Epidermal Growth Factor (EGF) - Oxford University Press

BIOLOGY OF REPRODUCTION (2014) 90(4):86, 1–10 Published online before print 19 February 2014. DOI 10.1095/biolreprod.113.115089

Epidermal Growth Factor (EGF) Sustains In Vitro Primordial Follicle Viability by Enhancing Stromal Cell Proliferation via MAPK and PI3K Pathways in the Prepubertal, but Not Adult, Cat Ovary1 Mayako Fujihara,2,3 Pierre Comizzoli,3 Carol L. Keefer,4 David E. Wildt,3 and Nucharin Songsasen3 3 4

Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, Virginia Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland

This study examined the influences of epidermal growth factor (EGF) and growth differentiation factor 9 (GDF9) on in vitro viability and activation of primordial follicles in the ovarian tissue of prepubertal (age, ,6 mo) versus adult (age, .8 mo) cats. Ovarian cortical slices were cultured in medium containing EGF and/or GDF9 for 14 days. EGF, but not GDF9, improved (P , 0.05) follicle viability in prepubertal donors in a dosedependent fashion. Neither EGF nor GDF9 enhanced follicle viability in ovarian tissue from adults, and neither factor activated primordial follicles regardless of age group. We then explored how EGF influenced primordial follicles in the prepubertal donors by coincubation with an inhibitor of EGF receptor (AG1478), mitogen-activated protein kinase (MAPK; U0126), or phosphoinositide 3-kinase (PI3K; LY294002). EGF enhanced (P , 0.05) MAPK and AKT phosphorylation, follicle viability, and stromal cell proliferation. These effects were suppressed (P , 0.05) when the tissue was cultured with this growth factor combined with each inhibitor. To identify the underlying influence of age in response to EGF, we assessed cell proliferation and discovered a greater thriving stromal cell population in prepubertal compared to adult tissue. We conclude that EGF plays a significant role in maintaining intraovarian primordial follicle viability (but without promoting activation) in the prepubertal cat. The mechanism of action is via stimulation of MAPK and PI3K signaling pathways that, in turn, promote ovarian cell proliferation. Particularly intriguing is that the ability of cat ovarian cells to multiply in reaction to EGF is age-dependent and highly responsive in prepubertal females. cat, folliculogenesis, in vitro culture, ovary, primordial follicle

INTRODUCTION At the time of birth, each mammalian ovary is comprised of thousands of primordial follicles, each sequestering an early stage, unfertilizable oocyte [1]. Although the majority of follicles initiate growth before reproductive senescence, most (;99%) degenerate at the primordial, primary, or secondary stage and never contribute to reproduction [2]. An ability to develop these premature follicles in vitro to the point where 1 Supported by a grant from the Smithsonian Institution Fellowship Program and a generous gift from Dr. Clinton and Missy Kelly. 2 Correspondence: Mayako Fujihara, Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, 1500 Remount Road, Front Royal, VA 22630. E-mail: [email protected]

Received: 17 October 2013. First decision: 21 November 2013. Accepted: 14 February 2014. Ó 2014 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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maturation-competent oocytes could be rescued holds enormous promise. For example, biopsy of ovarian tissue or recovery of the entire gonad combined with a successful culture protocol could offer a means of salvaging fertility in women, especially girls undergoing cancer therapy [3]. It also could provide vast amounts of germplasm, which is otherwise wasted, from genetically outstanding individuals, ranging from livestock to rare animal models to endangered species, especially from females that die before reaching sexual maturation or adequately reproducing. The production of embryos and offspring from oocytes derived from cultured follicles has been demonstrated. This milestone work involved incubating whole, newborn mouse ovaries to advance growth of primordial follicles to the primary and then secondary stage [4, 5]. While effective for this particular rodent, this same approach is impractical for larger mammals because ovarian mass is too great to culture as an intact unit. Therefore, most other studies that involve the human [4, 6], cow [7], sheep [8], goat [9], and baboon [10] have focused on culturing pieces of ovarian cortex. The result has been that some primordial follicles do grow to the primary or secondary stage in vitro, but to date and to our knowledge, with limited fertilization and no production of living young. Nonetheless, these investigations have been highly informative for beginning to understand follicle activation, including how locally produced factors and peptides stimulate progression beyond the primary stage independent of pituitary gonadotropins [5]. Although the mechanisms are not fully elucidated, it is well recognized that locally produced epidermal growth factor (EGF) and growth differentiation factor 9 (GDF9) are associated with primordial follicles. EGF is a known mitogenic polypeptide involved in regulating cell proliferation in mammals [11]. Specifically, EGF is detectable in the oocytes of human primordial, primary, and preantral follicles as well as granulosa and theca cells of later-stage follicles [12, 13]. EGF receptor (EGFR) also is expressed in the oocytes of human primordial and primary follicles [14]. EGF/EGFR signaling upregulates mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways that, in turn, stimulate proliferation, growth, and survival of various cell types, including cumulus cells and the oocyte itself [15]. For example, EGF has been shown to stimulate increased size in vitro of both the follicle and the oocyte of the goat during the transition from primordial to primary follicle [9]. A recent study in the rat has suggested that EGF/EGFR facilitates growth of primordial to secondary follicles via both MAPK and protein kinase C pathways [16]. A member of the transforming growth factor beta (TGFB)/ activin family, GDF9 is secreted by the oocyte [17], with its action mediated through Smad2/3 in multiple cell types, including granulosa cells [18, 19]. For the rat, GDF9 also is anti-apoptotic in preantral follicles via up-regulation of the

ABSTRACT

FUJIHARA ET AL. removed and immersed in L-15 medium containing 10 mM HEPES, 100 lg/ml of penicillin G sodium, and 100 lg/ml of streptomycin sulfate for transport to the laboratory at 48C within 1–6 h of surgery. Ovarian cortical slices (thickness, 0.5–1 mm) were dissected from the surface of each ovary and then sectioned in equal pieces (width, 1–1.5 mm). Tissue was incubated at 38.58C in 5% CO2 in humidified air under various culture conditions (see below) for 3 or 14 days. Half the volume of the culture medium was exchanged every 48 h throughout the study interval.

PI3K pathway [20]. Ovarian follicles of GDF9-deficient mice do not develop beyond the primary stage, suggesting that this growth factor plays key roles in transitioning from the primary to the secondary stage [21]. However, GDF9 also is involved in primordial follicle activation and early follicle development in vivo in the rat [22] and in vitro in the human [23], goat [24], cow [25], and hamster [26]. Our study model is the domestic cat because of its prevalence as a human companion species, the applicability of data to parallel studies of wild (often endangered) felids [27], and the usefulness for enhancing the field of fertility preservation, including in the human [3]. For example, follicle and oocyte size and nuclear configuration are remarkably similar between cats and women [28]. Investigations of the cat also have been useful for generating new insights into human infertility syndromes, including asynchronous and nuclear maturation [28] as well as ovarian hypersensitivity after gonadotropin therapy [29–31]. The cat also has become a valid model for exploring improved fertility preservation tools, including how to rescue the maternal genome via in vitro follicle development [32, 33]. However, little is known about what regulates primordial follicle viability or activation in any felid species. We know that EGF-positive cells exist in the cortex of the cat ovary and near primordial follicles (presumably in interstitial glands) [34]. Furthermore, EGFbinding sites are found on the granulosa cells of primary, secondary, and tertiary follicles as well as interstitial gland cells [34]. By contrast, GDF9 protein is localized in the cytoplasm of oocytes from both primary and small antral follicles in the cat ovary [35]. To date and to our knowledge, the relationship of ovarian donor age to its responsiveness to growth factors, including impact on follicle viability and activation, has not been explored in any species. We speculated that such a comparative study could provide novel insight into the regulatory mechanisms of primordial follicle development. The justification for taking this approach is derived from studies of fetal gonads. For example, primordial follicles that appear in the fetal bovine ovary by 90 days of gestation require a longer incubation period to be activated than when fetal ovaries are recovered at 140 days of gestation [36]. Likewise, when the gonads of neonatal piglets are xenografted into immunodeficient mice, the pig primordial follicles develop to the antral stage, in contrast to a no-development, static response using ovaries from 6-mo-old gilts [37]. Collectively, these findings suggest that a gap may exist between primordial follicle formation and acquisition of activation capacity that is dependent on age or sexual development. The present study had two purposes. The first was to determine if EGF and GDF9 play a significant role in primordial follicle viability and activation in the domestic cat and, if so, the potential mechanism(s). The second was to examine the influence of donor age on responsiveness to the growth factors, specifically between a prepubertal and adult female.

Assessment of Follicle Viability Follicle viability within the ovarian cortical tissue was evaluated in fresh (day of tissue excision and initial incubation) or after 14 days of culture using calcein-AM/ethidium homodimer-1 staining (Invitrogen) [33] and a fluorescent microscope (Olympus BX40; Olympus America, Inc.). Follicles were considered to be viable when the oocyte and surrounding granulosa cells fluoresced green. Because follicles were not distributed uniformly within the ovarian cortex, 10–200 follicles were observed for each ovarian piece, with all follicles in a given piece counted regardless of size.

Histological Analysis and Classification of Follicular Structure

Proliferation Analysis Assessment of proliferating cell nuclear antigen (PCNA) protein in mitotic cells is a commonly used staining approach for determining the extent of cellular proliferation [38, 39]. Because PCNA also is involved in cell repair [38, 39], we additionally evaluated cell growth using a newer tool, 5-ethynyl-2 0 deoxyuridine (EdU), which is incorporated into the DNA in place of thymidine with fluorescent dye [40]. Thus, the PCNA staining was used to identify proliferating cell types (i.e., granulosa or ovarian stromal cells), whereas the extent of EdU incorporation indicated proliferation level. The PCNA staining followed the routine immunohistochemistry procedure that relied on the ImmunoCruz mouse LSAB Staining System (Santa Cruz Biotechnology). Briefly, the fresh and cultured tissue was placed in Bouin fixative, embedded in paraffin, and subsequent sections (thickness, 5 lm) were dewaxed, rehydrated, and boiled for 20 min in a buffer (pH 6.2) containing 10 mM citric acid, 2 mM ethylenediaminetetra-acetic acid, and 0.1% Tween. Each section was incubated with 3% (v/v) H2O2 in PBS for 10 min, blocked with 1.5% goat serum and 1% (w/v) bovine serum albumin (BSA) in PBS for 30 min, and then incubated at 48C overnight in a moist chamber with mouse antiPCNA (1:1000) as a primary antibody in 0.3% (w/v) Triton-X. After washing with PBS, each section was incubated with anti-mouse immunoglobulin G (IgG) antibody conjugated with Biotin (Santa Cruz Biotechnology) as a secondary antibody for 1 h at room temperature (;228C) and then incubated with horseradish peroxidase (HRP)-streptavidin complex (Santa Cruz Biotechnology) for 1 h (;228C). Finally, each section was stained with HRP substrate (Santa Cruz Biotechnology), counterstained with hematoxylin, and observed under an Olympus BX40 microscope. For the negative controls, the primary antibody was omitted, and the section was incubated with normal mouse IgG (Santa Cruz Biotechnology) in PBS. To determine global cell proliferation, EdU staining was performed using a Click-iT EdU Alexa Fluor 488 Imaging Kit (Invitrogen) according to the manufacturer’s protocol. Ovarian tissue, either fresh or cultured for 2 or 13 days, was incubated with 10 lM EdU overnight (except for the EdU-negative control) and then fixed with 4% (v/v) paraformaldehyde in PBS for 30 min at

MATERIALS AND METHODS Chemicals All chemicals were purchased from Sigma-Aldrich unless otherwise indicated.

Collection and In Vitro Culture of Ovarian Cortical Tissues Ovaries were from prepubertal (age, 2.5–6 mo) and adult (age, 8 mo to 2 yr) domestic cats that underwent routine ovariohysterectomy at local veterinary clinics. Upon excision of the reproductive tract, each pair of ovaries was

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Pieces of fresh or cultured ovarian tissues were fixed in Bouin solution, maintained at 48C overnight, dehydrated in a graded series of ethanol solutions, and then embedded in paraffin. Serial sections (thickness, 5 lm) of each cortical piece were cut and stained with hematoxylin (Santa Cruz Biotechnology) and eosin. To avoid double counting, only follicles containing oocytes with a visible nucleus were included. The follicles within the cortical pieces were classified as primordial (one layer of flattened granulosa cells around the oocyte), primary (a single layer of cuboidal granulosa cells around the oocyte), or secondary (two or more layers of cuboidal granulosa cells) [33]. The follicle diameter was defined as the maximum diameter measured within the basal membrane. All follicles were further characterized as normal (when the nucleus of the oocyte and the surrounding granulosa cells were structurally intact) or abnormal (when the oocyte and/or granulosa cells contained a pyknotic, fragmented, or shrunken nucleus) [33]. Number of normal follicles was divided by total number evaluated to determine the proportion of follicle viability per treatment. To avoid double counting of follicles, three sections at 20-lm intervals were examined for each ovarian piece from three to six animals for each culture condition (see below).

EGF SUSTAINS CAT PRIMORDIAL FOLLICLE VIABILITY room temperature. After washing with 3% (w/v) BSA in PBS, the tissue was permeabilized with 0.5% (v/v) Triton X-100 in PBS for 30 min. Each section then was incubated with a Click-iT reaction cocktail containing reaction buffer plus buffer additive, CuSO4, and Alexa Fluor 488 azide for 1 h in the dark. For subsequent DNA staining, tissues were incubated for 1 h with 5 lg/ml of Hoechst 33342 and observed under the Olympus fluorescent microscope.

described for experiment 1 (at least two pieces/cat/culture treatment). Cell proliferation in fresh versus cultured tissue was evaluated by PCNA and EdU staining as described above. Study 3: Age dependence on ovarian cell proliferation influencing primordial follicle viability. To identify a potential cause for the variations in cell proliferation between prepubertal and adult donors, ovarian tissue was recovered from three cats of each age cohort, and prepubertal and adult ovaries were cultured with no factor or 100 ng/ml of EGF for 3 days. Fresh and cultured tissue were then examined for cell proliferation by EdU staining (at least two cortical pieces/cat/age group).

Western Blot Analysis Ovarian cortical tissues, either fresh and after 3 days of culture, were extracted in 23 Laemmli buffer (Bio-Rad) and b-mercaptoethanol with protease and phosphatase inhibitors, and the samples were processed on SDS-PAGE with the use of 4%–15% Mini-PROTEAN TGX Precast Gel (Bio-Rad). Gels were transferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore). Precision Plus Protein Dual Color Standards (Bio-Rad) were used as the molecular weight standard. Membranes were blocked with 3% (w/v) BSA in Tris-buffered saline containing 0.1% Tween (TBS-T) for 1 h (;228C) and incubated overnight at 48C with the respective primary antibody: rabbit antiphospho-p44/42 MAPK (1:500 in TBS-T; Thr202/Tyr204, p-MAPK; Cell Signaling Technology), rabbit anti-phospho-AKT (1:1000; Ser473, p-AKT; Cell Signaling Technology), or rabbit anti-b-actin (1:1000; Cell Signaling Technology). As a negative control, normal rabbit IgG (1:1000; Santa Cruz Biotechnology) was used in place of primary antibody to confirm the absence of nonspecific binding. Anti-rabbit IgG antibodies (1:2000; Cell Signaling Technology) conjugated with HRP was used as the secondary antibody, and blots were incubated for 45 min (;228C). Immunoreactivity was detected with Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Fisher Scientific).

Statistical Analysis Data are presented as the mean 6 SEM. A Shapiro-Wilk test was performed to evaluate normality of the data set, and a Bartlett test was used to confirm homogeneity of the variances. The proportions of each follicle stage between the two age groups were assessed by an unpaired, one-tailed Student ttest. Comparisons in follicle viability and morphology among groups were evaluated by analysis of variance followed by a Newman-Keuls multiplecomparison test. Differences were considered to be significant at P , 0.05 (GraphPad Prism ver 4.00; GraphPad Software).

RESULTS

Based on the calcein-AM/ethidium homodimer-1 staining, more than 87% of follicles in the freshly collected ovarian cortical tissue generally were viable regardless of donor age (Figs. 1 and 2). Histology revealed differences in follicular distribution between prepubertal and adult donors (P , 0.05). Specifically, prepubertal cat ovaries contained more primordial follicles as a proportion of the total but fewer primary and secondary stages than adult individuals (primordial, 79.0% 6 3.8% vs. 61.6% 6 5.3%; primary, 20.9% 6 3.9% vs. 34.0% 6 3.8%; secondary, 1.4% 6 1.4% vs. 10.7% 6 2.8%, respectively). An age-of-donor influence was found (P , 0.05) on the viability of ovarian follicles at 14 days of culture in the presence of EGF supplementation. Adding this growth factor improved follicle viability in a partially dose-dependent fashion in tissue from prepubertal, but not adult, donors (Fig. 2). Although no effect (P . 0.05) of the 50 ng/ml concentration was observed on the former cohort compared to controls (Fig. 2A), more than 78% of follicles exposed to 100 ng/ml were viable at 14 days, which was higher (P , 0.05) than the control value (Figs. 1 and 2A). The highest EGF concentration (200 ng/ml) offered no advantage, resulting in viability similar (P . 0.05) to that in the control. For ovarian cortex pieces from adult females, EGF provided no enhancement (P . 0.05) at any dosage (Fig. 2B); the highest concentration of 200 ng/ml actually appeared to be detrimental in that less than 2% of follicles were viable compared to higher values for untreated controls and groups supplemented with lower concentrations of EGF (control, 28.8% 6 8.6%; 50 ng/ml of EGF, 46.6% 6 10.0%; 100 ng/ml of EGF, 30.6% 6 9.7%; 200 ng/ml of EGF, 1.3% 6 0.5%) (Fig. 2B). Adding GDF9 to the culture environment at any concentration had no beneficial impact (P . 0.05) on follicle viability in tissue from either prepubertal (Figs. 1 and 2A) or adult (Fig. 2B) donors. Likewise, no synergistic benefits from combining the two factors as a supplement were observed (P . 0.05) (Fig. 2). By contrast, for the prepubertal cohort, it appeared that the addition of GDF9 negated the positive influence of EGF. For example, when GDF9 was supplemented at 50 ng/ml in medium containing 50 or 100 ng/ml of EGF, follicle viability was similar (P . 0.05) to that of using EGF alone. However,

Experimental Design Study 1: Influence of EGF and GDF9 on the viability and activation of primordial follicles in ovarian tissue from prepubertal versus adult cats. To investigate the influence of EGF and GDF9 on primordial follicles in vitro, ovarian cortical pieces from prepubertal versus adult donors were incubated separately on 1.5% (w/v) agarose gel blocks in protein-free medium within a 24-well culture plate (Corning Incorporated) as described previously [33]. The culture medium was Eagle Minimum Essential Medium (MEM) supplemented with 5.5 lg/ml of insulin, 5.5 lg/ml of transferrin, 6.7 ng/ml of selenium, 2 mM L-glutamine, 100 lg/ml of penicillin G sodium, 100 lg/ml of streptomycin sulfate, 0.05 mM ascorbic acid, 10 ng/ml of porcine follicle-stimulating hormone (FSH; Folltropin-V; Bioniche Animal Health), and 0.1% (w/v) polyvinyl alcohol. This culture medium was then supplemented with no factor (control) or with 50, 100, or 200 ng/ml of mouse EGF (64% homology with cat EGF) [41] or human GDF9 (74% homology with cat GDF9) alone or with a combination of EGF plus GDF9 at a concentration of either 50 or 100 ng/ml each. The EGF and GDF9 dose ranges were selected based on studies of ovarian tissue culture in other species [9, 16, 24, 26, 42]. Viability of ovarian follicles in the fresh and cultured tissue was assessed as described above. Based on these results, the fresh tissue and the tissue cultured with 0, 50, or 100 ng/ml of EGF supplementation were processed and examined histologically as described above. Cortical pieces from at least three prepubertal and three adult cats were included in each treatment group (five pieces/cat/culture treatment). Study 2: Mechanisms by which EGF enhances primordial follicle viability in cat ovarian tissue. Two experiments were conducted, with the design partially influenced by the findings from study 1. Experiment 1 examined downstream pathways for influencing primordial follicle viability. Ovarian cortical pieces from five prepubertal and four adult cats were allocated randomly into eight treatment groups (five pieces/cat/culture treatment): 1) no factor (control), 2) 100 ng/ml of EGF, 3) 100 ng/ml of EGF plus 0.1 lM AG1478 (EGFR inhibitor), 4) 100 ng/ml of EGF plus 1 lM AG1478, 5) 100 ng/ml of EGF plus 1 lM U0126 (MAPK inhibitor), 6) 100 ng/ml of EGF plus 10 lM U0126, 7) 100 ng/ml of EGF plus 1 lM LY294002 (PI3K inhibitor), or 8) 100 ng/ml of EGF plus 10 lM LY294002. Fresh ovarian tissue and tissue cultured for 14 days were evaluated for viability and morphology as described above. A minimum of four cat donors per treatment were used for the viability assessment and three for the morphological analysis. To confirm the downstream pathways of EGF/EGFR signaling on primordial follicle viability (solely for the prepubertal ovarian donors based on study 1), cortical pieces from three cats were cultured for 3 days under each of the eight culture conditions described above (five pieces/cat/culture treatment). After extracting proteins from fresh and cultured tissue, we examined phosphorylation of MAPK and AKT by Western blot analysis as described above. Experiment 2 entailed evaluating the influence of the EGF signaling pathway on ovarian cell proliferation. Cortical pieces from three prepubertal cats were cultured for 14 days under each of the eight culture conditions

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Study 1: Influence of EGF and GDF9 on the Viability and Activation of Primordial Follicles in Ovarian Tissue from Prepubertal Versus Adult Cats

FUJIHARA ET AL.

FIG. 1. Fluorescent staining with calcein-AM/ethidium homodimer-1 immediately after recovery of ovarian tissue from a prepubertal cat (fresh) or after culture in 0 (control) or 100 ng/ml of EGF or GDF9 for 14 days. Green fluorescence indicates viable follicles/cell. Bar ¼ 100 lm.

when GDF9 was added at 100 ng/ml to medium containing either 50 or 100 ng/ml of EGF, the proportion of viable follicles (39.2% 6 9.1% and 38.0 6 6.9%, respectively) was less (P , 0.05) than in cortices cultured with 100 ng/ml of EGF alone (78.9% 6 7.9%) (Fig. 2A).

Study 2: Mechanisms by which EGF Enhances Primordial Follicle Viability in Cat Ovarian Tissue For experiment 1, which examined downstream pathways potentially affecting primordial follicle viability, the simulta-

FIG. 2. Percentages (mean 6 SEM) of viable follicles in prepubertal (A) versus adult (B) cat ovarian tissue cultured for 0 (fresh) and 14 days with 0 (control), 50, 100, or 200 ng/ml of EGF (E) or GDF9 (G) or a combination of 50 or 100 ng/ml of EGF and GDF9 (three or more cats/age group, two ovarian pieces/cat/treatment group). Different letters indicate significant (P , 0.05) differences.

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Histological analysis confirmed the presence of structurally intact primordial and primary follicles in the ovarian cultures from prepubertal donors. As revealed in Figure 3, 68.0% 6 5.2% of these type follicles were categorized as normal in the freshly excised tissue compared to 67.9% 6 6.6% and 55.5% 6 7.4% for the EGF treatments at 50 and 100 ng/ml, respectively (P ¼ 0.32) after 14 days of culture. Furthermore, the total number of follicles per sectional area assessed in a given cortical piece (37–63 follicles/mm2) did not differ (P . 0.05) from the onset to the end of culture within any treatment group. Additionally, no differences were found in the proportion of primordial follicles (fresh, 79.1% 6 3.8%; control, 68.0% 6 9.1%; 50 ng/ml of EGF, 64.5% 6 13.9%; 100 ng/ml of EGF, 64.6% 6 8.6%; P ¼ 0.57) or primary follicles (fresh, 20.9% 6 3.9%; control, 31.1% 6 8.7%; 50 ng/ ml of EGF; 33.1% 6 11.5%; 100 ng/ml of EGF, 33.9% 6 7.5%; P ¼ 0.58) among groups. A marginal, albeit nonsignificant (P ¼ 0.26), increase in follicle diameter was observed in cultured tissues, especially with EGF supplementation for both primordial (fresh, 56.8 6 1.5 mm; control; 58.8 6 2.2 mm; 50 ng/ml of EGF, 63.1 6 4.4 mm; 100 ng/ml of EGF, 61.8 6 1.9 mm) and primary (fresh, 61.4 6 2.1 mm; control, 58.8 6 3.0 mm; 50 ng/ml of EGF, 70.6 6 4.7 mm; 100 ng/ml of EGF, 68.7 6 2.3 mm) follicles.

EGF SUSTAINS CAT PRIMORDIAL FOLLICLE VIABILITY

FIG. 3. Histomicrographs of prepubertal ovarian tissue cultured for 0 (fresh) or 14 days with 0 (control) or 50 versus 100 ng/ml of EGF. The letter ‘a’ indicates an abnormal follicle classified as having an oocyte and/or granulosa cells containing pyknotic, fragmented, or shrunken nuclei. Most follicles are at the primordial stage, with a few being primary (insert). Bar ¼ 50 lm.

FIG. 4. Analysis of downstream pathways of EGF for influencing primordial follicle viability in prepubertal (A) and adult (B) cat ovarian tissue cultured for 0 (fresh) and 14 days. Percentages (mean 6 SEM) of viable follicles in fresh versus tissue cultured with no EGF (control) or EGF plus AG1478 (0.1 or 1 lM), U0126 (1 or 10 lM), or LY294002 (1 or 10 lM) (four or more cats/treatment group). Different letters indicate a significant difference (P , 0.05).

Study 3: Age Dependence on Ovarian Cell Proliferation Influencing Primordial Follicle Viability Staining with EdU revealed that the cell proliferation was similar between the fresh ovarian tissues of prepubertal and 5

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adult donors (Fig. 7). However, the ability of ovarian cells within incubated tissue to proliferate depended on donor age. Specifically, a marked increase in proliferating cell number in the cultured ovarian pieces from prepubertal donors was observed compared to the fresh tissue controls (Fig. 7). On the contrary, the proportions of proliferating cells in cultured tissue from adult individuals remained the same as in the agematched, fresh controls (Fig. 7).

neous supplementation of AG1478 (EGFR inhibitor), U0126 (MAPK inhibitor), or LY294002 (PI3K inhibitor) to the ovarian cultures from prepubertal donors erased the beneficial effect of EGF. Percentages of viable follicles after 14 days of culture decreased (P , 0.05) from 60% for treatment with EGF alone to approximately 40% for treatment with EGF plus one of the three inhibitors (Fig. 4A). With the addition of each inhibitor, the proportions of viable EGF-treated follicles decreased to be comparable (P . 0.05) to the control (Fig. 4A). For the ovarian tissue from the adult counterparts, the percentages of viable follicles were the same (P . 0.05) for all treatment groups (Fig. 4B). For both age groups, the addition of each inhibitor had no influence (P . 0.05) on the proportions of morphologically normal follicles, their distribution at either the primordial or primary stage, or their diameter (data not shown). Analysis of the Western blots revealed that EGF stimulated phosphorylation of MAPK (p-MAPK) and AKT (p-AKT) in prepubertal ovarian tissues that were cultured for 3 days (Fig. 5A). Weak expressions were observed in tissue cultured without EGF (Fig. 5A). In the presence of EGFR, MAPK, and PI3K inhibitors in culture, the EGF enhancement effect on phosphorylation of MAPK and/or AKT was clearly reduced (Fig. 5B). The sizes of the proteins identified by all antibodies were expected and normal (p-MAPK, 42 and 44 kDa; p-AKT, 60 kDa; b-actin, 45 kDa). Collectively, these observations supported the notion that EGF was activating the MAPK and PI3K signaling pathways in the ovaries of prepubertal donors. For experiment 2, PCNA expression was detected in some granulosa cells of primordial and primary follicles in fresh tissue of prepubertal donors as well as that cultured for 14 days. This occurred regardless of EGF treatment (Fig. 6A), suggesting that developing follicles in incubated ovarian tissue retained proliferative ability. However, only a few PCNApositive cells were observed in the ovarian stroma of fresh tissue or the control group compared to abundant PCNApositive stromal cells in tissue supplemented with EGF (Fig. 6A). These latter groups also contained more EdU-positive cells than the control (Fig. 6B). Moreover, the number of EdUpositive cells decreased in the tissue cultures that were supplemented with EGF plus one each of the three inhibitors (Fig. 6B). Although it was not possible to objectively quantify, the suppression of EdU-positive cells by the inhibitors appeared to occur in a dose-dependent fashion (Fig. 6B).

FUJIHARA ET AL.

FIG. 5. Analysis of downstream pathways of EGF in cultured prepubertal ovarian tissue. A) Western blot analysis for MAPK and AKT phosphorylation in prepubertal ovarian tissue cultured for 0 (fresh) and 3 days with 0 (control) or 100 ng/ml of EGF (three cats/treatment group). B) Western blot analysis for MAPK and AKT phosphorylation in prepubertal ovarian tissue cultured for 3 days in 100 ng/ml of EGF alone or EGF plus AG1478 (0.1 or 1 lM), U0126 (1 or 10 lM), or LY294002 (1 or 10 lM) (three cats/ treatment group).

DISCUSSION We used an ovarian culture system developed earlier in our laboratory [33] to examine the influence of the two local regulatory factors, EGF and GDF9, on early stage cat follicle viability in vitro. From studies of other species, both factors are known to be secreted by the oocyte and surrounding somatic cells [12, 13, 17–19] and are suggested to be associated with primordial follicles [9, 16, 22–26, 42]. We also explored, to our knowledge for the first time, the effect of age—specifically, differences in growth factor impacts on ovarian cultures from prepubertal versus adult donors. The present study had three major findings. First, EGF played a dose-dependent role in maintaining viability, but not promoting activation, of primordial follicles, but only in ovarian tissue from prepubertal cats. The mechanism was by stimulating the MAPK and PI3K signaling pathways that, in turn, enhanced proliferation of ovarian stromal cells. Second, this biological event was strongly age dependent and failed to occur in ovarian cultures from adult donors. Third, based on the evaluations and metrics measured, GDF9 had no beneficial influence on tissues

" FIG. 6. Influence of EGF and its signaling pathways on cell proliferation in cultured prepubertal ovarian tissue. A) Histomicrographs of primordial, primary, and stromal cells in cat prepubertal ovarian tissue cultured for 0 (fresh) or 14 days with 0 (control) or 100 ng/ml of EGF. Arrowheads indicate PCNA-positive cells. Bar ¼ 50 lm. B) Florescent micrographs of prepubertal ovarian tissue cultured for 14 days with 0 (control) or 100 ng/ml of EGF alone or EGF plus AG1478 (0.1 or 1 lM), U0126 (1 or 10 lM), or LY294002 (1 or 10 lM) (three cats/treatment group). Green fluorescence indicates EdUpositive cells. Blue color is tissue counterstained with Hoechst 33342. Bar ¼ 100 lm.

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cultured from either age group and, thus, had a negligible impact on follicle viability in the domestic cat. Earlier examples in a variety of species indicated how EGF appears to be involved in follicle development at the primordial [9, 16, 42] or preantral [7] stage as well as assisting in oocyte maturation [43]. Although we observed no influence on primordial follicle activation in the cat, EGF promoted follicle viability and proliferation of ovarian stromal cells, but only in the younger donor group. Two explanations for this agedependent response are possible. One relates simply to the potential inherent differences in proliferation capacity of stromal cells between the two age groups. The present results actually support this possibility because more proliferating cells were observed in prepubertal compared to adult cultured tissues. Prepubertal cat ovaries are markedly smaller in size than adult counterparts [44], but they are comprised of stromal cells exquisitely positioned to respond, multiply, and grow when provoked by the appropriate growth factor(s). The second possibility is that the variation displayed between prepubertal and adult ovarian tissue regarding EGF was linked to innate differences in extracellular matrix rigidity. It is well established that elasticity is decreased and inflexibility is increased in human epithelial cells during protracted intervals of in vitro culture [45]. Furthermore, artificially modifying the rigidity of the microenvironment influences the responsiveness of cells to growth factors and cell signaling [46]. Specifically, culturing tumorigenic cells on polyacrylamide gels with decreasing matrix rigidity inhibits PI3K/AKT activity that leads to increased TGFB1-induced apoptosis as a tumor suppressor. By contrast, increasing the rigidity of the microenvironment switches TGFB1 actions to be more tumorigenic [46]. Whereas both of these explanations were plausible for explaining reaction differences between the age groups, it clearly appears that a near-term priority should be more study on the uniqueness of the prepubertal ovary. Because stromal cells of the latter were so receptive to activation, the cat model may be particularly useful for testing the hypothesis that extracellular matrix rigidity of the ovarian cortex is a limiting factor for stimulating successful primordial follicle survival. Given earlier evidence that EGF appeared to be involved in primordial follicle activation in the rat [16] and goat [9], the lack of an analogous response in our cat study using the same factor dosage over a 14-day culture was somewhat surprising. Although no definitive evidence demonstrates that EGF is a primordial follicle activator, this finding in the cat was unexpected because Western blot analysis revealed that EGF/ EGFR signaling indeed stimulated the MAPK and PI3K pathways, which have been shown to be involved in activating rodent [4, 16, 47, 48] and human [4] primordial follicles. Previous studies of the cat have demonstrated, first, that EGF is localized in theca cells and small single cells in the ovarian cortex that are adjacent to primordial follicles [34] and, second, that EGFR is detected in the granulosa cells of primary, secondary, and tertiary follicles as well as interstitial gland cells [34]. At the same time, primordial follicles in the cat do not express EGF or EGFR [34], unlike in the human, where both



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FIG. 7. Florescent micrographs of prepubertal and adult ovarian tissue cultured for 0 (fresh) or 3 days with 0 (control) and 100 ng/ml of EGF (three cats/ treatment group). Green fluorescence indicates EdU-positive cells. Blue color is tissue counterstained with Hoechst 33342. Bar ¼ 100 lm.

a low EGF concentration promotes proliferation, whereas a high dose induces cell-cycle arrest and apoptosis [56]. A dosedependence finding also was found when examining GDF9 influence in the cat ovarian cultures, with the highest concentration (200 ng/ml) being detrimental to follicle viability. Interestingly, this same dose promotes primordial to secondary follicular growth in the hamster [26] and improves follicle viability in the goat [24]. GDF9 also boosts proliferation of granulosa cells of early antral and preovulatory rat follicles while simultaneously suppressing FSH-stimulated estradiol and progesterone production [57]. Because the latter two steroid hormones regulate cell survival in many cell types [58], this could be the mechanism whereby an inordinately high GDF9 concentration adversely influences follicle viability. Additionally, although the low GDF9 treatment (50 ng/ml) did not influence the impact of EGF on follicle viability, the higher concentration (100 ng/ml) was detrimental in prepubertal animals. It is known from studying mouse preovulatory follicles that oocyte-secreted GDF9 stimulates EGFR expression in granulosa cells [59]. Furthermore, some tumor cell lines producing hyperlevels of EGFR also produce excessive EGF that, in turn, inhibits cell proliferation and promotes apoptosis. Additionally, transfection evidence in the hamster suggests that the extent of EGF-induced apoptosis is associated positively with EGFR expression levels [56]. Therefore, the elevated GDF9 concentration may have exerted its negative impact on the normally positive influence of EGF by overexpressing EGFR in the ovarian stromal/follicular cells that, in turn, upregulated signaling pathways that increase apoptosis. In summary, the present findings revealed a role for EGF, but not GDF9, in the culture of ovarian cortical pieces from the domestic cat. The former has a marked influence on primordial follicle viability via stimulation of the MAPK and PI3K signaling pathways that boost ovarian stromal cell growth and production. Most interesting was determining that this effect only occurred in younger, prepubertal ovarian donors. This observation importantly emphasized, to our knowledge for the first time, that reproductive status—in this case, prepubertal versus adult—influenced the ability of primordial follicles to respond to this exogenous growth factor in vitro. Thus, we can conclude, at least for the cat, that distinctively different 8

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factors are located in the oocyte and granulosa cells of primordial follicles. This significant species difference no doubt explained the lack of beneficial effect of EGF on cat primordial follicle activation. Rather, the influence of EGF appeared to be related to up-regulating the MAPK and PI3K pathways to promote healthy ovarian stromal cell proliferation, which we suspect was a prerequisite to ensuring follicle viability. For example, it is well established that stromal cells secrete growth factors, such as Kit ligand, that can regulate primordial follicle activation and/or survival [49]. Thus, for the cat, EGF likely is exerting its impact indirectly, not directly, on primordial follicle viability. Substantial evidence indicates that GDF9 plays a role in progression of early stage follicular growth, including primordial follicle activation, in a variety of commonly studied species [22–26]. For example, GDF9 supplementations to ovarian cortical incubations increase proportions of viable follicles at 14 days in vitro in the human [23] and at 7 days in the goat [24]. Additionally, GDF9 is expressed only in the oocyte of primary and later-stage follicles in the human [50], rat [51], and goat [52]. In the cat ovary, location of this growth factor appears to be restricted to oocytes of primary and antral follicles, whereas the GDF9 receptor (BMPR2) is mainly abundant in primordial and primary stages [35]. Thus, whereas others suggest a role of GDF9 in regulating early stages of follicle development, we found no evidence that this factor was influencing primordial follicle viability in the prepubertal or adult cat ovary. GDF9 may be irrelevant in the cat or, more likely, occurring at later, perhaps transitional stages of development. For example, the exposure of rat ovarian tissue to this factor in vitro promotes follicular growth only after the primary developmental stage [51, 53]. It was noteworthy that the factor dosage offered in vitro influenced primordial follicle viability, sometimes adversely (e.g., the highest EGF concentration of 200 ng/ml). Although EGFR signaling boosts cell proliferation [54], evidence exists of an opposing effect of EGF on cell persistence, depending on species and the dose provided. For example, EGF is a promoter of survival for neonatal kidney cells of the rat, but it encourages apoptosis under the same conditions in newborn mice [55]. In a transfected hamster cell line expressing EGFR,


ACKNOWLEDGMENT The authors thank veterinary hospitals in the Front Royal, Stephens City, Harrisonburg, and Purcellville, Virginia, areas for providing cat ovaries. The authors also acknowledge Dr. Budhan Pukazhenthi (Smithsonian Conservation Biology Institute) for technical advice and Ms. Lei Li (Maryland University) for technical assistance with Western blot analysis.

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