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a Department of Biological Sciences, Kent State University, Kent, Ohio 44242
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) is an intraovarian cytokine that may play a role in ovarian development and function. Effects of TNF are mediated by binding to at least one of two TNF receptor subtypes (with molecular masses of approximately 60 and 80 kDa); therefore, the overall goal of this study was to determine whether rat ovaries have TNF receptors during critical times in development. Two approaches were used: 1) demonstration of specific binding of radiolabeled TNF to ovarian cells and 2) semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis for each of the two TNF receptors. Ovarian cells were obtained on Embryonic Day 19, day of birth (Day 0), and Days 2, 5, 10, and 20. TNF binding was present on all days, with significantly greater binding on Day 20. Messenger RNA for both receptor subtypes was detected on all days using RT-PCR analysis but was significantly greater for the 60-kDa receptor on Day 20. In conclusion, rat ovaries contained receptors capable of binding TNF and mRNA for both receptor subtypes. Identification of ovarian TNF receptors provides support for a role of TNF in ovarian development and function.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). TNF is a 17.3-kDa protein that was first identified as a product of activated macrophages [1, 2] that induces necrosis of certain tumors in vivo [3]. TNF has been identified in the ovaries of many different species, including rats [4], mice [5], rabbits [6], cows [7], and humans [8]. Sites of TNF localization in the rat ovary include oocytes [4], granulosa cells [7], corpora lutea [9], and macrophages [9]; however, oocytes appeared to be the most predominant source of TNF immunostaining [4]. In adult rats, immunoreactive TNF was present in the cytoplasm of oocytes within follicles at all stages of development from primordial follicles through preovulatory follicles, and also in ovulated eggs. Oocytic expression of TNF was observed in neonatal rats as early as two days after birth; however, fetal oocytes one day before birth did not contain immunoreactive TNF [4]. TNF in resident ovarian macrophages, granulosa cells, and oocytes may be involved in regulating ovarian function (reviewed in [10, 11]).
There are two major types of TNF receptors: type I, which is approximately 60 kDa (p60) and type II, which is approximately 80 kDa (p80) [12, 13]. Signaling through the p60 receptor is necessary for many biological functions of TNF, including cytotoxicity, growth stimulation of fibroblast cells, induction of manganous-superoxide dismutase, antiviral activity, and endotoxic shock (reviewed in [14]). The role of the p80 receptor in TNF signaling is poorly understood; however, it has been shown that the p80 receptor was involved in the growth stimulation signal of lymphoid cells [14]. These observations have led to the idea that TNF binding to the p60 receptor results in cytotoxicity, whereas binding to both the p60 and p80 receptors results in differentiation of cells that possess both receptor subtypes [14].
Clearly, functional receptors must be present if TNF is to play a role in ovarian function or development. The purpose of this study was to begin characterization of TNF receptors during critical points in ovarian development. Embryonic Day 19 (E19), day of birth (Day 0), and Days 2, 5, 10, and 20 were chosen for evaluation because they span developmental milestones. For example, on E19, oocytes begin to enter meiotic arrest, and by Day 0, all oocytes have entered meiotic arrest [15]. Between Days 0 and 2, primordial follicles are formed [16] and oocytes begin to produce TNF [4]. Between Days 2 and 5, primordial follicles are activated and primary follicles are formed [17]. Between Days 5 and 10, granulosa cells begin to proliferate, producing multilaminar follicles; granulosa cells acquire FSH receptors; and follicles first become steroidogenically active [18]. Between Days 10 and 20, follicles become fully mature, developing to the preovulatory stage [19].
Currently there is very limited information on ovarian TNF receptors. Human oocytes and cumulus cells express both TNF itself and the p80 TNF receptor at both the mRNA and protein levels [20]. In human ovarian cancer, both mRNA and protein for the p60 receptor have been localized to the tumor epithelium and the tumor itself, and the p80 receptor was found on macrophages infiltrating the ovary [21]. The coexpression of TNF and its receptors in various ovarian cells suggests the capacity for autocrine and/or paracrine actions of TNF. Previous investigators have also demonstrated specific high-affinity, low-capacity saturable binding sites for TNF on porcine granulosa cells using competitive receptor binding assays [22]. Effects of TNF on cultured granulosa and theca cells include alterations of steroidogenesis [22–28] and cellular morphology [28]. In addition to its effects on ovarian steroidogenesis, responses to TNF range from cytotoxicity to proliferative responses, presumably depending on the type(s) of receptors and signal transduction machinery present within individual cell types. For example, TNF can cause cell proliferation or cytotoxicity in various ovarian cancer cells [29, 30]. TNF has also been shown to stimulate apoptosis in cultured hen granulosa cells [31] and cultured antral follicles from diethylstilbestrol (DES)-treated immature rats [32]. A greater understanding of ovarian TNF receptors should provide important support for the overall hypothesis that TNF plays an important role in ovarian physiology.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reverse transcription (RT)-polymerase chain reaction (PCR) reagents were purchased from Perkin Elmer (Branchburg, NJ). Taq polymerase, guanidium thiocyanate, sodium citrate, sarkosyl, chloroform, agarose, tryptone, BSA, yeast extract, and glycerol were purchased from Fisher Scientific (Pittsburgh, PA). PCR primers, buffer-saturated phenol, McCoy's 5A medium, penicillin/streptomycin, deoxyribonuclease (DNase), collagenase, fetal calf serum, the CloneAmp pAMP1 System for Cloning of Amplification Products kit, and Subcloning Efficiency DH5 competent cells were purchased from Gibco/BRL (Gaithersburg, MD). Restriction enzymes were purchased from Promega (Madison, WI). Isoamyl and isopropanol alcohols, 2-mercaptoethanol, ethidium bromide, Tris-acetate, Tris-HCl, EDTA, ampicillin, isopropyl-1-thio-ß-D-galactopyranoside (IPTG), G-25 Sephadex, and MgCl2 were purchased from Sigma (St. Louis, MO). [32P]dCTP and 125I were purchased from Dupont NEN (Wilmington, DE). A Cy5-labeled primer kit was purchased from Pharmacia Biotech (Piscataway, NJ). Iodogen was purchased from Pierce (Rockford, IL). Recombinant murine TNF was purchased from Genzyme (Cambridge, MA). Twenty-four-well culture plates were purchased from Costar (Cambridge, MA). A Qiaquick PCR purification kit was purchased from Qiagen (Chatsworth, CA). Ecolite scintillation fluid was purchased from ICN (Costa Mesa, CA).
Animals
Sprague-Dawley rats obtained from a departmental colony were housed in a temperature- and light-controlled room with a 12L:12D light cycle (lights-on at 0730 h) in an American Association of Laboratory Animal Care (AALAC)-approved facility. The animals had unlimited access to food and water, and were housed with one male and two females per cage. Females were checked daily for the presence of a sperm plug (considered Embryonic Day 0). To obtain E19 ovaries from fetuses, the pregnant females were first anesthetized with Metofane (methoxyflurane; Mallinckrodt Veterinary, Inc., Mundelein, IL) and decapitated, and then the pups were removed. Fetuses and neonates from E19, Day 0, and Days 2, 5, 10, and 20 postpartum were gassed with CO2 and decapitated, and the ovaries were removed. Ovaries for binding assays were used immediately, and ovaries for RNA analyses were frozen immediately at -70°C. All animal procedures were carried out in accordance with protocols approved by the Kent State University Animal Care and Use Committee and in accordance with the Guiding Principles for the Care and Use of Research Animals as set forth by the Society for the Study of Reproduction.
Competitive Receptor Binding Assays
Recombinant murine TNF was radiolabeled with 125I using the Iodogen method according to the protocol provided by the manufacturer and was then purified using G-25 Sephadex columns. Competitive receptor binding assays were performed as previously described for porcine granulosa cells [22], with minor modifications. Ovaries from each age group were cleaned and dispersed into single cells in McCoy's 5A medium containing 100 U/ml penicillin, 100 µg/ml streptomycin, 5 mg/ml BSA, 5 mg/ml DNase, and 5 mg/ml collagenase. One hundred thousand cells per well were allowed to attach to a 24-well plate in medium containing 10% fetal calf serum, for 16–18 h at 37°C. Previous validation of this method of dispersing and attaching rat ovarian cells demonstrated more than 95% cell viability using trypan blue exclusion. After overnight plating of the cells, the excess medium was removed, and the cells were incubated with 0.025 M Tris-HCl containing 10 mM MgCl2 and 0.1% BSA and either 125I-TNF alone (total binding) or 125I-TNF plus a 500-fold excess of unlabeled TNF (nonspecific binding) in six replicate wells. To obtain sufficient cells to perform 6 replicate wells per dose of radiolabeled TNF, ovaries from 15 pups (Day E19), 9 pups (Days 0 and 2), 5 pups (Day 5), and 2 pups (Days 10 and 20) were pooled. The concentrations of 125I-TNF used for initial dose-response assays were 0.1 ng/ml, 0.5 ng/ml, 1.0 ng/ml, 2.5 ng/ml, 5.0 ng/ml, 10.0 ng/ml, and 20.0 ng/ml. After cells had been incubated for 4 h at 4°C, the media containing unbound TNF were removed, and the cells were washed twice with 0.025 M Tris-HCl containing 10 mM MgCl2 and 0.1% BSA. The cells were solubilized in 1.0% SDS, and the amount of 125I-TNF bound to the cells was measured in a gamma counter. Specific binding was determined by subtracting nonspecific binding from total binding.
It would be ideal to precisely determine the number and affinity of receptors on all days of development. Unfortunately, this would require using a wide range of doses and would necessitate using at least 100–200 of the youngest pups (E19, 0, and 2 days old) per experiment. Therefore, subsequent assays were performed at the saturating dose of 125I-TNF (10.0 ng/ml) to determine relative changes in specific binding between the days examined.
RNA Isolation
Total RNA was extracted using the guanidium isothiocyanate-phenol-chloroform isolation method [33]. Briefly, the dissected ovaries were homogenized in denaturing solution containing 4 M guanidium thiocyanate; 25 mM sodium citrate (pH 7.0); 0.5% sarkosyl; sterile distilled, deionized H2O; and 0.1 M 2-mercaptoethanol. The following reagents were added in order: 0.1 vol 2 M sodium acetate (pH 4.0), 1 vol buffer-saturated phenol, and 0.2 vol chloroform:isoamyl alcohol (49:1 ratio). The RNA was precipitated twice with isopropanol and washed with 70% ethanol. The RNA pellets were vacuum-dried, dissolved in diethyl pyrocarbonate (DEPC)-H2O, and heated at 65°C for 10 min. The concentration of total RNA was determined by measuring A260.
Semiquantitative RT-PCR
For mRNA analysis, each reaction contained primers for either the p60 or p80 TNF receptor subtype mRNA plus primers for cyclophilin, which was used as an internal control. Cyclophilin is constitutively expressed in the ovary and has been successfully used to examine changes in expression of ovarian mRNAs during developmental time points similar to those examined here [34]. The primers that were used to amplify the p60 mRNA consisted of an upstream primer complementary to nucleotides 195–215 of the rat TNF p60 receptor [35] and a downstream primer complementary to nucleotides 710–730 of the rat p60 receptor [35] (Table 1). Amplification of the p80 receptor mRNA consisted of an upstream primer complementary to nucleotides 485–505 of the mouse p80 TNF receptor [35] and a downstream primer complementary to nucleotides 992–1012 of the mouse p80 receptor [35] (Table 1). The upstream primer for cyclophilin is complementary to nucleotides 1–30 and the downstream primer to nucleotides 323–350 [36] (Table 1). One microgram of total RNA from each sample was reverse-transcribed for 1 h at 42°C using 100 IU Moloney murine leukemia virus (MuLV) reverse transcriptase, 5 mM of each nucleotide base (dATP, dCTP, dGTP, dCTP), and 200 mM random cDNA primers. PCR was then performed using 5 IU Taq polymerase and 1 µM of specific primers, with the addition of 1.5 µCi of [-32P]dCTP (specific activity 3000 Ci/nmol) to each reaction. Each cycle included denaturation at 92°C for 20 sec, annealing at 60°C for 1 min, and extension at 72°C for 30 sec. Amplification of p60 plus cyclophilin was performed for 30 cycles, and that of p80 plus cyclophilin was carried out for 40 cycles. Validation of the semiquantitative RT-PCR method included demonstration of linear amplification with increasing RNA concentration and increasing number of cycles (data not shown). The numbers of cycles used were within the linear range of amplification for both the cyclophilin and receptor cDNAs. The final PCR products were electrophoresed on a 2% agarose gel in single-strength Tris-acetate-EDTA buffer (0.04 M Tris-acetate, 1 mM EDTA, pH 8.0), stained with 0.5 µg/ml ethidium bromide, and visualized under UV light. Bands of 536 (p60), 527 (p80), and 350 (cyclophilin) base pairs (bp) were excised from the gels and placed in vials containing 5 ml Ecolite scintillation fluid; and the amount of radioactivity was determined on a scintillation counter. The data for p60 and p80 were normalized to the cyclophilin values by dividing receptor cpm by cyclophilin cpm.
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Cloning and Sequencing
PCR products were cloned using the CloneAmp pAMP1 System. For cloning purposes, RT-PCR was performed as described above with the use of modified primers synthesized according to CloneAmp instructions, which allowed insertion into plasmid DNA. The final PCR products were purified using a Qiaquick PCR purification kit. The purified PCR products were annealed to a pAMP1 vector DNA and then transformed into DH5 cells according to the manufacturer's instructions. The plasmid DNA for the p60 receptor was linearized with EcoRI and the p80 plasmid DNA with BamHI. The linearized plasmid DNA for each receptor subtype was sequenced by the chain termination method using a Cy5-labeled primer kit and an ALFexpress DNA sequencer (Pharmacia Biotech).
Statistical Analysis
Values in all figures are given as mean ± SEM. Statistical analysis was performed on data by one-way ANOVA, followed by Fisher's post-hoc test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine whether the amount of binding changed over the course of ovarian development, competitive receptor binding assays were performed on dispersed ovarian cells from Days E19, 0, 2, 5, 10, and 20. Initial dose-response assays using increasing concentrations of labeled TNF were performed on Days 5, 10, and 20 (data not shown). In these studies, there was significant specific binding at all doses of TNF (0.1–20.0 ng/ml) for each time point, and binding increased in a dose-dependent manner. There were no significant differences (P > 0.05) in specific binding between 10.0 and 20.0 ng/ml of 125I-TNF (Fig. 1, Day 20); therefore, 10.0 ng/ml of 125I-TNF was used as the saturating dose for subsequent experiments including Days E19, 0, and 2. Ovaries on all days of development demonstrated specific binding at 10 ng/ml of TNF, with significantly greater binding on Day 20 (P < 0.001) compared to the earlier days of development (Fig. 2) and with no differences between binding on Days E19, 0, 2, 5, and 10 (P > 0.05).
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Semiquantitative PCR/Sequencing of cDNA
The sequence of PCR products confirmed their identity as p60 and p80 receptor cDNA. The experimental rat p60 cDNA, corresponding to nucleotides 195–730, was 99% homologous to two published rat cDNA sequences (GenBank Accession Numbers M63122 and M75682). The experimental rat p80 cDNA (Accession Number AF142499) was 98% homologous to 54 overlapping bases of rat p80 cDNA previously reported (Accession Number U55819) and 90% and 48% homologous to the corresponding regions of the mouse (GenBank Accession Number M59377) and human (GenBank Accession Number S63368) sequences.
To determine whether the relative amounts of mRNA for the p60 and p80 TNF receptor changed over the course of development, semiquantitative PCR was performed using cyclophilin as an internal control. RT-PCR products of 536 bp, corresponding to p60, and of 350 bp, corresponding to cyclophilin, were detected in ovarian samples on all days of development. The relative amount of p60 mRNA (normalized to cyclophilin) significantly increased on Day 20 (P < 0.001) compared to all other days examined (Fig. 3). Coamplification of p80 and cyclophilin resulted in two bands at 527 bp (p80) and 350 bp (cyclophilin) for all days examined. Unlike p60 results, there were no significant differences between relative amounts of p80 mRNA on the different days examined (P > 0.05; Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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receptors, and that rat ovarian cells from all days examined were capable of binding radiolabeled TNF. This suggests that the ovaries may contain both types of TNF receptors and provides further evidence that the ovary contains at least one functional TNF receptor.
The significant findings of the competitive receptor binding assays were 1) the demonstration of TNF binding to ovarian cells on all days of development studied and 2) the increase in TNF binding on Day 20 compared to all other days. These results are supported by previous studies that have demonstrated specific binding of radiolabeled TNF to porcine granulosa cells [22] and bovine luteal cells [37]. Nonspecific binding in the current study was similar to that observed in cultured porcine granulosa cells [22], using binding assay methods very similar to ours. Those authors concluded that TNF binding to granulosa cells was saturable, specific, and of high affinity. Our studies have extended these findings by examining TNF binding at several important stages of development in the rat ovary and also showed both saturable and specific binding. Additional studies in the human ovary have immunolocalized p60 to ovarian tumors [21] and p80 to ovarian macrophages [21], oocytes, and cumulus cells [20]. Thus, there is a growing body of evidence supporting the hypothesis that ovarian cells possess functional TNF receptors.
There are several hypotheses that may explain the increased binding on Day 20 compared to the earlier days examined. The comparable increases (1.5- to 2-fold) in TNF binding and p60 mRNA suggest the strong possibility that increased numbers of p60 receptors may account for the increase in binding. In contrast, p80 mRNA levels remained constant on all days of development; therefore, the increased binding on Day 20 was most likely due to an increase in p60 rather than p80 receptor numbers. It is unknown whether the increased binding (and presumably the increased number) of p60 receptors occurred in a cell-specific manner. It is possible that there may be either an overall increase in the number of receptors on all ovarian cells, or on a specific cell population. For example, it is possible that theca cells may have more TNF receptors than other cell types in the ovary. If that were indeed the situation, then ovaries with many large antral/preovulatory follicles that contain a highly developed thecal layer (Day 20) would be expected to have greater binding than those with relatively fewer large follicles (Day 10) or those lacking thecal cells altogether (Days E19, 0, 2, and 5). It has previously been shown that FSH treatment increased specific binding of TNF to cultured swine granulosa cells [22]. Therefore, increased expression of FSH receptors or an increased number of FSH receptor-containing granulosa cells may be involved in increasing TNF binding on Day 20. It is also possible that increased TNF binding and expression of p60 mRNA may be related to the increased incidence of atresia on Day 20 [38]. Previous investigators have demonstrated that TNF can stimulate apoptosis of granulosa cells through its second messenger, ceramide [31, 32], and effects of TNF mediated through ceramide have been coupled to the p60 receptor [39]. If TNF binding to p60 receptors is an important component of follicular atresia, it is reasonable to hypothesize that induction of TNF receptors may be involved in follicular atresia. Furthermore, macrophage infiltration during advanced follicular atresia [38] may contribute to the increased TNF binding as macrophages are known to possess TNF receptors [21]. A final possibility is that an increase in binding affinity rather than receptor numbers may be responsible for increased TNF binding on Day 20. Although this is possible, the literature contains few or no references to changes in binding affinity that are developmentally regulated, and treatments, such as insulin and FSH, that altered TNF binding to porcine granulosa cells had no effect on binding affinity.
There are two possible explanations for the increase in p60 mRNA on Day 20: an increase in p60 transcription or an increase in p60 mRNA stability. Few studies have directly examined regulation of p60 transcription, although the p60 promoter in both mice [40] and humans [41] contains an Sp1 site, and the mouse promoter contains additional sites resembling those found in interferon and major histocompatibility complex II promoters [40]. It is not known whether these specific promoter sites are involved in the increase in p60 mRNA, but the changing profile of steroid hormones, growth factors, and peptides (reviewed in [42]) associated with follicular development may alter the p60 transcription rate. Little is known regarding regulation of p60 mRNA stability, although previous investigators showed a significant decrease in p60 mRNA half-life during phorbol ester-induced differentiation of myelomonocytic cell lines [43].
The presence of both biologically active TNF [4] and TNF receptor binding provides support for an autocrine or paracrine effect of TNF throughout folliculogenesis. There is a dramatic increase in immunoreactive TNF within rat oocytes coincident with the onset of follicle assembly, and TNF persists within the oocyte until after ovulation [4]. Furthermore, TNF has been shown to affect a variety of ovarian cell types in the rat and other species, including granulosa cells, theca cells, and luteal cells (reviewed in [11]). Effects of TNF on isolated granulosa cells vary somewhat depending upon the species investigated and the stage of follicular maturity. In general, however, TNF appears to suppress gonadotropin-stimulated steroidogenesis in pig [22], mouse [44], chicken [45], and rat [23–25, 46] granulosa cells. Suppression of gonadotropin-stimulated androgen production has also been reported for theca-interstitial cells in the rat [27, 28]. Similar to its effects on follicular compartments, TNF can also inhibit luteal progesterone production in several species (reviewed in [11]).
In addition to its effects on steroidogenesis, TNF may also affect ovarian cell proliferation and apoptosis. For example, the addition of TNF stimulates proliferation of cultured mouse primordial germ cells [47]. Another example of TNF-increased cell proliferation is in a study using granulosa cells from immature rats treated with either eCG or DES. These cells responded to FSH with a decrease in cell proliferation—an effect that is reversed by coincubation with TNF [48]. In contrast, TNF has been shown to induce apoptosis in cultured intact antral follicles from DES-treated immature rats [32] and in cultured hen granulosa cells [31]. In similar studies of nonovarian cells, TNF has also been shown to down-regulate the rate of cellular proliferation of rat trophoblasts [49, 50] and blastocysts [35]. Furthermore, antisense oligodeoxyribonucleotides against the p60 receptor protected the blastocysts from TNF's inhibitory effects on cell proliferation [35], suggesting that these anti-proliferative effects may be mediated via the p60 receptor.
TNF may also be involved in ovulation [51], a process that has been likened to an inflammatory reaction [52]. This is supported by 1) an increase in the concentration of TNF in bovine follicular fluid at ovulation [53]; 2) TNF-induced synthesis of ovulatory mediators, such as progesterone and prostaglandin, in the thecal layer of incubated preovulatory rat follicles [26]; 3) release of TNF from the ovulating rat ovary [54]; and 4) the enhanced ovulation rate of rat ovaries perfused with TNF [51].
A final, intriguing possibility is that TNF may play a role in follicle assembly or maintenance of follicular integrity. The appearance of immunoreactive, bioactive TNF within the oocyte shortly after birth is temporally coincident with follicle assembly [4], suggesting the interesting possibility that these two events may be related. For example, TNF may influence follicle assembly through promoting intracellular adhesion or cell migration. It has been shown that TNF induces the aggregation of thecal cells in vitro [28] and can induce expression of adhesion molecules such as intercellular adhesion molecule-1 [55]. Other investigators have demonstrated that TNF inhibited tissue-type plasminogen activator (tPA) and stimulated urokinase-type plasminogen activator (uPA) activity in preantral rat follicles [48]. These authors suggested that TNF's effects on the two plasminogen activators are important because tPA is associated with follicle rupture, whereas uPA may be more important in the extracellular matrix remodeling that occurs during ovarian cell proliferation. Thus, TNF may act on preantral follicles to maintain follicle integrity, thereby preventing premature follicle rupture.
In conclusion, the identification of mRNA for both receptor subtypes and specific binding of TNF provide pivotal evidence supporting a role for TNF in ovarian development and function.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Jennifer L. Marcinkiewicz, 256 Cunningham Hall, Department of Biological Sciences, Kent State University, Kent, OH 44242. FAX: 330 672 3713; jmarcink{at}kent.edu
Accepted: July 20, 1999.
Received: May 3, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in follicular development, ovulation, and the life span of the corpus luteum. Domest Anim Endocrinol 1997; 14:1–15.[CrossRef][Medline]). J Biol Chem 1989; 264:14927–14934. and its receptors type I and type II in human oocytes. Mol Reprod Dev 1997; 47:127–133.[CrossRef][Medline] receptors in bovine corpora lutea in the estrous cycle. Biol Reprod 1998; 58(suppl 1):79 (abstract 26). inhibits follicle-stimulating hormone-induced differentiation in cultured rat granulosa cells. Biochem Biophys Res Commun 1989; 163:1038–1046.[CrossRef][Medline] (TNF-) stimulates proliferation of mouse primordial germ cells in culture. Dev Biol 1994; 161:91–95.[CrossRef][Medline]This article has been cited by other articles:
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  C. R. Greenfeld, K. F. Roby, M. E. Pepling, J. K. Babus, P. F. Terranova, and J. A. Flaws Tumor Necrosis Factor (TNF) Receptor Type 2 Is an Important Mediator of TNF alpha Function in the Mouse Ovary Biol Reprod, February 1, 2007; 76(2): 224 - 231. [Abstract] [Full Text] [PDF]
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L. J. Morrison and J. L. Marcinkiewicz Tumor Necrosis Factor {alpha} Enhances Oocyte/Follicle Apoptosis in the Neonatal Rat Ovary Biol Reprod, February 1, 2002; 66(2): 450 - 457. [Abstract] [Full Text] [PDF]
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J. Prange-Kiel, C. Kreutzkamm, U. Wehrenberg, and G. M. Rune Role of Tumor Necrosis Factor in Preovulatory Follicles of Swine Biol Reprod, September 1, 2001; 65(3): 928 - 935. [Abstract] [Full Text] [PDF]
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