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a Department of Animal Science, Cornell University, Ithaca, New York 14853
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and interferon-
(IFN) were studied because of previous demonstrations of their role in Fas-mediated apoptosis in other cell types. Fas antigen mRNA was detectable in cultured granulosa and theca cells, and expression was increased by treatment with IFN but not TNF. Granulosa and theca cells were resistant to FasL-induced killing unless pretreated with IFN. TNF had no effect on FasL-induced killing. Granulosa and theca cell cultures in which killing occurred in response to FasL stained positively for annexin V, an early marker for cells undergoing apoptosis. These results provide a basis for further studies using the bovine ovary to examine the role of the Fas antigen in follicular atresia.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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While knowledge of the expression of Fas antigen mRNA and protein is important, it is also critical to know whether Fas antigen can trigger apoptosis in ovarian cells. Cultured ovarian cells from the human and mouse were resistant to killing by cytotoxic Fas mAb unless the cells were pretreated with the cytokines, interferon (IFN) or IFN+TNF, or with cycloheximide [3,10,11]. Studies of Fas expression and function in the ovary have been limited primarily to human and murine cells by the lack of suitable reagents, especially specific agonistic antibodies for Fas antigen in other species. Furthermore, studies in the human are usually limited to granulosa/luteal cells obtained secondary to in vitro fertilization. Studies in the mouse are hampered by its size, which limits the ability to obtain sufficient tissue from individual follicles. Recombinant soluble human FasL has recently become available. This reagent kills susceptible cells by ligation to the Fas antigen. It is reported by the manufacturer to be effective in numerous mammalian species including the human, mouse, and rat.
Ovarian function in the cow has been studied extensively. Follicles grow and regress in well-defined waves at predictable times during the estrous cycle [12], allowing the investigator to obtain significant amounts of follicular tissues at specific times of development with a high degree of accuracy. Thus the cow may be an excellent model for studying the role of the Fas antigen in follicular atresia. Here we describe the expression of Fas antigen in cultured bovine granulosa and theca cells. We also describe the killing of these cells by FasL following pretreatment with cytokines. The data show that the cow is a suitable model for studying the role of the Fas pathway in the ovary.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All culture media reagents and murine TNF were obtained from Gibco BRL (Grand Island, NY). Tissue culture plates were obtained from Corning-Costar (Cambridge, MA), except that slide-well chambers were from Nunc-Intermed (Naperville, IL). Avian myeloblastosis virus reverse transcriptase was obtained from Promega (Madison, WI), random hexamer from Pharmacia (Piscataway, NJ), and Taq polymerase from Fisher (Pittsburgh, PA). MTT (3-[4,5-dimethylthiazol-2yl]2,5-diphenyltetrazolium bromide) was obtained from Sigma Chemical Co. (St. Louis, MO). Bovine IFN and TNF were graciously provided by Dr. Dale Godson, Veterinary Infectious Disease Organization (Saskatoon, SK, Canada). Soluble recombinant human FasL was obtained from Upstate Biotechnology (Lake Placid, NY).
Functional Analysis of Fas Antigen in Cultured Bovine Granulosa Cells
Freshly excised cow ovaries were obtained from an abattoir, transported in saline at room temperature (approximately 1.5 h), and processed immediately. Granulosa cells were obtained by aspiration of 5- to 10-mm follicles with a 21-gauge needle, followed by flushing of the follicles with Dulbecco's modified Eagle's medium (DMEM) containing 0.002 M EDTA. Cells were collected by centrifugation, washed, counted, and plated in basal medium (DMEM-F12 medium supplemented with 1 mM pyruvate, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone) containing 10% fetal bovine serum (FBS). Cells were plated at 5 x 104 cells per well in 96-well plates for analysis of FasL-induced apoptosis, or 0.8 to 1 x 106 cells per well in 6-well plates for analysis of RNA.
Granulosa cells were pretreated 24 h after plating with no cytokines (control), 10 ng/ml murine TNF, 200 U/ml bovine IFN, or 200 U/ml IFN+10 ng/ml TNF in basal medium containing 5% FBS. Doses were based on maximal induction of Fas antigen mRNA expression in preliminary dose response studies using bovine and murine TNF and bovine IFN (not shown). At 48 h, cells in 6-well plates were frozen for analysis of Fas antigen mRNA. Cells in 96-well plates from each cytokine pretreatment were given the same cytokine treatment with or without 100 ng/ml FasL. At 72 h, cells were assessed qualitatively for viability by phase-contrast microscopy by noting the relative abundance of attached versus floating cells and the presence of blebbed membranes and apoptotic bodies. Cell number was then assessed quantitatively at 72 h of culture by MTT assay [13]. The assay detects a change in optical density (OD570) caused by reduction of MTT to formazan by live cells containing active mitochondria. Percentage killing was calculated for each FasL treatment compared to the control treatment with the same cytokine pretreatment.
Functional Analysis of Fas Antigen in Cultured Bovine Theca Cells
To obtain theca cells for culture, follicles were dissected out of abattoir ovaries and bisected. The inner follicle wall, composed of theca interna and attached granulosa cells, was peeled, scraped vigorously to remove adherent granulosa cells, and digested with collagenase and dispase for 1 h as described previously [14]. Roberts and Skinner [14] demonstrated that theca cells prepared using this method were contaminated at a low level with endothelial cells (4 ± 2%). The isolation procedure for theca cells was assessed for its potential to result in contamination with granulosa cells by measuring aromatase mRNA using reverse transcription-polymerase chain reaction (RT-PCR). Granulosa cells from healthy follicles are known to express aromatase while theca cells do not. Theca samples from healthy follicles contained 0.08 ± 0.02% of the aromatase mRNA found in granulosa cells [15]. Dispersed theca cells were collected by centrifugation, washed, and plated in either 96-well or 6-well plates at the same concentrations as granulosa cells in basal medium containing 10% FBS. At 24 h, medium was changed to basal medium containing 5% FBS, and the cells were incubated for a further 24 h to insure recovery from enzymatic digestion. At 48 h, cells were pretreated with no cytokines (control), 10 ng/ml murine TNF, 200 U/ml bovine IFN, or 200 U/ml IFN+10 ng/ml TNF in basal medium containing 5% FBS. At 72 h, cells in 6-well plates were frozen for analysis of Fas antigen mRNA, while cells in 96-well plates from each cytokine pretreatment were given the same cytokine treatment with or without 100 ng/ml FasL. At 96 h, visual observations were made and viability was assessed by MTT assay as described for granulosa cells.
Detection of Membrane-Associated Phosphatidylserine
Detection of phosphatidylserine on the outside of the cell membrane, a unique and early marker for apoptosis [16], was performed using a commercial kit (Vybrant Apoptosis Assay Kit #2; Molecular Probes, Eugene, OR). Cells were cultured as described above, pretreated with 200 U/ml IFN, treated with or without FasL, and tested 6 h later. Binding of annexin V-Alexa-488 conjugate and propidium iodide (PI) was performed according to the manufacturer's instructions. After binding and washing, cells were fixed in acetone at -20°C for 5 min, hydrated for 5 min, and coverslipped. Cells were observed under phase contrast and epifluorescent illumination using a 495-nm excitation filter and a 520-nm absorption filter for annexin V-Alexa 488 and a 546-nm excitation filter and a 590-nm absorption filter for PI. Healthy cells were unstained by either dye; cells in early stages of apoptosis were stained only by annexin V, while dead cells were stained by annexin V and PI. The assay was repeated on 3 separate granulosa and theca cell preparations.
Analysis of Fas Antigen mRNA
Fas antigen mRNA was quantified by a competitive RT-PCR assay described previously for measurement of mouse Fas antigen mRNA [11] and modified for use with bovine-specific primers and controls. RNA was reverse transcribed in the presence of various concentrations of an internal standard RNA. The internal standard RNA was prepared by in vitro transcription of a 206-base pair (bp) fragment of mutated bovine Fas antigen cDNA in the plasmid pALTER-1 (Promega) containing a 50-bp deletion internal to the PCR primer binding sites (positions 591–640; numbering according to [17]). Complementary DNA in the RT reaction was amplified by PCR in the presence of [32P]dCTP as described previously [11]. Primers were designed to generate a 206-bp fragment for the test RNA and a 156-bp fragment for the internal RNA standard (positions of 5' and 3' primers were from 468 to 490 in exons 3 and 4 and from 673 to 634 in exon 6, respectively). RT-PCR products were fractionated on a 2% agarose gel. The gel was dried, and radioactive signal was quantified on a Fuji BAS1000 (Tokyo, Japan) phosphorimager. The concentration of Fas antigen mRNA in each sample was calculated by regression of the log (sample signal/standard signal) versus log standard concentration. Samples from the same culture preparation were assayed together along with an RNA pool and water blank. Between-assay coefficient of variation was 10.4%.
Statistical Analyses
Data were analyzed by a randomized complete block ANOVA. Duncan's New Multiple Range test was used for comparison of means when overall significance was observed. Messenger RNA concentration was log transformed prior to analysis. To assess effects of FasL on cell viability, both OD570 and percentage killing were analyzed statistically.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fas antigen mRNA levels were measured using a quantitative competitive RT-PCR assay (a typical assay is shown in Fig. 1). Fas antigen mRNA was detectable in granulosa cells cultured for 48 h and was increased 5.3-fold by treatment with IFN for 24 h (Figs. 1 and 2). Treatment with TNF had no effect on Fas antigen mRNA levels and did not alter effects of IFN.
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FasL-Mediated Killing in Bovine Granulosa Cells
In order to assess whether the Fas antigen pathway was active in cultured granulosa cells, cultures were pretreated with or without cytokines for 24 h and then treated with FasL for 24 h in the presence or absence of cytokines. Cell number was assessed by OD570 values in an MTT assay. Percentage killing was calculated from OD570 values for each FasL treatment in comparison to controls receiving the same cytokine pretreatment. Granulosa cells not pretreated with cytokines or pretreated with TNF were resistant to FasL-mediated killing (6.8 ± 2.6% and 1.5 ± 2.7% killing, respectively, P > 0.05, Fig. 3). However, 56 ± 11% killing was observed when cells were treated with FasL after IFN pretreatment (P < 0.05). FasL-induced killing after pretreatment with IFN+TNF was 67 ± 11% and was not different from FasL-induced killing following pretreatment with IFN (P > 0.05). A qualitative assessment of granulosa cell cultures undergoing FasL-induced killing by phase-contrast microscopy showed massive loss of cells (Fig. 4, f and h), and characteristic features of apoptotic cell death, including membrane blebbing and formation of apoptotic bodies, were visible at higher magnification (not shown). Cultures not treated with FasL and given various cytokine pretreatments appeared healthy and well attached to the culture dish with apparently the same number of dying cells, or fewer, than in cultures given no pretreatments (Fig. 4, a, c, e, and g). Despite a similar qualitative appearance of control cultures (no FasL) receiving different cytokine pretreatments (Fig. 4), significant differences in cell numbers as assessed by MTT assay were observed; OD570 values of cells pretreated with IFN and IFN+TNF were 86% and 68% those for cells given no pretreatment, respectively (P < 0.05, Fig. 3).
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FasL-Mediated Killing of Granulosa Cells Occurs by Apoptosis
A specific and early marker of cells undergoing apoptosis is the translocation of phosphatidylserine from the inside to the outside of the cell membrane, a process observed by the specific binding of fluorescent annexin V to the external membrane of cells with intact membranes (i.e., those excluding the nuclear dye PI). Six hours after FasL treatment of granulosa cell cultures pretreated with IFN+TNF (Fig. 5, top panels) or IFN (not shown), numerous cells displayed bright staining with annexin V, especially those cells showing formation of apoptotic bodies and cellular condensation. Staining with PI was minimal because cells in later stages of apoptosis lost membrane integrity and eventually became detached from the culture dish. Cultures pretreated with IFN+TNF (Fig. 5, bottom panels) or IFN (not shown) in the absence of FasL displayed only sporadic staining with annexin V.
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Fas Antigen mRNA Expression in Bovine Theca Cells
Fas antigen mRNA was expressed in bovine thecal cells cultured for 72 h and was increased 4.3-fold by treatment with IFN for 24 h. Treatment with TNF had no effect on Fas antigen mRNA levels and did not alter the effects of IFN (Fig. 6).
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FasL-Mediated Killing of Bovine Theca Cells
Theca cells not pretreated with cytokines or pretreated with TNF were resistant to FasL-mediated killing (-10.5 ± 4.3% and -1.5 ± 4.8% killing, respectively, Fig. 7). When cells were pretreated with IFN, FasL induced 42.4 ± 5.0% killing (P < 0.05). Killing due to FasL increased to 72.8 ± 2.9% when cells were pretreated with IFN+TNF (P < 0.05 vs. killing following all other pretreatments). Qualitative assessment of viability by phase-contrast microscopy indicated that untreated theca cultures were generally healthy (Fig. 8a). In contrast, cultures treated with FasL in the presence of IFN or IFN+TNF exhibited massive loss of attached cells and numerous dying cells (Fig. 8, f and h) with membrane blebbing and formation of apoptotic bodies visible at higher magnification (not shown). Significant differences in cell number, as assessed by MTT assay, were observed between control cultures (no FasL) receiving no pretreatment versus cultures receiving pretreatment with IFN+TNF (Fig. 7). OD570 values of cells pretreated with IFN+TNF were 86% those of untreated cells (P < 0.05). Control cultures pretreated with IFN had a similar but nonsignificant reduction in OD570. Despite the difference in OD570 between control cultures given no pretreatment and cultures pretreated with IFN+TNF, cultures appeared similarly healthy when assessed qualitatively by phase-contrast microscopy (Fig. 8, a vs. g).
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FasL-Mediated Killing of Theca Cells Occurred by Apoptosis
In cultures pretreated with IFN+TNF (Fig. 9, top panels) or IFN (not shown), numerous cells displayed bright staining with annexin V 6 h after treatment with FasL. Cells undergoing processes associated with apoptosis (formation of apoptotic bodies and cellular condensation) stained prominently. Staining with PI was minimal. Cultures treated with IFN+TNF (Fig. 9, bottom panels) or IFN (not shown) in the absence of FasL displayed only sporadic staining with annexin V.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The increase in responsiveness of follicle cells to FasL after pretreatment with IFN may be related to the 4- to 5-fold increase in expression of Fas antigen mRNA induced by IFN in granulosa and theca cells. Methods to measure bovine Fas antigen protein have not been developed. Therefore, it is not known whether an increase in Fas antigen protein occurs concomitantly with increased Fas antigen mRNA. Our previous studies using the mouse showed that treatment with IFN+TNF increased responsiveness of granulosa cells to Fas-mediated killing and was correlated with increased expression of both Fas antigen mRNA and protein [10]. Despite these correlations, it is unlikely that the level of Fas antigen expression is the only factor determining cellular responsiveness. Responsiveness of mouse granulosa cells to Fas-mediated killing was also induced by treatment with cycloheximide, suggesting that basal levels of Fas antigen expression are adequate to mediate killing when labile protein inhibitors are removed [10]. In ovarian surface epithelial cells of the mouse, both IFN [11,18] and TNF [18] induced equivalent (2-fold) increases in expression of Fas antigen mRNA and increased immunostaining for Fas antigen protein, but only IFN induced responsiveness to Fas-mediated killing. Studies using non-ovarian cells suggest that the Fas pathway is tightly regulated by inhibitory factors. Cycloheximide treatment promotes Fas-mediated killing in otherwise resistant cell types [19]. Furthermore, at least one specific inhibitor of the Fas pathway has been identified, FAP-1, a protein tyrosine phosphatase that binds to the death domain of the Fas antigen [20] and is expressed in the ovary [10]. One class of proteins, known as inhibitors of apoptosis (IAPs), prevents apoptosis in response to a variety of stimuli apparently by inhibiting caspase activity [21,22]. IAPs are expressed in ovarian cells at relatively higher levels in healthy compared to atretic follicles [23]. Therefore, it is likely that FasL alone fails to induce apoptosis in granulosa and theca cells under conditions in which the cells are healthy and survival pathways within the cell, which oppose Fas-mediated killing, are operational.
Both IFN and TNF have been shown to stimulate Fas antigen expression and Fas-mediated killing in a variety of non-ovarian cell types (reviewed in [10]). It remains to be determined whether IFN is a physiological regulator of the Fas pathway in the ovary. However, IFN is present in follicular fluid [24], and lymphocytes that infiltrate the ovary could provide a source of IFN [25,26]. IFN has been shown to inhibit LH receptor induction [27] and gonadotropin-stimulated steroidogenesis [27–29], to alter inhibin production by granulosa cells [29], and to inhibit progesterone secretion by bovine luteal cells [30]. Further studies are required to determine factors relevant to the modulation of the Fas pathway in ovarian cells in vivo.
TNF is produced by a variety of cells in the ovary including macrophages, endothelial cells, granulosa cells, theca cells, and oocytes [31]. TNF has diverse effects on ovarian cells including inhibition of steroidogenesis [31], stimulation of proliferation [32,33], and induction of apoptosis [34,35]. Variable reports on the effects of TNF on cell proliferation and viability from different studies may be due to differences in the doses of TNF and the culture conditions used, and in the species studied. In the current studies, TNF had no effect on levels of Fas antigen expression or on FasL-induced killing of bovine granulosa or theca cells. In previous studies using mouse granulosa cells, cotreatment with TNF plus IFN was essential for Fas-mediated apoptosis, while TNF alone had no effect [10]. In the current studies, the number of live cells, as assessed by optical density in an MTT assay, was reduced in control cultures of granulosa cells (no FasL treatment) pretreated with TNF or IFN+TNF, and in theca cells pretreated with IFN+TNF. IFN alone had no effect on cell numbers as assessed by MTT assay. Qualitative assessment of cultures for the presence of apoptotic cells using phase-contrast microscopy and staining with annexin V indicated that the reduction in cell numbers in cultures pretreated with cytokines (in the absence of FasL) was not caused by apoptosis. It is possible that cytokines inhibited growth of cells in culture, resulting in reduced numbers of cells before initiation of FasL treatments.
Ovarian follicle atresia occurs by apoptosis of follicle cells [1]. Granulosa cells appear to be the first cells within the follicle to undergo apoptosis, and at least some of the cells within the theca layer undergo apoptosis at a slower rate. In situ end-labeling of DNA in rat ovarian sections revealed DNA fragmentation in atretic follicles that was pronounced in the granulosa cell layer and more scattered [36] or rare [9] in the thecal cell layer. End-labeling of DNA and separation by gel electrophoresis showed internucleosomal cleavage of DNA within both the granulosa and theca layers of regressing follicles in the pig and chicken [37,38]. In the results described here, binding of annexin V to the outer cell membrane, an early and specific marker for apoptosis [16], demonstrates that both granulosa cells and theca cells undergo Fas-mediated apoptosis, at least when pretreated with IFN.
In summary, bovine granulosa and theca cells express Fas antigen and are resistant to FasL-induced apoptosis in vitro in the absence of IFN. The results provide a basis for further studies using the cow on the role of the Fas antigen in the ovary.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grants from the USDA (98–35203–6220) and NIH (HD 32535). 
2 Correspondence: Susan M. Quirk, 258 Morrison Hall, Cornell University, Ithaca, NY 14853. FAX: 607 255 9829; smq1{at}cornell.edu
Accepted: August 17, 1999.
Received: April 5, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, tumor necrosis factor- and cycloheximide. Endocrinology 1998; 139:4860–4869.. J Endocrinol 1992; 133:131–139. in follicular development, ovulation and the life span of the corpus luteum. Domest Anim Endocrinol 1997; 14:1–15.[CrossRef][Medline] in the human ovary: presence in follicular fluid and effects on cell proliferation and prostaglandin production. Fertil Steril 1992; 58:934–940.[Medline] alters steroidogenesis and stimulates proliferation of human ovarian granulosal cells in vitro. Fertil Steril 1993; 59:332–338.[Medline] and its second messenger, ceramide, stimulate apoptosis in cultured ovarian follicles. Endocrinology 1996; 137:4864–4870.[Abstract]This article has been cited by other articles:
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  T. Mazoochi, M. Salehnia, S. Pourbeiranvand, M. Forouzandeh, S. J. Mowla, and E. Hajizadeh Analysis of apoptosis and expression of genes related to apoptosis in cultures of follicles derived from vitrified and non-vitrified ovaries Mol. Hum. Reprod., March 1, 2009; 15(3): 155 - 164. [Abstract] [Full Text] [PDF]
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 S. M Quirk, R. G Cowan, and R. M Harman The susceptibility of granulosa cells to apoptosis is influenced by oestradiol and the cell cycle. J. Endocrinol., June 1, 2006; 189(3): 441 - 453. [Abstract] [Full Text] [PDF]
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 K. A Margalit, R. G Cowan, R. M Harman, and S. M Quirk Apoptosis of bovine ovarian surface epithelial cells by Fas antigen/Fas ligand signaling Reproduction, November 1, 2005; 130(5): 751 - 758. [Abstract] [Full Text] [PDF]
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 K. Okuda, A. Korzekwa, M. Shibaya, S. Murakami, R. Nishimura, M. Tsubouchi, I. Woclawek-Potocka, and D. J. Skarzynski Progesterone Is a Suppressor of Apoptosis in Bovine Luteal Cells Biol Reprod, December 1, 2004; 71(6): 2065 - 2071. [Abstract] [Full Text] [PDF]
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 S. M. Quirk, R. G. Cowan, and R. M. Harman Progesterone Receptor and the Cell Cycle Modulate Apoptosis in Granulosa Cells Endocrinology, November 1, 2004; 145(11): 5033 - 5043. [Abstract] [Full Text] [PDF]
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F. J. Rubio Pomar, B. A.J. Roelen, K. A. Slot, H. T.A. van Tol, B. Colenbrander, and K. J. Teerds Role of Fas-Mediated Apoptosis and Follicle-Stimulating Hormone on the Developmental Capacity of Bovine Cumulus Oocyte Complexes In Vitro Biol Reprod, September 1, 2004; 71(3): 790 - 796. [Abstract] [Full Text] [PDF]
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J.T. Bridgham and A.L. Johnson Alternatively Spliced Variants of Gallus gallus TNFRSF23 Are Expressed in the Ovary and Differentially Regulated by Cell Signaling Pathways Biol Reprod, April 1, 2004; 70(4): 972 - 979. [Abstract] [Full Text] [PDF]
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 S. M. Quirk, R. G. Cowan, R. M. Harman, C.-L. Hu, and D. A. Porter Ovarian follicular growth and atresia: The relationship between cell proliferation and survival J Anim Sci, January 1, 2004; 82(13_suppl): E40 - 52. [Abstract] [Full Text] [PDF]
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J. K. Pru, I. R. Hendry, J. S. Davis, and B. R. Rueda Soluble Fas Ligand Activates the Sphingomyelin Pathway and Induces Apoptosis in Luteal Steroidogenic Cells Independently of Stress-Activated p38MAPK Endocrinology, November 1, 2002; 143(11): 4350 - 4357. [Abstract] [Full Text] [PDF]
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H. Taniguchi, Y. Yokomizo, and K. Okuda Fas-Fas Ligand System Mediates Luteal Cell Death in Bovine Corpus Luteum Biol Reprod, March 1, 2002; 66(3): 754 - 759. [Abstract] [Full Text] [PDF]
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J.T. Bridgham and A.L. Johnson Expression and Regulation of Fas Antigen and Tumor Necrosis Factor Receptor Type I in Hen Granulosa Cells Biol Reprod, September 1, 2001; 65(3): 733 - 739. [Abstract] [Full Text] [PDF]
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C.-L. Hu, R. G. Cowan, R. M. Harman, D. A. Porter, and S. M. Quirk Apoptosis of Bovine Granulosa Cells After Serum Withdrawal Is Mediated by Fas Antigen (CD95) and Fas Ligand Biol Reprod, February 1, 2001; 64(2): 518 - 526. [Abstract] [Full Text]
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S. M. Quirk, R. M. Harman, and R. G. Cowan Regulation of Fas Antigen (Fas, CD95)-Mediated Apoptosis of Bovine Granulosa Cells by Serum and Growth Factors Biol Reprod, November 1, 2000; 63(5): 1278 - 1284. [Abstract] [Full Text]
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 N. A. Cataldo, D. A. Dumesic, P. C. Goldsmith, and R. B. Jaffe Immunolocalization of Fas and Fas ligand in the ovaries of women with polycystic ovary syndrome: relationship to apoptosis Hum. Reprod., September 1, 2000; 15(9): 1889 - 1897. [Abstract] [Full Text] [PDF]
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S. M. Quirk, R. M. Harman, S. C. Huber, and R. G. Cowan Responsiveness of Mouse Corpora Luteal Cells to Fas Antigen (CD95)-Mediated Apoptosis Biol Reprod, July 1, 2000; 63(1): 49 - 56. [Abstract] [Full Text]
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D. A. Porter, S. L. Vickers, R. G. Cowan, S. C. Huber, and S. M. Quirk Expression and Function of Fas Antigen Vary in Bovine Granulosa and Theca Cells During Ovarian Follicular Development and Atresia Biol Reprod, January 1, 2000; 62(1): 62 - 66. [Abstract] [Full Text]
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