Hum. Reprod. Advance Access originally published online on February 9, 2007
Human Reproduction 2007 22(5):1247-1252; doi:10.1093/humrep/del519
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Regulation of granulosa cell proliferation and EGF-like ligands during the periovulatory interval in monkeys
1 Department of Physiology, Medical College of Georgia, Augusta, GA, USA 2 Department of Obstetrics, Gynecology and Reproductive Sciences, University of Maryland School of Medicine, Baltimore, MD, USA 3 California National Primate Research Center, Davis, CA, USA
4 To whom correspondence should be addressed at: Department of Obstetrics, Gynecology and Reproductive Sciences, University of Maryland School of Medicine, Baltimore, MD, 21201, USA. E-mail: cchaffin{at}upi.umaryland.edu
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Abstract |
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BACKGROUND: This study seeks to clarify cell cycle dynamics of granulosa cells following hCG and elucidate the expression of epidermal growth factor (EGF)-like ligands during luteinization.
METHODS: Granulosa cells were obtained from rhesus macaques undergoing controlled ovarian stimulation protocols before or after an ovulatory hCG bolus. Cell cycle characteristics were determined by flow cytometry and levels of EGF receptor (EGFR), amphiregulin (AREG), epiregulin (EREG) and betacellulin (BTC) mRNAs were measured by real-time RT-PCR.
RESULTS: The proportion of cells in S-phase was 7.5% prior to hCG and did not decline until 24 h after hCG (3.1%). EGFR protein and BTC mRNA did not change following hCG, whereas AREG and EREG mRNA increased starting at 3 and 12 h post-hCG, respectively, and remained elevated thereafter.
CONCLUSIONS: Cell cycle transit of macaque granulosa cells does not change until 24 h after an ovulatory stimulus, whereas the EGF-like ligands EREG and AREG are increased rapidly. This suggests that luteinizing granulosa cells are refractory to mitogenic stimulation by EGFR ligands.
Key words: non-human primate/granulosa cell/proliferation/luteinization/epidermal growth factor
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Introduction |
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The midcycle surge of luteinizing hormone (LH) in humans and non-human primates initiates a cascade of ‘periovulatory’ events culminating in the extrusion of a fertilizable oocyte and remodelling of the follicle into a functional corpus luteum (CL). In addition, formation of the CL has classically been considered to consist at least in part of the differentiation of granulosa into luteal cells, and with that, has been associated with the abrupt exit of granulosa cells from the cell cycle (Robker and Richards, 1998a
,b; Chaffin et al., 2001; Quirk et al., 2004). However, the dynamics and regulation of granulosa cell proliferation during the early portion of the periovulatory interval in primates remain poorly defined.
The periovulatory interval is associated with the expression of numerous mitogenic growth factors, including members of the epidermal growth factor (EGF) family (Freimann et al., 2004; Freimann et al., 2005; Conti et al., 2006). In rodents, expression of the EGF-like ligands epiregulin (EREG), amphiregulin (AREG) and betacellulin (BTC) increases rapidly in response to an ovulatory gonadotrophin stimulus (Park et al., 2004; Sekiguchi et al., 2004; Ashkenazi et al., 2005), and it is clear in several species that EGF-like ligands mediate expansion of the cumulus—oocyte complex (COC) (Prochazka et al., 2003; Park et al., 2004; Bolamba et al., 2006; Conti et al., 2006). EGFs may also promote steroidogenesis by cumulus cells (Jamnongjit et al., 2005; Shimada et al., 2006), although the actions of EGFs on mural granulosa cell steroidogenesis are not clear and may depend on the degree of differentiation (Stevenson, 2000; Hernandez and Bahr, 2003; Rusovici et al., 2005).
EGFs are potent granulosa cell mitogens (Tapanainen et al., 1987; Roy, 1993; Vitale et al., 2006). EGF receptor (EGFR) protein expression is evident in pre-ovulatory follicles in hamster, rat, porcine and human ovaries, and is detectable at low levels in CL throughout the luteal phase (Fujinaga et al., 1992; Assarsson et al., 1995; Tamura et al., 1995; Garnett et al., 2002; Akayama et al., 2005). In addition, there is cross-talk between EGF and other signalling systems, including insulin and insulin-like growth factors (Roudabush et al., 2000; Adams et al., 2004). Thus, although EGFs are likely to play a central role in periovuatory processes, the expression of these factors during the early stages of luteinization in primate granulosa cells is not known. In general, the potential interaction between mitogenic growth factors such as EGF-like ligands and cell cycle dynamics during luteinization and CL formation remains a poorly understood, albeit potentially important, area.
Studies were undertaken therefore to elucidate the effects of an ovulatory hCG bolus given to rhesus monkeys undergoing controlled ovarian stimulation on the proliferation and EGF-like ligand expression by mural granulosa cells. Two specific hypotheses were tested: (i) an ovulatory stimulus leads to rapid withdrawal from the cell cycle and (ii) an ovulatory stimulus induces expression of EREG, AREG and BTC.
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Materials and methods |
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Animals
Adult female rhesus macaques (Macaca mulatta) were housed at the California National Primate Research Center (CNPRC) as described (VandeVoort and Tarantal, 1991
). Animal protocols and experiments were approved by the University of California, Davis Animal Care and Use Committee, and studies were conducted in accordance with Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). Following the onset of menstruation, adult female rhesus monkeys were treated with recombinant human follicle stimulating hormone (r-hFSH, Organon, West Orange, NJ, USA; 37.5 IU i.m. bid) for 7 days. The GnRH antagonist Antide (Ares-Serono, Randolph, MA, USA, 5 mg/kg body weight, s.c., sid) was administered on days 6–7 to prevent endogenous gonadotrophin secretion. Follicles were aspirated the morning after the last dose of r-hFSH by an ultrasound-guided procedure as described previously (VandeVoort and Tarantal, 1991), and the characteristics of the follicular cohort in this model have been described (VandeVoort et al., 2003). The resulting cells are referred to as non-luteinized granulosa cells (NLGC). A subset of animals received an ovulatory bolus of r-hCG (1000 IU, s.c., Ares-Serono) on the morning of day 8 and follicles were aspirated before (0 h), 3, 6, 12 and 24 h after hCG (n 
3 per time point). Aspirates represented the pooled contents of multiple follicles from each animal. These were maintained at 
35°C within a temperature-controlled isolette containing a fixed volume of media at all times. COCs were removed by transferring the mixture of media, follicular fluid and cellular aspirate to a 24 mm diameter, 70-µM filter (Netwell Inserts #3479, Corning, Inc., Acton, MA, USA); granulosa cells were filtered through the mesh and COC retained. To collect granulosa cells for the corresponding time points, the tube was rinsed with fresh tyrode lactate (TL)-HEPES/polyvinylalcohol (PVA) medium (TL-HEPES/0.1 mg ml–1 PVA) that was also poured through the filter. The filter was further rinsed with fresh TL-Hepes-PVA medium until blood cells were removed. The rinse from the filter was saved for recovery of granulosa cells for various experiments (see below).
Preparation of macaque granulosa cells
Granulosa cells were recovered from the filter rinse by a modification of the method previously described (Stewart and Vandevoort, 1997). Briefly, the cell suspension was centrifuged for 5 min at 300g to pellet the red cells; this was then increased to 500g for an additional 5 min, resulting in a thin layer of granulosa cells over the red cell pellet. The supernatant was removed, and the layer of granulosa cells was transferred to a 40% Percoll gradient in medium 199 (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged for 30 min at 500g. The supernatant was removed, and the granulosa cells were recovered from the surface of the Percoll with a Pasteur pipette and washed once with TL-HEPES/PVA. The cell pellet was resuspended in 1 ml TL-HEPES/PVA and counted on hemocytometer. An additional 14 ml TL-HEPES/PVA supplemented with 5 µg ml–1 r-hFSH was added to the cell suspension. Cells were placed in a biohazard shipping container and were shipped from the CNPRC to the University of Maryland Baltimore by overnight delivery at ambient temperature from September to June. Upon receipt, cells were recovered by centrifugation (Chaffin et al., 2003).
Flow cytometric analysis
Cell cycle analysis was undertaken for granulosa cells aspirated from follicles before and after hCG (n = 8, 6, 4, 5, 18, respectively) by flow cytometry. 1 x 106 granulosa cells ml–1 were fixed with 70% ice-cold ETOH for >24 h. Samples were stained with 1 ml propidium iodide (PI) solution containing RNAse A (100 U ml–1). Stained cells were held at room temperature for at least 1 h before fluorescence-activated cell sorter analysis. Immediately before analysis, cells were syringed up and down several times using a 1 cc insulin syringe (BD Biosciences, Bedford, MA, USA), then passed through a Falcon 35 µM nylon mesh cell strainer cap (BD Biosciences) to obtain a single cell suspension and remove aggregated cells. Subsequent cell staging was performed on either a FACSCalibur or FACSVantage flow cytometry system.
Real time RT–PCR
Total granulosa cell RNA was extracted from cells using the RNAqeous Micro kit (Ambion Inc., Austin, TX, USA) and reverse transcribed using moloney murine leukaemia virus reverse transcriptase (Invitrogen, Carlsbad, CA, USA). EGFR Primers and 6FAM-labelled probes were synthesized by Applied Biosystems (Foster City, CA, USA) as well as primers and VIC-labelled probe for internal control ribosomal protein L19 (RPL19). Target gene and RPL19 were detected in the same reaction. Semi-quantitative real time experiments using Assays-on-Demand Gene Expression products (Applied Biosystems) for AREG (Hs00155832_m1), EREG (Hs00154995_m1) and BTC (Hs00156140_m1) were performed per manufacturer's instructions. The protocol consisted of 40 cycles of denaturing at 95°C for 15 s and annealing/extending at 60°C for 1 min per cycle. Detection of gene expression was performed during the second step in a two-step RT-PCR protocol. To relatively quantify mRNA levels, a standard curve was constructed using pooled macaque granulosa cell cDNA. Data were analysed as the inverse log [(Ct-Y intercept)/slope of the standard curve] and expressed as a ratio of target gene: endogenous control. Additional primer and probe sequences are as shown in Table I.
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Western blot
Cells were sonicated briefly in cell lysis buffer (Cell Signalling technology, Danvers, MA). Supernatants were cleared by centrifugation at 16 000g x 10 min and protein concentration determined by BCA protein assay kit (Pierce, Rockford, IL, USA). Proteins (35 µg) were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% milk in Tris buffered saline containing 0.05% Tween, and probed with a rabbit anti-EGFR antibody (ab2430, Abcam, Cambridge, MA, USA) diluated 1:600 in blocking buffer. Secondary anti-rabbit antibody (Pierce, Rockford, IL, USA) was used at 1:1000 dilution. Membranes were reprobed with an antibody against total ERK (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as a loading control. Protein bands were visualized with Supersignal West Dura chemiluminescent detection reagents (Pierce) per manufacturer's directions.
Statistical analysis
Data are presented as mean ± SEM. Bartlett's 
2 was used to test for heterogeneity of variance, and data were logarithmically transformed (log + 2) if variances were significantly different. Data were analysed by one-way ANOVA followed by a Student–Newman–Keuls means comparison test. Means were considered significantly different if P < 0.05. N 3 animals per treatment unless indicated.
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Results |
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Concentrations of intra-follicular progesterone increased 44-fold by 6 h post-hCG, and remained elevated through 24 h, verifying luteinization in response to hCG (Fru et al., 2006
). Granulosa cells aspirated before (0 h), 3, 6, 12 and 24 h after an ovulatory hCG bolus were analysed by flow cytometry for stage of the cell cycle. The percentage of cells in S-phase did not decline until 24 h post-hCG (0 h: 7.5% ± 1.3; 24 h: 3.1% ± 0.36; P < 0.05; Figure 1). No significant differences were observed for cells in G0/G1 or G2/M phases of the cell cycle.
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The expression of EGFR mRNA in periovulatory granulosa cells declined 9-fold (P < 0.05) within 3 h of the ovulatory hCG injection and partially returned to pre-hCG levels thereafter (Figure 2). However, levels of total EGFR protein were not altered significantly within 12 h after hCG administration (Figure 2).
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The expression of AREG, EREG and BTC mRNA was examined before and up to 24 h after hCG. AREG mRNA was undetectable by RT–PCR in any sample prior to the ovulatory bolus, but was induced starting 3 h post-hCG (P < 0.05; Figure 3). The expression of EREG mRNA increased 12-fold by 12 h following hCG treatment (P < 0.05) and remained elevated at 24 h (11-fold versus 0 h; P < 0.05; Figure 3). In contrast, BTC mRNA tended to decrease 3 h post-hCG (2.9-fold, P = 0.07, not significant; Figure 3) and was not different than 0 h values at any time point following hCG.
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Discussion |
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Granulosa cell proliferation during the periovulatory interval remains poorly understood. The overall goal of this project was to discern changes in granulosa cell proliferation following an ovulatory gonadotrophin bolus to rhesus monkeys undergoing controlled ovarian stimulation and to elucidate the expression of EGF-like ligands during the periovulatory interval. Granulosa cell proliferation in rats has been reported to be sharply reduced following an ovulatory stimulus whereas the expression of EGF family members increases (Robker and Richards, 1998a
,b; Quirk et al., 2004; Conti et al., 2006), suggesting a possible paradox in the regulation of granulosa cell proliferation. EGFs are also potential survival signals for granulosa cells (Tilly et al., 1992; Mao et al., 2004), creating a complex situation in which the potentially anti-apoptotic effects of EGFs are balanced against their mitogenic actions during luteinization.
The first aim of this project was to begin to clarify cell cycle dynamics of primate granulosa cells following an ovulatory stimulus. The transit of pre-ovulatory (0 h) granulosa cells through S-phase is at a low rate (7.5%), consistent with the hypothesis that proliferation in pre-ovulatory follicles is reduced relative to that seen in developing antral follicles (Hirshfield, 1991; Gougeon, 1998; Cannon et al., 2005). Previous studies of rhesus monkeys undergoing a similar controlled ovarian stimulation paradigm used Ki-67 immunostaining as a marker of proliferation and found nearly 50% of granulosa cells in pre-hCG (0 h) follicles positive for the antigen (Chaffin et al., 2001). However, Ki-67 is expressed in all phases of the cell cycle except G0, potentially inflating estimates of proliferation (Gerdes, 1990; Brenner et al., 2003). Following hCG, the percentage of cells in S-phase is maintained for 12 h before a modest decline occurs at 24 h. These data are in contrast to bovine and rat, in which an ovulatory stimulus induces a rapid exit of mural granulosa cells from the cell cycle (Robker and Richards, 1998a,b; Quirk et al., 2004; Cannon et al., 2005). The reason for this discrepancy is not clear, but could indicate that the limited proportion (<10%) of S-phase cells during the periovulatory interval represents a basal rate of proliferation. Consistent with this hypothesis, 5% of steroidogenic luteal cells in the primate continue to proliferate during the luteal phase (Christenson and Stouffer, 1996; Gaytan et al., 1998; Young et al., 2000). This low level of persistent proliferation may be due to the presence of mitogenic growth factors such as EGFs.
EGFR protein is expressed in the luteinizing follicle as well as the CL (Tamura et al., 1995; Garnett et al., 2002; Prochazka et al., 2003), underscoring a potentially important role for this receptor in the ovary. While an ovulatory stimulus rapidly suppresses EGFR mRNA, levels of total EGFR protein do not change following hCG, suggesting that mural granulosa cells retain the ability to respond to EGFs during the early portion of the periovulatory interval. Given the limited sample availability, phospho-EGFR was not assessed. However, recent data suggest that inhibition of EGFR in human luteinized granulosa cells causes apoptosis (Khan et al., 2005), suggesting that the EGFR is functional and capable of being activated during the periovulatory interval. Importantly, BTC mRNA is present before and after an ovulatory hCG stimulus, suggesting that this particular EGFR ligand plays a role in pre-ovulatory follicles. It is interesting to note that BTC mRNA is not expressed at appreciable levels prior to hCG in rats (Park et al., 2004; Ashkenazi et al., 2005), denoting a potentially important species difference in the function of EGFs. AREG and EREG mRNAs, in contrast, are induced by the ovulatory stimulus, suggesting their likely importance during the periovulatory interval. Other studies have shown increases in AREG and EREG by human granulosa-lutein cells in response to a cAMP stimulus as well as prostaglandin E2 (Freimann et al., 2004; Ben-Ami et al., 2006), indicating the convergence of multiple signalling pathways in the regulation of these genes. Recently, EREG and AREG mRNA expression has been reported in mouse cumulus cells (Hernandez-Gonzalez et al., 2006; Shimada et al., 2006), whereas EGFR protein is present in cumulus cells of the primates during in vitro maturation protocols (unpublished observation), suggesting an autocrine/paracrine role for EGF-like ligands on cumulus cells that may be independent of actions on mural granulosa cells. Although it remains likely that hCG-induced EGFs play a role in cumulus cell expansion and meiotic resumption in primates as has been reported for mice and rats (Ashkenazi et al., 2005; Jamnongjit et al., 2005; Conti et al., 2006), this remains to be experimentally tested.
The mitogenic actions of EGFs on granulosa cells are clear (Chaffkin et al., 1993), and given the presence of EGFR protein along with the putative increase in EGF-like ligands following hCG, it is not apparent why the proportion of granulosa cells in S-phase does not increase. Clearly, some yet to be defined mechanism exists to prevent transit through the cell cycle, possibly LH/CG induction of cyclin-dependent kinase inhibitors such as p21 and p27 (Chaffin et al., 2001). This would suggest that EGFs have critical actions outside their traditional role in growth promotion, possibly in steroidogenesis. EGFs have been reported to regulate steroidogenesis in human granulosa cells, although the effects of EGFs on early periovulatory steroidogenesis are not known (Stevenson, 2000). Interestingly, Jamnongjit et al. (2005) showed that although EGFs were only modestly able to stimulate progesterone synthesis by isolated mouse follicles relative to LH, treatment with an EGFR antagonist in conjunction with LH completely attenuated progesterone synthesis, suggesting an interaction between the ovulatory gonadotrophin stimulus and the EGFR.
In summary, an ovulatory hCG bolus to rhesus monkeys undergoing controlled ovarian stimulation does not result in dramatic changes in mural granulosa cell proliferation, although AREG and EREG mRNAs are rapidly induced, and BTC mRNA and EGFR protein expression is maintained. The data suggest that luteinizing primate granulosa cells may be refractory to strong mitogenic signals such as from EGFR ligands.
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The authors thank Dr Jennifer Cannon for technical assistance. They are grateful to Organon Inc., West Orange, NJ, USA, for the generous supply of recombinant human FSH and Serono laboratories (Ares Advanced Technology), Randolph, MA, USA, for the gift of recombinant hCG. This research was supported in part by NIH HD043358 (CLC), RR13439 (CAV), RR00169 (CNPRC), NIH HD047964 (KNF) and UNCF/Merck fellowship (KNF).
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Adams TE, McKern NM, Ward CW. (2004) Signalling by the type 1 insulin-like growth factor receptor: interplay with the epidermal growth factor receptor. Growth Factors 22:89–95.[CrossRef][Web of Science][Medline]
Akayama Y, Takekida S, Ohara N, Tateiwa H, Chen W, Nakabayashi K, Maruo T. (2005) Gene expression and immunolocalization of heparin-binding epidermal growth factor-like growth factor and human epidermal growth factor receptors in human corpus luteum. Hum Reprod 20:2708–2714.
Ashkenazi H, Cao X, Motola S, Popliker M, Conti M, Tsafriri A. (2005) Epidermal growth factor family members: endogenous mediators of the ovulatory response. Endocrinology 146:77–84.
Assarsson B, Nilsson I, Selstam G. (1995) Ovarian levels of epidermal growth factor receptor mRNA in the rat—a postovulatory decrease. Acta Physiol Scand 154:177–183.[Web of Science][Medline]
Ben-Ami I, Freimann S, Armon L, Dantes A, Strassburger D, Friedler S, Raziel A, Seger R, Ron-El R, Amsterdam A. (2006) PGE2 up-regulates EGF-like growth factor biosynthesis in human granulosa cells: new insights into the coordination between PGE2 and LH in ovulation. Mol Hum Reprod 12:593–599.
Bolamba D, Russ KD, Harper SA, Sandler JL, Durrant BS. (2006) Effects of epidermal growth factor and hormones on granulosa expansion and nuclear maturation of dog oocytes in vitro. Theriogenology 65:1037–1047.[CrossRef][Web of Science][Medline]
Brenner RM, Slayden OD, Rodgers WH, Critchley HO, Carroll R, Nie XJ, Mah K. (2003) Immunocytochemical assessment of mitotic activity with an antibody to phosphorylated histone H3 in the macaque and human endometrium. Hum Reprod 18:1185–1193.
Cannon JD, Cherian-Shaw M, Chaffin CL. (2005) Proliferation of rat granulosa cells during the periovulatory interval. Endocrinology 146:414–422.
Chaffin CL, Schwinof KM, Stouffer RL. (2001) Gonadotropin and steroid control of granulosa cell proliferation during the periovulatory interval in rhesus monkeys. Biol Reprod 65:755–762.
Chaffin CL, Brogan RS, Stouffer RL, VandeVoort CA. (2003) Dynamics of Myc/Max/Mad expression during luteinization of primate granulosa cells in vitro: association with periovulatory proliferation. Endocrinology 144:1249–1256.
Chaffkin LM, Luciano AA, Peluso JJ. (1993) The role of progesterone in regulating human granulosa cell proliferation and differentiation in vitro. J Clin Endocrinol Metab 76:696–700.[Abstract]
Christenson LK and Stouffer RL. (1996) Proliferation of microvascular endothelial cells in the primate corpus luteum during the menstrual cycle and simulated early pregnancy. Endocrinology 137:367–374.[Abstract]
Conti M, Hsieh M, Park JY, Su YQ. (2006) Role of the epidermal growth factor network in ovarian follicles. Mol Endocrinol 20:715–723.
Freimann S, Ben-Ami I, Dantes A, Ron-El R, Amsterdam A. (2004) EGF-like factor epiregulin and amphiregulin expression is regulated by gonadotropins/cAMP in human ovarian follicular cells. Biochem Biophys Res Commun 324:829–834.[CrossRef][Web of Science][Medline]
Freimann S, Ben-Ami I, Dantes A, Armon L, Ben Ya'cov-Klein A, Ron-El R, Amsterdam A. (2005) Differential expression of genes coding for EGF-like factors and ADAMTS1 following gonadotropin stimulation in normal and transformed human granulosa cells. Biochem Biophys Res Commun 333:935–943.[CrossRef][Web of Science][Medline]
Fru KN, Vandevoort CA, Chaffin CL. (2006) Mineralocorticoid synthesis during the periovulatory interval in macaques. Biol Reprod 75:568–574.
Fujinaga H, Yamoto M, Nakano R, Shima K. (1992) Epidermal growth factor binding sites in porcine granulosa cells and their regulation by follicle-stimulating hormone 10.1095/biolreprod46.4.705. Biol Reprod 46:705–709.[Abstract]
Garnett K, Wang J, Roy SK. (2002) Spatiotemporal expression of epidermal growth factor receptor messenger RNA and protein in the hamster ovary: follicle stage-specific differential modulation by follicle-stimulating hormone, luteinizing hormone, estradiol, and progesterone. Biol Reprod 67:1593–1604.
Gaytan F, Morales C, Garcia-Pardo L, Reymundo C, Bellido C, Sanchez-Criado JE. (1998) Macrophages, cell proliferation, and cell death in the human menstrual corpus luteum. Biol Reprod 59:417–425.
Gerdes J. (1990) Ki-67 and other proliferation markers useful for immunohistological diagnostic and prognostic evaluations in human malignancies. Semin Cancer Biol 1:199–206.[Medline]
Gougeon A. (1998) Ovarian follicular growth in humans: ovarian ageing and population of growing follicles. Maturitas 30:137–142.[CrossRef][Web of Science][Medline]
Hernandez A and Bahr J. (2003) Role of FSH and epidermal growth factor (EGF) in the initiation of steroidogenesis in granulosa cells associated with follicular selection in chicken ovaries. Reproduction 125:683–691.[Abstract]
Hernandez-Gonzalez I, Gonzalez-Robayna I, Shimada M, Wayne CM, Ochsner SA, White L, Richards JS. (2006) Gene expression profiles of cumulus cell oocyte complexes (COCs) during ovulation reveal cumulus cells express neuronal and immune-related genes: Does this expand their role in the ovulation process? Mol Endocrinol 20:1300–1321.
Hirshfield AN. (1991) Development of follicles in the mammalian ovary. Int Rev Cytol 124:43–101.[Web of Science][Medline]
Jamnongjit M, Gill A, Hammes SR. (2005) Epidermal growth factor receptor signaling is required for normal ovarian steroidogenesis and oocyte maturation. Proc Natl Acad Sci U S A 102:16257–16262.
Khan SM, Oliver RH, Yeh J. (2005) Epidermal growth factor receptor inhibition by tyrphostin 51 induces apoptosis in luteinized granulosa cells 10.1210/jc.2004-0454. J Clin Endocrinol Metab 90:469–473.
Mao J, Smith MF, Rucker EB, Wu GM, McCauley TC, Cantley TC, Prather RS, Didion BA, Day BN. (2004) Effect of epidermal growth factor and insulin-like growth factor I on porcine preantral follicular growth, antrum formation, and stimulation of granulosal cell proliferation and suppression of apoptosis in vitro. J Anim Sci 82:1967–1975.
National Research Council. (1996) Guide for the Care and Use of Laboratory Animals(National Academy Press, Washington, DC).
Park J-Y, Su Y-Q, Ariga M, Law E, Jin S-LC, Conti M. (2004) EGF-like growth factors as mediators of LH action in the ovulatory follicle. Science 303:682–684.
Prochazka R, Kalab P, Nagyova E. (2003) Epidermal growth factor-receptor tyrosine kinase activity regulates expansion of porcine oocyte-cumulus cell complexes in vitro. Biol Reprod 68:797–803.
Quirk SM, Cowan RG, Harman RM. (2004) Progesterone receptor and the cell cycle modulate apoptosis in granulosa cells. Endocrinology 145:5033–5043.
Robker RL and Richards JS. (1998a) Hormonal control of the cell cycle in ovarian cells: proliferation versus differentiation. Biol Reprod 59:476–482.
Robker RL and Richards JS. (1998b) Hormone-induced proliferation and differentiation of granulosa cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27Kip1. Mol Endocrinol 12:924–940.
Roudabush FL, Pierce KL, Maudsley S, Khan KD, Luttrell LM. (2000) Transactivation of the EGF receptor mediates IGF-1-stimulated Shc phosphorylation and ERK1/2 activation in COS-7 cells. J Biol Chem 275:22583–22589.
Roy S. (1993) Epidermal growth factor and transforming growth factor-beta modulation of follicle-stimulating hormone-induced deoxyribonucleic acid synthesis in hamster preantral and early antral follicles. Biol Reprod 48:552–557.[Abstract]
Rusovici R, Hui YY, Lavoie HA. (2005) Epidermal growth factor-mediated inhibition of follicle-stimulating hormone-stimulated StAR gene expression in porcine granulosa cells is associated with reduced histone H3 acetylation. Biol Reprod 72:862–871.
Sekiguchi T, Mizutani T, Yamada K, Kajitani T, Yazawa T, Yoshino M, Miyamoto K. (2004) Expression of epiregulin and amphiregulin in the rat ovary. J Mol Endocrinol 33:281–291.[Abstract]
Shimada M, Hernandez-Gonzalez I, Gonzalez-Robayna I, Richards JS. (2006) Paracrine and autocrine regulation of epidermal growth factor-like factors in cumulus oocyte complexes and granulosa cells: key roles for prostaglandin synthase 2 and progesterone receptor. Mol Endocrinol 20:1352–1365.
Stevenson AF. (2000) Human granulosa cells in vitro: characteristics of growth, morphology and influence of some cytokines on steroidogenesis. Indian J Exp Biol 38:1183–1191.[Medline]
Stewart DR and Vandevoort CA. (1997) Simulation of human luteal endocrine function with granulosa lutein cell culture. J Clin Endocrinol Metab 82:3078–3083.
Tamura M, Sasano H, Suzuki T, Fukaya T, Funayama Y, Takayama K, Takaya R, Yajima A. (1995) Expression of epidermal growth factors and epidermal growth factor receptor in normal cycling human ovaries. Hum Reprod 10:1891–1896.
Tapanainen J, Leinonen PJ, Tapanainen P, Yamamoto M, Jaffe RB. (1987) Regulation of human granulosa-luteal cell progesterone production and proliferation by gonadotropins and growth factors. Fertil Steril 48:576–580.[Web of Science][Medline]
Tilly JL, Billig H, Kowalski KI, Hsueh AJ. (1992) Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism. Mol Endocrinol 6:1942–1950.
VandeVoort CA and Tarantal AF. (1991) The macaque model for in vitro fertilization: superovulation techniques and ultrasound-guided follicular aspiration. J Med Primatol 20:110–116.[Web of Science][Medline]
VandeVoort CA, Leibo SP, Tarantal AF. (2003) Improved collection and developmental competence of immature macaque oocytes. Theriogenology 59:699–707.[CrossRef][Web of Science][Medline]
Vitale AM, Abramovich D, Peluffo MC, Meresman G, Tesone M. (2006) Effect of gonadotropin-releasing hormone agonist and antagonist on proliferation and apoptosis of human luteinized granulosa cells. Fertil Steril 85:1064–1067.[CrossRef][Web of Science][Medline]
Young FM, Rodger FE, Illingworth PJ, Fraser HM. (2000) Cell proliferation and vascular morphology in the marmoset corpus luteum. Hum Reprod 15:557–566.
Submitted on August 16, 2006; resubmitted on October 25, 2006; resubmitted on November 14, 2006; accepted on November 29, 2006.
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 I. Ben-Ami, L. Armon, S. Freimann, D. Strassburger, R. Ron-El, and A. Amsterdam EGF-like growth factors as LH mediators in the human corpus luteum Hum. Reprod., January 1, 2009; 24(1): 176 - 184. [Abstract] [Full Text] [PDF]
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