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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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(IFN). IFN has been shown to induce responsiveness to Fas antigen-mediated apoptosis in other cell types. In the presence of IFN, killing of granulosa cells by FasL was greater in subordinate compared to healthy dominant follicles on Day 5, did not differ between healthy and atretic dominant follicles, and was similar in theca among all follicles. Granulosa cells from preovulatory follicles, which had been exposed to the LH surge in vivo, were completely resistant to FasL-induced killing. In summary, Fas antigen expression, and responsiveness to Fas antigen-mediated apoptosis, vary during follicular development.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN) [4–7]. IFN has been shown to induce Fas antigen expression and enhances Fas antigen-mediated cell death in many cell types. Expression of Fas antigen and the existence of a functional Fas antigen pathway in follicle cells suggest a role for Fas antigen in follicular development and atresia. The cow provides an excellent model to study the role of Fas antigen in the ovary. Follicular dynamics have been well characterized using real-time transrectal ultrasonography [8]. Follicles develop in waves in which 3 to 6 follicles begin to grow larger than 5 mm in response to FSH. One of these follicles is selected for dominance and continues growing, while the subordinate follicles stop growing and become atretic. Cows exhibit 2 or 3 waves of follicle growth during a typical estrous cycle, but only the dominant follicle present during the follicular phase, in the absence of a functional corpus luteum, ovulates. Otherwise, the dominant follicle stops growing and becomes atretic. To examine whether Fas antigen is involved in follicular atresia, Fas antigen mRNA levels were measured in granulosa and theca interna cells from individual healthy and atretic bovine follicles isolated during the first follicular wave, and responsiveness of cells from these follicles to Fas antigen-mediated killing was tested. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Prostaglandin F2
(PGF2) was obtained from Upjohn Co. (Lutalyse; Kalamozoo, MI). Culture media, fetal bovine serum (FBS), penicillin, streptomycin, and fungizone were obtained from Gibco BRL (Grand Island, NY). Tissue culture plates were obtained from Corning-Costar (Cambridge, MA). Recombinant IFN was provided by Dr. Dale Godson, Veterinary Infectious Disease Organization (Saskatoon, SK, Canada). Soluble recombinant human FasL was obtained from Upstate Biotechnology (Lake Placid, NY). MTT (3-[4,5-dimethylthiazol-2yl]2,5-diphenyltetrazolium bromide) was obtained from Sigma Chemical Co. (St. Louis, MO). Avian myeloblastosis virus (AMV) reverse transcriptase (RT) was obtained from Promega (Madison, WI), random hexamer from Pharmacia (Piscataway, NJ), and Taq polymerase from Fisher (Pittsburgh, PA).
Animals
Ovaries were obtained from cycling, nonlactating Holstein cattle (n = 16). All procedures were approved by the Cornell University Institutional Animal Care and Use Committee and conducted in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. Animals were injected with a luteolytic dose of PGF2 between Days 7 and 14 of the estrous cycle and observed for estrous behavior twice daily starting 2 days after injection. After the onset of estrus, animals were examined daily by transrectal ultrasonography to monitor the onset of the first wave of follicular growth and identify the dominant and 2 largest subordinate follicles in that wave. Ovaries were removed from three groups of animals by colpotomy [9] as follows: from the first group (D5) 5 days after estrus when one follicle was clearly dominant (
9 mm) and the subordinate follicles were atretic and had stopped growing; from the second group (D11) after the dominant follicle in the first wave had not grown for 4 consecutive days; and from the third group (D1) the morning after observation of standing estrus between 1800 h and 2200 h.
Tissue Preparation
The dominant and 2 largest subordinate follicles were dissected from D5 ovaries. Dominant and preovulatory follicles were dissected from D11 and D1 ovaries, respectively. Follicular diameter was measured, and follicular fluid was aspirated using an 18-gauge needle and frozen. Granulosa cells were isolated by bisecting the follicle in Dulbecco's modified Eagle's medium (DMEM)-F12 medium, removing the follicle wall composed of theca interna and attached granulosa cells, and scraping granulosa cells from the theca interna using a finely drawn Pasteur pipette. Granulosa cells were collected by centrifugation, counted, and either frozen in liquid nitrogen for analysis of Fas antigen mRNA (n = 3 per time point) or cultured (n = 3 per time point). Theca interna were thoroughly scraped to remove adhering granulosa cells and were frozen in liquid nitrogen for analysis of Fas antigen mRNA (n = 3 per time point) or prepared for culture as previously described (n = 3 per time point) [7].
In preovulatory follicles, sufficient tissue was obtained for both RNA analysis and cell culture. Cells from subordinate follicles in each animal were pooled for cell culture experiments. Estradiol concentration in follicular fluid samples was measured by RIA as previously described [10], and the between-assay coefficient of variation (n = 4) was 3.1%. Dissected follicle diameter and follicular fluid estradiol concentration data were examined by one-way ANOVA followed by Fisher's multiple comparisons [11].
Cell Culture
Granulosa cells were plated (T = 0 h) at a concentration of 5 x 104 cells per well in 96-well culture dishes in DMEM-F12 medium containing 5% FBS plus 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml fungizone, 1 mM pyruvate, and 2 mM glutamine. To assess the ability of FasL to induce cell death, granulosa cells were treated at T = 0 h with or without 100 ng/ml FasL and with or without 200 U/ml IFN. Treatments were performed in triplicate. The relative number of live cells at T = 24 h was assessed by optical density using an MTT assay as previously reported [12]. FasL-induced killing of bovine granulosa and theca cells was previously characterized as apoptosis based on staining of outer cell membranes with annexin V [7].
Theca cells were plated (T = 0 h) at concentrations of 3 x 104 to 4 x 104 cells per well in 96-well culture dishes in Ham's F-12 medium containing 10% FBS plus antibiotics. Theca cells were treated at T = 24 h in DMEM-F12 medium containing 5% FBS with or without 100 ng/ml FasL and with or without 200 U/ml IFN. Treatments were performed in triplicate. Relative cell numbers were determined by MTT assay at T = 48 h.
Percentage killing was examined by two-way randomized block ANOVA to confirm overall significance, with dominant and subordinate follicles collected on D5 as one block and dominant and preovulatory follicles (D5, D11, and D1) as another block. Within blocks, percentage killing was examined by a one-way randomized block design ANOVA with treatment as the random variable, and comparisons of means were performed by Duncan's New Multiple Range test [11].
Analysis of Fas Antigen mRNA
Fas antigen mRNA was quantified by a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay as previously described [7]. Briefly, RNA was reverse transcribed in the presence of increasing concentrations of an internal Fas antigen standard RNA. Complementary DNA was amplified by PCR in the presence of [32P]dCTP. The concentration of Fas antigen mRNA in each sample was calculated as previously described [7]. Each assay included a sample of each follicle type and an RNA pool, used to calculate the between-assay coefficient of variation (8.8%, n = 8 assays).
Data were examined by one-way ANOVA. After overall significance was confirmed, dominant and subordinate follicle samples from D5 were compared by paired t-tests, while dominant and preovulatory samples were examined by one-way ANOVA followed by Fisher's multiple comparisons. Comparisons of D5 subordinate to D11 or D1 follicles were performed by 2-sample t-tests [11].
Analysis of Aromatase mRNA
Granulosa cells from healthy dominant follicles express aromatase whereas theca cells do not [13]. Relative levels of aromatase mRNA in granulosa versus theca cells isolated from D5 healthy dominant follicles (n = 2) were compared by RT-PCR in order to assess potential contamination of the theca cell layer with granulosa cells prepared using our dissection technique. Theca (0.6 µg) and a serial dilution of 0.6 µg–0.96 ng granulosa cell RNA were reverse transcribed using AMV reverse transcriptase and random hexamer primer. Complementary DNA in the RT reactions was amplified by PCR in the presence of [32P]dCTP using primers designed to generate a 200-base pair fragment (positions of 5' and 3' primers were from 214 to 238 in exon 2 and from 413 to 388 in exon 3, respectively [14]). Amplification consisted of a preincubation at 94°C for 5 min before addition of Taq polymerase and then 25 cycles at 94°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec. RT-PCR products were fractionated on a 2% agarose gel, and signals were quantified as previously described [7]. Percentage contamination of theca cells by granulosa cells was estimated by determining the amount of granulosa cell RNA that produced an aromatase signal equivalent to that produced by 0.6 µg theca cell RNA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Standing estrus was observed 2–4 days after animals were injected with PGF2. On Day 5 after estrus, one follicle in each animal had become dominant and was larger than the second largest follicle of the same wave by at least 4 mm (Table 1). All subordinate follicles analyzed were atretic and had stopped growing before Day 5 after estrus. Dominant follicles stopped growing 6–8 days after estrus. These follicles were isolated, after 4 days of no growth, on Day 10 (n = 4), 11 (n = 2), or 12 (n = 1) of the estrous cycle, and are referred to as D11 follicles. Preovulatory (D1) follicles were collected 10–14 h after estrus was observed.
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Estradiol concentration confirmed whether follicles isolated at different stages of development were healthy or atretic. Follicular fluid from D5 dominant and preovulatory follicles had high levels of estradiol, while follicular fluid from D5 subordinate and D11 dominant follicles had low levels of estradiol (P < 0.05; Table 1).
Expression of Fas Antigen mRNA
Fas antigen expression was detected by competitive RT-PCR in granulosa (Fig. 1) and theca (Fig. 2) cell fractions isolated from all follicles. Granulosa cells from atretic subordinate follicles expressed 5.3-fold more Fas antigen mRNA than granulosa cells from the healthy dominant follicles on Day 5 (P < 0.05). Granulosa cells from D11 atretic dominant follicles expressed Fas antigen mRNA at levels similar to those observed in granulosa cells from healthy dominant follicles on Day 5 or preovulatory follicles on Day 1.
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Theca isolated from healthy dominant follicles on Day 5 expressed less Fas antigen mRNA than subordinate follicles, and they expressed less Fas antigen mRNA than theca from atretic D11 dominant and D1 preovulatory follicles (P < 0.05). Fas antigen mRNA levels in theca were approximately 5% of levels observed in granulosa cells. To test whether Fas antigen mRNA in theca was due to contamination with granulosa cells, aromatase mRNA levels were measured in granulosa and theca cells isolated from healthy D5 dominant follicles by RT-PCR. Granulosa cells from healthy follicles are known to express aromatase while theca do not [13]. Theca samples contained 0.08 ± 0.02% (n = 2 follicles) of the aromatase message found in granulosa cells, indicating that contamination of theca samples with granulosa cells was not significant.
FasL-Induced Killing of Follicle Cells
In experiments testing responsiveness of cells to FasL-induced apoptosis, cells were treated with or without IFN, as our previous studies had shown that IFN stimulates Fas antigen expression in bovine granulosa and theca cells and sensitizes cells to FasL-induced apoptosis in vitro [7]. FasL killed granulosa cells isolated from all first-wave follicles (D5 and D11 dominant and subordinate follicles) in the presence but not absence of IFN treatment (Fig. 3). Percentage killing of granulosa cells by FasL, relative to that in control cultures, was greater in subordinate compared to dominant follicles on Day 5 (65 ± 6 vs. 41 ± 4%; P < 0.05), but was similar in healthy D5 (41.4 ± 4%) and atretic D11 (36 ± 7%) dominant follicles. In contrast, granulosa cells from preovulatory follicles were completely resistant to Fas-mediated killing even when treated with IFN (Fig. 3).
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FasL was not cytotoxic to theca in the absence of IFN. FasL killed 28–42% of theca cells treated with IFN, but no differences in responsiveness to Fas-mediated killing were observed among follicles (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Interestingly, granulosa cells from healthy preovulatory follicles were completely resistant to FasL-induced killing in the presence of IFN, while theca cells from the same follicles were susceptible. Both cell types expressed Fas antigen mRNA, but Fas antigen protein expression was not tested. These follicles were isolated 10–14 h after observation of estrus. Since the LH surge occurs approximately 2–3 h after the onset of estrus [18], these follicles were exposed to a preovulatory surge of LH in vivo. We postulate that the further differentiation of granulosa cells to the preovulatory stage renders them resistant to Fas antigen-mediated killing and that this change does not occur in the theca cells. Previous reports have shown that the preovulatory LH surge induces dramatic changes in granulosa cell function including changes in steroidogenic enzyme expression, increased capacity to secrete oxytocin [19], and induction of prostaglandin endoperoxide synthase-2 expression [20]. In the hen, susceptibility of granulosa cells to apoptosis is dependent upon the stage of follicle development. Avian granulosa cells are inherently susceptible to apoptosis before selection into the preovulatory hierarchy but become apoptosis resistant after selection [21]. Further experiments are required to test whether exposure to an LH surge in vivo or LH treatment in vitro renders granulosa cells resistant to FasL-induced killing.
The expression of Fas antigen mRNA within the theca layer was 5% of that in the granulosa cell layer. Despite this difference, the percentage of theca cells susceptible to FasL-induced killing in the presence of IFN in vitro was within the range observed for granulosa cells isolated during the first follicular wave. The distribution of Fas antigen mRNA and protein within the theca cell layer should be assessed in future studies to determine whether the majority of theca cells express Fas antigen at a relatively low level or whether only a small percentage of theca cells express Fas antigen. Additional studies are necessary to determine the relative importance of the Fas antigen pathway in regression of the granulosa versus theca cells during follicle atresia.
IFN rendered cultured cells from some follicles susceptible to FasL-induced killing. It is unclear whether these cells are normally resistant in vivo or exactly how IFN increases responsiveness to FasL-induced killing. It may be related to IFN's induction of Fas antigen expression, since IFN induced 5.3- and 4.3-fold increases in Fas antigen mRNA in bovine granulosa and theca cells, respectively [7]. However, IFN may have additional effects on the cell death pathway. IFN has been shown to induce expression of pro-apoptotic factors including interleukin-1ß-converting enzyme (Ice) family members (caspases) in human lung epithelial cells [22] and in the human colon adenocarcinoma cell line (HT-29) [23]. IFN induces expression of pro-apoptotic Bcl-2 family members Bak in HT-29 cells [23], and Bax in a human colorectal carcinoma cell line (COLO 201) while reducing expression of anti-apoptotic Bcl-2 in COLO 201 cells [24]. Although IFN has been measured in follicular fluid [25,26] and affects ovarian cell steroidogenesis and inhibin production in vitro (reviewed in [7]), it is not known whether IFN is a physiological regulator of cell death in the ovary. Other unidentified factors may be required for activation of Fas antigen-mediated death in the ovary.
Follicle cell resistance to Fas antigen-mediated apoptosis may be regulated by labile protein inhibitors. Inhibiting protein synthesis with cycloheximide potentiates Fas antigen-mediated killing in ovarian and other cell types [6,27]. In addition, specific inhibitors of apoptosis have been identified in ovarian and other cell types [6,28–31]. Several of these inhibitors have been shown to inhibit Fas antigen-mediated cell death [29,31]. Growth factors and cytokines involved in follicle cell signaling suppress apoptosis in many cell types [1,32] and may also modulate Fas antigen-mediated killing [33].
These results emphasize the importance of studying the functional activity of the Fas antigen pathway in addition to Fas antigen expression. Differences in expression of Fas antigen mRNA and cellular responsiveness to FasL-induced death detected in follicles at various stages of development are consistent with a potential role for the Fas antigen pathway in ovarian follicular atresia.
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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: July 19, 1999.
Received: April 5, 1999.
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, tumor necrosis factor- and cycloheximide. Endocrinology 1998; 139:4860–4869.-hydroxylase, and cytochrome P450 aromatase in bovine follicles during the first follicular wave. Endocrinology 1995; 136:981–989.[Abstract] and Fas. Am J Physiol 1997; 273:L921-L929. modulates a p53-independent apoptotic pathway and apoptosis-related gene expression. J Biol Chem 1997; 272:16351–16357.) and/or interferon-gamma (IFN-), resulting from down-regulation of Bcl-2 expression. Clin Exp Immunol 1998; 111:211–218.[CrossRef][Medline]This article has been cited by other articles:
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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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J. W Kim, R. P Rhoads, N. Segoale, N. B Kristensen, D. E Bauman, and Y. R Boisclair Isolation of the cDNA encoding the acid labile subunit (ALS) of the 150 kDa IGF-binding protein complex in cattle and ALS regulation during the transition from pregnancy to lactation. J. Endocrinol., June 1, 2006; 189(3): 583 - 593. [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. E Valdez, S P. Cuneo, and A. M Turzillo Regulation of apoptosis in the atresia of dominant bovine follicles of the first follicular wave following ovulation Reproduction, July 1, 2005; 130(1): 71 - 81. [Abstract] [Full Text] [PDF]
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 R. Z. Spaczynski, J. L. Tilly, A. Mansour, and A. J. Duleba Insulin and insulin-like growth factors inhibit and luteinizing hormone augments ovarian theca-interstitial cell apoptosis Mol. Hum. Reprod., May 1, 2005; 11(5): 319 - 324. [Abstract] [Full Text] [PDF]
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A.C.O. Evans, J.L.H. Ireland, M.E. Winn, P. Lonergan, G.W. Smith, P.M. Coussens, and J.J. Ireland Identification of Genes Involved in Apoptosis and Dominant Follicle Development During Follicular Waves in Cattle Biol Reprod, May 1, 2004; 70(5): 1475 - 1484. [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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H. F. Irving-Rodgers, M. Krupa, and R. J. Rodgers Cholesterol Side-Chain Cleavage Cytochrome P450 and 3{beta}-Hydroxysteroid Dehydrogenase Expression and the Concentrations of Steroid Hormones in the Follicular Fluids of Different Phenotypes of Healthy and Atretic Bovine Ovarian Follicles Biol Reprod, December 1, 2003; 69(6): 2022 - 2028. [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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J. S. Richards, S. C. Sharma, A. E. Falender, and Y. H. Lo Expression of FKHR, FKHRL1, and AFX Genes in the Rodent Ovary: Evidence for Regulation by IGF-I, Estrogen, and the Gonadotropins Mol. Endocrinol., March 1, 2002; 16(3): 580 - 599. [Abstract] [Full Text] [PDF]
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 J. S. Richards, D. L. Russell, S. Ochsner, M. Hsieh, K. H. Doyle, A. E. Falender, Y. K. Lo, and S. C. Sharma Novel Signaling Pathways That Control Ovarian Follicular Development, Ovulation, and Luteinization Recent Prog. Horm. Res., January 1, 2002; 57(1): 195 - 220. [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. L. Vickers, R. G. Cowan, R. M. Harman, D. A. Porter, and S. M. Quirk Expression and Activity of the Fas Antigen in Bovine Ovarian Follicle Cells Biol Reprod, January 1, 2000; 62(1): 54 - 61. [Abstract] [Full Text]
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