for 1 h at room temperature, and then chemiluminescence was visualized using an enhanced chemiluminescence system (Amersham) according to the manufacturer's protocol. Chemiluminescence was recorded with a digital recorder (LAS-1000; Fiji Film, Tokyo, Japan) and then protein expression levels were quantified using ImageGauge (Fiji Film) on a Macintosh computer.
As described previously [12, 13], for RT-PCR analysis total RNA was extracted from granulosa cell samples using an RNeasy mini kit (Qiagen, Chatsworth, CA) treated with an RNase-free DNase kit (Qiagen), and then reverse-transcribed using a T-primed first-strand kit (Amersham) to synthesize cDNA. After RT, cDNA was mixed with the PCR mixture containing 10x PCR buffer, 0.1 mM dNTP mixture, 1.5 mM MgCl2, 0.5 µM of each primer pair, and 0.00625 units of platinum Taq DNA polymerase (Gibco BRL, Grand Island, NY). Primer pairs specific for partial cDNA sequences of TRAF2 (GenBank accession number HSU12597) were used: forward, 5'-AGATT GAAGC CCTGA GTAGC AAGG-3' and reverse, 5'-ATCAC CACAA AGAAG AGGGA CAGG-3'. The expected PCR product size was 304 base pairs (bp). Glyceraldehyde dehydrogenase (GAPDH; GenBank accession number AF017079) was amplified as an intrinsic control using the following primers: forward, 5'-GATGG TGAAG GTCGG AGTG-3' and reverse, 5'-CGAAG TTGTC ATGGA TGACC-3'; expected PCR product size, 500 bp. The mixture was subjected to PCR in a thermal cycler (GeneAmp PCR Systems 2400; PE Applied Biosystems, Foster City, CA). Hot start-PCR conditions for TRAF2 were 96°C for 4 min, 40 cycles at 94°C for 45 sec, 63°C for 45 sec, 72°C for 1 min, and then 1 cycle at 72°C for 5 min. Those for GAPDH were 2 cycles at 96°C for 1 min, 63°C for 4 min, 25 cycles at 94°C for 1 min, 63°C for 2.5 min, 72°C for 1 min, and then 1 cycle at 72°C for 5 min. PCR products were electrophoresed in 2% agarose gels and stained with ethidium bromide (Wako). A ready-load 100 bp DNA ladder (Gibco) was used as a molecular weight marker for electrophoresis. After electrophoresis the gels were recorded with a digital recorder, and then mRNA expression levels were semiquantified using ImageGauge on a Macintosh computer. The relative abundance of specific mRNA was normalized to the relative abundance of GAPDH mRNA. To confirm the expression of TRAF2 mRNA, the DNA sequence of the PCR product was determined using an automatic DNA sequencer (ABI prism 340; PE Applied Biosystems) according to the manufacturer's protocol.
Statistical Analysis
All experiments involving follicle isolation were repeated with separate groups (nine sows/group) for independent observation. ANOVA with the Fisher least significant differences test for biochemical data and the Wilcoxon signed-rank tests for histological estimation were carried out using StatView IV on a Macintosh computer. Differences at P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In healthy follicles, many PCNA-positive proliferating cells were observed in the granulosa layer, especially in the outer surface zone (Fig. 1A). Some PCNA-positive cells were detected among cumulus cells and in theca interna and externa layers (data not shown). In atretic follicles, however, no PCNA-positive cells were seen in the granulosa layer (Fig. 1B). In contrast, no TUNEL-positive apoptotic cells were observed in healthy follicles (Fig. 1C). Similar to our previous studies [9–11], apoptosis occurred in granulosa cells located on the inner surface of the follicular wall in the follicles at the early stage of atresia (Fig. 1D).
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In healthy follicles, strong immunoreactivity for TNF was observed in the outer surface zone of the granulosa layer (Fig. 1E) and in the theca externa layer, and weak immunoreactivity was observed in the theca interna layer. Decreased immunoreactivity was observed in early and progressed atretic follicles (Fig. 1F). Similar staining patterns were observed for the localization of TRAF2 (Fig. 1, G and H). In healthy follicles, strong immunoreactivity with anti-TRAF2 antibody was observed on the outer surface zone of the granulosa layer and theca externa layer (Fig. 1G). In follicles at the early atretic stage, no immunoreactivity for TRAF2 was observed in the granulosa layer but strong signals were observed in the theca interna layer (Fig. 1H). No positive staining for TRAF2 was observed in progressed atretic follicles (data not shown).
In healthy follicles, positive signals for TNF mRNA detected by in situ hybridization were observed in granulosa cells and theca interna cells (Fig. 2A). Strong staining was observed in the outer surface zone of the granulosa layer and in theca interna cells located close to the basement membrane. Trace or no expression of TNF mRNA was observed in granulosa cells located on the inner surface of the follicular wall. In atretic follicles, no positive staining for TNF mRNA was observed in granulosa cells, and trace or weak staining was observed in the theca interna layer (Fig. 2B). The same expression pattern was observed for TRAF2 mRNA (Fig. 2, C and D). In healthy follicles, strong staining for TRAF2 mRNA was observed in the outer surface zone of the granulosa layer and in the inner surface zone of the theca interna layer (Fig. 2C). No positive staining for TRAF2 mRNA was demonstrated in the granulosa cells of atretic follicles, and trace staining was observed in the theca interna layer (Fig. 2D).
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Western Blot Analysis
Equal protein loading per lane was verified by staining the membranes with Ponceau S (Fig. 3B). TNFR2 was found in granulosa cells prepared from healthy follicles, and slightly decreased expression was observed in those from early atretic follicles (Fig. 3A). In granulosa cells from progressed atretic follicles, however, TNFR2 was not detected. Changes in the chemiluminescence levels quantified using an automatic image analyzer showed that TNFR2 expression decreased and finally disappeared during follicular atresia (Fig. 3C).
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RT-PCR Analysis
The sequence of the PCR product for the corresponding domain of TRAF2 was determined using an automatic DNA sequencer, indicating that the PCR product corresponded to TRAF2. TRAF2 and GAPDH mRNAs were detected in granulosa cells of healthy, early atretic, and progressed atretic follicles (Fig. 4A). Semiquantitative RT-PCR analysis for TRAF2 mRNA in isolated granulosa cells was performed (Fig. 4B). A high TRAF2:GAPDH ratio was observed in granulosa cells of healthy follicles. Decreased expression of TRAF2 mRNA was observed in those of early atretic follicles, and extremely low expression of TRAF2 mRNA was demonstrated in progressed atretic follicles.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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is produced by activated monocytes/macrophages, lymphocytes, and endothelial cells; it binds with TNFR1, which has been shown to be present in various normal cells, or TNFR2, which has been detected only in specific cell types, and can induce both cell death and cell proliferation [1, 2]. Prange-Kiel et al. [8] studied the effect of TNF on cultured granulosa cells obtained from preovulatory follicles of pig ovaries and showed that TNF treatment up-regulates TNFR type I expression and down-regulates the expression of transforming growth factor ß receptor type II, a marker of luteinization, and that TNF leads to increased proliferation, and at the same time, it induces apoptosis. Thus, their results indicated that TNF exerts an inhibitory influence on luteinization and that TNF influences the balance between follicular growth (proliferation) and atresia (apoptosis). TNF inhibits estradiol-17ß secretion by FSH-stimulated human granulosa cells [14]. Expression of TNFR2, but not TNFR1, was demonstrated in human ovaries [15]. We detected expression of TNFR2 in granulosa cells of healthy follicles of pig ovaries, and TNFR2 disappeared during follicular atresia. Recent studies have indicated that TRAF proteins, which are recruited by both TNFR1 and TNFR2, control TNF function [3–7]. The effector proteins of TRAF proteins are transcription factors (NF-B and the AP-1 family) that can turn on numerous genes involved in various aspects of cellular and immune functions, and that activation of NF-B and AP-1 protects the cells from apoptosis via the transcription of antiapoptotic genes. TRAF2, a member of the TRAF protein family, is involved in TNFR-mediated activation of NF-B and JNK, and the resulting survival/antiapoptotic signal is transduced [3–7, 15–17], and TRAF2 expression is a good marker of TNF-dependent cell proliferation in TNFR1 and TNFR2 signaling cascades.
To date there has been no firm evidence regarding whether TNF acts as a survival factor in the follicles of pig ovaries. Moreover, no information is available regarding whether the TNFR-TRAF signal transduction system exists in porcine follicles. In the present study, the same pattern of localization of TNF and TRAF2, an antiapoptotic molecule involved in intracellular TNF signal transduction [7] in proliferating cells, was demonstrated histochemically in follicles, especially in the outer zone of the granulosa layer where the apoptotic cell frequency is extremely low [11]. Moreover, TNFR2 and TRAF2 expression decreased during follicular atresia. The present findings would tend to support our hypothesis that TNF may act as a survival/proliferating factor in granulosa cells of porcine ovaries, as follows: 1) TNF binds to the extracellular domain of TNFR2, 2) the intracellular region of the receptor binds with TRAF2, 3) NF-B is activated, and 4) consequently, expression of survival genes is up-regulated, resulting in proliferation of granulosa cells.
In vitro study of bovine thecal cells showed that TNF inhibits androstendione production but induces cell proliferation [18]. However, TNF does not affect steroidogenesis or other physiological functions, such as LH responsiveness, in porcine cultured thecal cells [19]. The role of TNF on thecal cells has not been revealed. In the present study, positive immunostaining for TNF was observed in the theca externa layer of healthy follicles, and decreased staining was observed in early and progressed atretic follicles. Moreover, a similar pattern of immunohistochemical reactivity was observed for TRAF2, which is a good indicator of TNF-dependent cell proliferation [7]. We presumed that TNF also acts as a survival/proliferating factor in the theca externa layer. Further work, however, is needed to clear up the detailed function of TNF.
Although the biological roles of TNF and its receptor system in ovarian tissues are largely unknown, this system may dominantly contribute to the selective survival of necessary cells under physiological conditions [1, 2]. Our findings propose that TNF and its receptor system play an important role in induction of survival/proliferating signals in granulosa cells during follicular growth in porcine ovaries. Further studies are necessary to determine which molecular system regulates the disappearance of TNF receptor-associated proteins in granulosa cells at the early stage of atresia, and which intracellular signal transduction pathway dominantly causes granulosa cell survival in the selection of follicles.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: FAX: 81 75 753 6345; manabe{at}kais.kyoto-u.ac.jp
Received: 20 February 2002.
First decision: 17 March 2002.
Accepted: 16 August 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B activation. Cell 1995 81:495-504[CrossRef][Medline]2,6-sialyltransferase messenger ribonucleic acid increases glycoconjugates containing 2,6-linked sialic acid residues in granulosa cells during follicular atresia of porcine ovaries. Biol Reprod 1999 60:1475-1482 and its receptors type I and type II in human oocytes. Mol Reprod Dev 1997 47:127-133[CrossRef][Medline] (TNF-) inhibits steroidogenesis of bovine ovarian granulosa and thecal cells in vitro. Endocrine 1998 8:105-109This article has been cited by other articles:
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