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BOR - Papers in Press, published online ahead of print October 3, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.058529
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BIOLOGY OF REPRODUCTION 78, 167–175 (2008)
DOI: 10.1095/biolreprod.106.058529
© 2008 by the Society for the Study of Reproduction, Inc.

Kit Ligand 2 Promotes Murine Oocyte Growth In Vitro1

Fiona H Thomas , Rubina S Ismail , Jin-Yi Jiang , and Barbara C Vanderhyden 2

Department of Cellular and Molecular Medicine, University of Ottawa and Centre for Cancer Therapeutics, Ottawa Health Research Institute, Ottawa, Ontario, Canada K1H 8L6

ABSTRACT

Oocyte-granulosa cell communication, mediated by paracrine factors, is essential for oocyte development. Kit ligand (KITL) is expressed in granulosa cells as soluble (KITL1) or membrane-associated (KITL2) proteins. However, the relative biopotency of each isoform during oocyte development is unknown. Our initial results showed that Kitl2 was down-regulated in cultured granulosa cells. To determine the effect of the two isoforms of KITL on oocyte growth, Kitl-deficient fibroblasts were transfected with constructs expressing either KITL1 or KITL2, and growing oocytes were isolated from 12-day-old mice and cultured on the transfected fibroblasts for 2 days. At the end of culture, oocyte diameters were measured, the incidence of spontaneous germinal vesicle breakdown (GVBD) was noted, and oocytes were analyzed for KIT receptor expression. Oocyte growth occurred only in the presence of the KITL2-producing fibroblasts, and suppression of KITL2 expression impaired oocyte growth. Up-regulation of KIT expression occurred in the presence of KITL2 but not KITL1. The presence of KITL2 inhibited spontaneous GVBD. Meiosis inhibitors did not attenuate the GVBD that occurred in the absence of KITL2, suggesting that this process reflects oocyte degeneration rather than meiotic progression. These results indicate that KITL2 is the principal KITL isoform required for oocyte growth and survival in vitro.

follicle, follicular development, growth factors, oocyte development

INTRODUCTION

The KIT tyrosine kinase receptor and its ligand (KITL) serve essential functions in the development of the ovary during embryogenesis by mediating signals that promote the proliferation and survival of primordial germ cells [1]. In the postnatal ovary, KIT continues to be expressed in increasing amounts on the surface of primordial, growing, and fully grown oocytes [2–5], and KITL is expressed by granulosa cells [4–6]. Strong evidence for the importance of these ligand-receptor interactions is provided by studies showing that female mice with naturally occurring mutations in Kitl or Kit are infertile because of developmental abnormalities [1–4].

KITL is expressed as either a membrane-associated or soluble protein that arises from alternatively spliced mRNAs [7]. Although six different transcripts have been reported [7–10], two major mRNA species, Kitl1 and Kitl2, have been shown to have tissue-specific patterns of expression [7]. Soluble KITL (KITL1) can be cleaved because of the presence of an 84-bp exon (exon 6), which encodes a proteolytic cleavage site, allowing the extracellular domain to be released as a soluble product. Membrane-bound KITL (KITL2) lacks this exon, is not efficiently cleaved, and thus remains more stably on the membrane [7]. The ratio of Kitl1:Kitl2 mRNA differs between tissues [7], between ovaries of mice of different ages [11], and between granulosa cells of preovulatory and ovulatory rat follicles [5], suggesting that these transcripts are differentially regulated.

Several lines of evidence support the physiological importance of membrane-bound KITL2 in vivo. The most compelling evidence is provided by the viable KitlSl-d mutants in which the genomic regions encoding the transmembrane and cytoplasmic domains of KITL are deleted; these mice can only produce the secreted form of KITL, but they show all the pleiotropic defects seen in KitlSl mutants, which lack expression of both isoforms [12, 13]. Both soluble and membrane-bound KITL have similar binding affinities for KIT receptors [14], but it has been demonstrated in mast cells that because KITL2 is membrane anchored, it may prevent subsequent down-regulation and internalization of activated KIT [15]. KITL2 has also been reported to induce a more persistent activation of KIT receptor kinase than the soluble form of KITL [16, 17].

Binding of KITL to the KIT receptor leads to the phosphorylation of a set of cellular proteins via the kinase domain of the KIT receptor on its cytoplasmic tail [18–20]. Consequently, several signaling pathways regulating apoptosis are activated via factors including RAS, RAF, mitogen-activated protein kinase, and AKT [21, 22]. KITL stimulation also induces activation of phosphatidylinositol (PI) 3-kinase (PI3K), which is required for KITL-induced mitogenesis and survival in hematopoietic cells [23]. The roles of KIT in oogenesis have been extensively reviewed [24, 25], and one of the most important downstream effectors of KIT activation in oocytes is the PI3K/AKT module, through which the signal is transduced into changes in expression of BAX and BCL2L1, important players in the apoptotic pathway [26]. Blockage of KIT activity by injection of neutralizing antibodies impairs early follicle development [27]. Selective PI3K inhibitors block the anti-apoptotic effect of KITL in germ cells during fetal oogenesis [28], and mice expressing a mutant KIT receptor (KITY719F), which fails to interact with PI3K, have impaired follicle development at the early preantral stage [29]. From these studies, it has been proposed that KITL-induced KIT activity and downstream PI3K/AKT signaling regulate oocyte growth and survival. The relative contributions of KITL1 and KITL2 to these processes have yet to be determined.

Previous research in our laboratory has shown that, in vivo, KITL2 expression is increased in rat granulosa cells in response to eCG [5], suggesting a role in follicular development. Our subsequent studies have shown that in the presence of a low concentration of FSH, there is a decrease in the ratio of steady-state Kitl1:Kitl2 mRNA in murine oocyte-granulosa cell complexes grown in vitro, due to an increase in Kitl2 mRNA levels [30]. Importantly, this expression pattern was also associated with increased oocyte growth in culture, suggesting that KITL2 is important for oocyte growth and/or that the correct balance of KITL1/KITL2 production is necessary for optimum oocyte growth in vitro. Thus, in addition to the evidence indicating a role for KITL in regulating oocyte survival [26], there is evidence to suggest that KITL2 is the principal isoform regulating oocyte growth [30]. Although soluble KITL1 can promote early oocyte growth (in 8-day-old mice) in vitro [31], the growth was limited, and the specific effects of KITL1 and KITL2 on oocyte growth and survival were not investigated. Moreover, the role of each isoform in regulating the expression of the KIT receptor in oocytes is unknown.

During oocyte growth, oocytes are arrested at the diplotene stage of the first meiotic division, and normal oocyte growth and acquisition of developmental competence require this maintenance of meiotic arrest. When fully grown oocytes are removed from follicles and placed into culture, they will spontaneously resume meiosis [32, 33], which has led to the hypothesis that meiosis-inhibitory factors found within the follicle maintain oocytes in an arrested state until the time of ovulation. Our previous findings have demonstrated that soluble KITL1 acts as a negative regulator of meiosis through activation of KIT receptors in fully grown rat oocytes [6]. Therefore, to focus on the contributions of each KITL isoform to oocyte growth without the potentially confounding issues associated with meiosis, this study used oocytes in the growth phase before the acquisition of meiotic competence.

To investigate the role of soluble KITL1 and membrane-bound KITL2 during oocyte development, an initial set of experiments was carried out to determine the expression of Kitl1 and Kitl2 mRNA in rat granulosa cells and to assess the suitability of cultured granulosa cells as a model system for the sustained expression of each KITL isoform. In addition, a novel method was devised in which murine oocytes were cocultured with KITL-deficient murine fibroblasts that were transfected with constructs expressing either Kitl1 or Kitl2. These fibroblasts were then used to address the aims of this study, which were 1) to investigate the effects of KITL1 and KITL2 on oocyte growth, 2) to confirm that the KITL actions were mediated by KIT receptors, and 3) to investigate the effects of KITL1 and KITL2 on the regulation of oocyte KIT receptor expression in vitro.

MATERIALS AND METHODS

Isolation of Rat Granulosa Cells

Prepubertal female Sprague-Dawley rats were obtained from Charles River Canada Inc. (St. Constant, QB, Canada), and all experiments were performed in accordance with Canadian Council on Animal Care guidelines. Animals were allowed free access to food and water, and lighting was provided for 14 h daily. Animals were injected i.p. at 26 days of age with 7.5 IU of eCG (Equinex; Ayerst, Montreal, QB, Canada). Animals were killed at 40 h after eCG injection, and granulosa cells isolated from ovarian follicles were used for extraction of total RNA. To examine the expression of Kitl in cultured granulosa cells, ovaries were excised and transferred into Waymouth MB 752/1 medium (WAY; Sigma-Aldrich Ltd., Oakville, ON, Canada) supplemented with penicillin (75 mg/L), streptomycin (50 mg/L), sodium pyruvate (25 mg/L; all from Sigma) and BSA (3 mg/ml; ICN Biomedical, Costa Mesa, CA) for granulosa cell isolation. Antral follicles were punctured with 25-gauge needles to extrude their contents; ovary remnants, including oocytes, were removed from the media; and granulosa cells were isolated by centrifugation (3000 x g for 4 min).

Northern Analysis of Kitl Transcripts in Cultured Rat Granulosa Cells

Granulosa cells were seeded at a density of 0.5 x 106 cells/ml of WAY/fetal bovine serum (FBS) in 100-mm tissue culture-treated dishes (Falcon). The next day, the cell monolayers were washed to remove any nonadherent cells and replaced with either WAY/FBS or WAY/BSA supplemented with penicillin (75 µg/ml); streptomycin (10 µg/ml); 5 µg/ml each of insulin and transferrin; and sodium selenite (5 ng/ml). Freshly isolated granulosa cells and granulosa cells cultured for 48 h were immediately immersed in 3 M LiCl/8 M urea and were homogenized, and RNA was precipitated overnight at 4°C. RNA was extracted, and Northern analysis was performed as described previously [6]. RNA from NIH 3T3 fibroblasts or Kitl-deficient fibroblasts was used as positive [7] and negative [34] hybridization controls, respectively.

Sal1 fragment (2.06 kb) isolated from a plasmid containing a mouse Kitl cDNA [34] were used to probe the Northern blots. The signal was assessed by exposing blots to phosphor screens, which were scanned with a PSI phosphoimager (Molecular Dynamics, Sunnyvale, CA). After hybridization to detect Kitl signal, blots were reprobed with [32P]-labeled 28S rRNA cDNA probes to control for differences in RNA loading. Intensity of the bands on the autoradiographic film were estimated by densitometry with an Ultrascan XL Enhance Laser Densitometer (LKB; Bromma, Sweden), and the ratios of Kitl:28S RNA were calculated and standardized to untreated (control) values that were arbitrarily set to 1.0 for each Northern blot.

Expression of Alternatively Spliced Kitl Transcripts in Granulosa Cells and Kit in Oocytes

RT-PCR was used to determine the relative proportion of Kitl1 and Kitl2 transcripts in granulosa cell total RNA preparations by a procedure described previously [5]. Some PCR reactions were performed with water instead of cDNA to serve as negative controls. C13, a cell line that produces predominantly Kitl1 transcripts, was used as a positive control.

For quantitative measurement of Kitl1, Kitl2, and Kit transcripts, total RNA was extracted with the RNeasy Mini kit (Qiagen Inc., Mississauga, ON, Canada) according to the manufacturer's instructions. RNAs were reverse transcribed in a final volume of 20 µl of solution with an RT kit (Ambion, Inc., Austin, TX) and 1 µg of total RNA from cultured cells or total RNA of 80–240 oocytes. Real-time quantitative PCR analysis was performed with a LightCycler 2.0 System (Roche Diagnostic Corporation, Indianapolis, IN) as described previously [35]. The 5' forward primers of Kitl1 and Kitl2 were 5'-TGCAGCCAGTTCCCTTAGGA-3' and 5'-TCATGGTGGCATCTGACACTAGT-3', respectively. The 3' reverse primer for both Kitl isoforms was 5'-TGTAGGCCTGGGTCTTCA-3'. The 5' forward and 3' reverse primers of Kit were 5'-AGGAGATAAATGGAAACAATTATGT-3' and 5'-TTGATCATCTTTACAGCGACAGTCA-3', respectively, as reported previously [30]. Transcript levels were normalized on the basis of the level of transcripts for Rn18s (5' forward primer; 5'-CGCGGTTCTATTTTGTTGGT-3', 3' reverse primer; 5'-AGTCGGCATCGTTTATGGTC-3'; Invitrogen Canada Inc., Burlington, ON, Canada). The amplification reaction was then performed with the QuantiTect SYBR Green PCR kit (Qiagen). The thermal cycling conditions were composed of an initial denaturation step at 95°C for 15 min and 40 cycles (for all except Kit for 60 cycles) at 95°C for 15 sec, 56°C for 20 sec (for all except Kit at 52°C), and 72°C for 30 sec. Transcript levels were expressed as a ratio to Rn18s values.

Stable Transfection of Murine Fibroblasts with Kitl1 or Kitl2 cDNA Constructs

Murine Kitl1 (1.28 kb) and Kitl2 (820 bp) cDNAs, cloned into a PGEM7 plasmid vector, were obtained from Amgen Ltd. (Thousand Oaks, CA). Kitl-deficient (Sl4) fibroblasts (Amgen) were grown to 70% confluence (4 x 105 cells) in 5 ml of Alpha Minimal Essential Medium in 60-mm dishes (Falcon) and transfected with 6 µg of vector (containing the Kitl1 or Kitl2 fragment) and Lipofectamine (Invitrogen). After 24 h, cells were diluted 1:10 and cultured under antibiotic selective pressure with hygromycin (600 µg/ml). Individual colonies were isolated and expanded for cryopreservation, and expression of each isoform by the fibroblasts was confirmed by RT-PCR. Briefly, total RNA was extracted from the fibroblasts, and cDNA was synthesized as described previously [30]. Kitl1 and Kitl2 were amplified by PCR with specific primers [30] and visualized by agarose gel electrophoresis.

Oocyte Isolation and Culture on KITL-Deficient, KITL1-Producing, or KITL2-Producing Fibroblasts

KITL-deficient, KITL1-producing, and KITL2-producing fibroblasts were plated at a density of 2.6 x 104 cells/ml in 96-well plates and cultured overnight in 150 µl of Dulbecco modified Eagle medium containing 10% FBS (Sigma). This medium was replaced with serum-free culture medium for the oocyte coculture (Waymouth MB752 medium; Gibco, Life Technologies Inc., Burlington, ON, Canada) supplemented with BSA (0.3%); penicillin (75 µg/ml); streptomycin (10 µg/ml); 5 µg/ml each of insulin and transferrin; and 5 ng/ml of sodium selenite and sodium pyruvate (25 µg/ml); all from Sigma. Fibroblasts were 80% confluent at the start of the coculture with oocytes.

Oocyte-granulosa cell complexes were isolated from 12-day-old CD1 mice (Charles River) by collagenase and DNase digestion (Sigma) [36]. Oocytes were isolated from the complexes by repeated pipetting and briefly submerged in acid tyrodes (NaCl, 8.0 g/L; KCl, 0.2 g/L; CaCl2, 0.24 g/L; and glucose 1 g/L, pH 2.5) to dissolve the zona pellucida and facilitate attachment to the fibroblasts. Groups of four zona-free oocytes per well were incubated on the KITL-deficient, KITL1-producing, or KITL2-producing fibroblasts (n = approximately 30 oocytes per group). Isolated immature oocytes (51 ± 0.45 µm at the time of collection), all containing an intact germinal vesicle, were assigned randomly to each group and were incubated for 2 days in a sterile atmosphere of humidified air with 5% CO2 at 37°C.

Assessment of Oocyte Growth and GVBD after Culture on KITL-Deficient, KITL1-Producing, or KITL2-Producing Fibroblasts

At the end of the culture period, oocyte diameters (taken as the average of the two largest diameter measurements) were measured with an inverted microscope (Leitz Diavert, Wetzlar, Germany), and the proportion of oocytes that had undergone germinal vesicle breakdown (GVBD) was recorded.

The first set of experiments (n = 3) was set up to determine oocyte growth and GVBD during a 2-day culture on KITL-deficient, KITL1-producing, and KITL2-producing fibroblasts. Because an increased KITL1:KITL2 ratio has been previously shown to be detrimental to oocyte growth [30], the effect of increasing the KITL1:KITL2 ratio by the addition of exogenous soluble KITL1 (10 ng/ml; Genzyme, Mississauga, ON, Canada) to the oocytes cultured with the KITL2-producing fibroblasts was also investigated. In a second set of experiments (n = 3), Gleevec, an inhibitor of KIT activity [37] (5 µM; Novartis, Basel, Switzerland), and the anti-KIT neutralizing antibody ACK2 (1 µg/ml; eBioscience, San Diego, CA) were used to confirm the specificity of any KIT-mediated effects.

Because GVBD is a marker of oocyte degeneration as well as resumption of meiosis [38], a set of experiments (n = 3) was conducted with a membrane-permeable analogue of cAMP (dbcAMP; 0.3 mM; Sigma) or PMA (phorbol 12-myristate 12-acetate, an activator of protein kinase C; 0.1 nM; Calbiochem, Mississauga, ON, Canada) as treatments added to oocytes cultured in the presence of KITL-deficient fibroblasts. These factors have been previously reported to maintain meiotically competent oocytes in meiotic arrest in vitro [39, 40].

In a final set of experiments, Wortmannin (100 nM; Calbiochem), an inhibitor of PI3K, was added to the oocyte-fibroblast cocultures to investigate the involvement of this signaling pathway in KITL-stimulated oocyte growth.

Assessment of KIT Expression in Oocytes after Culture on KITL-Deficient, KITL1-Producing, or KITL2-Producing Fibroblasts

Zona-free oocytes were removed from the fibroblasts following culture, collected in supplemented Waymouth medium, and stained for KIT receptors as described by Ismail et al. [5], except that 2 µg/ml KIT (2B8) rat monoclonal immunoglobulin G2b (IgG2b) antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was used, as well as a 1:100 dilution of fluorescein isothiocyanate-conjugated anti-rat IgG (DAKO Cytomation Inc., Mississauga, ON, Canada). Whole mounted zona-free oocytes were exposed to KIT antibodies and subsequently visualized with a fluorescence microscope.

Statistical Analyses

All results are presented as means ± SEM of at least three independent experiments, unless stated otherwise. Statistical comparisons involving multiple groups were made by an analysis of variance, followed by the Dunnett or Tukey posttest for multiple comparisons. Comparisons between two treatments were made by an unpaired t-test (Prism 3.0 statistical software; GraphPad, San Diego, CA). Mean oocyte diameters were compared between experimental groups by a one-way analysis of variance, followed by the Bonferroni adjustment for multiple comparisons. For a comparison of four groups, P values < 0.0083 were accepted as statistically significant [41]. The number of oocytes showing GVBD was compared by chi-square analysis, followed by the calculation of 95% confidence intervals for the difference in sample proportions. Statistical significance was inferred at P < 0.05.

RESULTS

Effect of Culture on Kitl Expression in eCG-Primed Granulosa Cells

To determine if Kitl isoform expression was maintained when granulosa cells were placed in culture, granulosa cells were isolated from rat ovaries after eCG priming and cultured for 48 h. Northern blot analysis showed that Kitl mRNA levels were 13.0 ± 0.4 fold lower in cultured granulosa cells relative to those in which RNA was extracted immediately after isolation (Fig. 1A). This decrease in Kitl levels occurred regardless of whether the cells were cultured in the presence or absence of 5% FBS. To determine if there was equal loss of both Kitl1 and Kitl2 mRNA in cultured cells, RT-PCR reactions were performed on the granulosa cell RNA samples (Fig. 1B). As expected, there was a predominance of Kitl2 mRNA in extracts of freshly isolated granulosa cells. However, in cultured cells, there was a dramatic shift in the Kitl1:Kitl2 ratio, due primarily to a significant reduction in Kitl2 expression. Expression of both isoforms was reduced when the cells were cultured in serum-free medium. Quantification of the changes in expression of Kitl2 mRNA by real-time PCR confirmed a 3-fold decrease (Fig. 1C).


Figure 01
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  		FIG. 1 Kitl mRNA abundance in granulosa cells before or after 48 h of culture. Granulosa cells were isolated from eCG-primed rats and were either flash frozen (0 h) or cultured for a total of 48 h in WAY/FBS (48-a). In some cases, granulosa cells were cultured for the second 24 h in WAY/BSA (48-b). A) Results of Northern blot analyses are presented as the ratio of Kitl:28S RNA band intensity, standardized to uncultured (0 h) levels that were arbitrarily set to 1.0. B) RT-PCR for Kitl detected Kitl1 (287 bp) and Kitl2 (203 bp) transcripts. The positive control is C13 (labeled +ve control), a cell line that produces predominantly Kitl1 transcripts; water was used instead of cDNA as a negative control. C) Real-time PCR quantification of Kitl1 and Kitl2 mRNA abundance in rat granulosa cells (GCs) with (+) or without (–) culture for 48 h. Bars indicated with different letters are significantly different (P < 0.01 in A; P < 0.05 in C, n = 3), and error bars represent mean ± SEM.

The culture-associated decreases in Kitl expression suggested that granulosa cell culture would not offer a stable system in which to investigate the effects of KITL1 and KITL2 on oocyte growth. Consequently, an alternative system was developed in which KITL-deficient murine fibroblasts were transfected to stably express either KITL1 or KITL2 (Fig. 2A). Quantitative analysis showed mRNA expression levels 20- to 50-fold higher than freshly isolated rat granulosa cells (Fig. 2B). These cells were then used for coculture with oocytes from which the zona pellucida had been removed to facilitate attachment of the oocytes to the fibroblasts for the duration of the culture period.


Figure 02
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  		FIG. 2 Kitl mRNA abundance in Kitl-deficient fibroblasts and fibroblasts transfected with constructs expressing either Kitl1 or Kitl2. A) RT-PCR analysis shows expression of Kitl2 (471 bp) and Kitl1 (192 bp), or no Kitl is shown in duplicate samples from the appropriate fibroblasts. B) Real-time PCR analysis comparing Kitl expression in granulosa cells isolated from diethylstilbestrol- (GC1) and eCG-primed (GC2) rats and from Sl4 (no Kitl) fibroblasts transfected with Kitl1 and Kitl2. For each figure, bars indicated with different letters are significantly different (P < 0.01, n = 3), and error bars represent mean ± SEM.

Oocyte Growth after Culture on KITL-Deficient and KITL-Producing Fibroblasts

Oocyte diameter was significantly increased after a 2-day culture on KITL2-producing fibroblasts compared with oocytes cultured on KITL-deficient or KITL1-producing fibroblasts (P < 0.008; Fig. 3A). Addition of exogenous KITL1 to the cultures containing KITL2-producing fibroblasts inhibited oocyte growth. Specificity of KITL2 action was confirmed by results showing that the increased oocyte diameter in the presence of KITL2 was suppressed by Gleevec, an inhibitor of KIT activity, as well as ACK2, an anti-KIT antibody (P < 0.008; Fig. 3B). Rather surprisingly, oocytes cultured on KITL-deficient fibroblasts showed a high percentage (70%) undergoing premature and spontaneous GVBD (Table 1). This proportion was reduced in oocytes grown on KITL2-producing fibroblasts (P < 0.05) but not on KITL1-producing fibroblasts. Furthermore, the addition of Gleevec to the KITL2-producing fibroblasts attenuated the KITL2-associated inhibition of GVBD, as did the presence of exogenous KITL1.


Figure 03
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  		FIG. 3 Oocyte diameters measured after a 2-day incubation in the presence of KITL-deficient (n = 32), KITL1-producing (n = 32), or KITL2-producing fibroblasts (n = 30). A) The effect of increasing the KITL1:KITL2 ratio was tested by adding exogenous soluble KITL1 (exog KITL1; 10 ng/ml) to the oocytes on KITL2-producing fibroblasts (n = 29). In (B), Gleevec (Glv), an inhibitor of KIT activity (n = 15; 5 µM), and the anti-KIT antibody ACK2 (n = 13; 1 µg/ml) were used to confirm the specificity of any KIT-mediated effects. Values are mean ± SEM. Bars indicated with different letters are significantly different (P < 0.0083).


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  		TABLE 1 Percentage of oocytes that had undergone GVBD after a 2-day incubation in the presence of KITL-deficient, KITL1-producing, or KITL2-producing fibroblasts.

Effect of dbcAMP and PMA on Oocyte GVBD

To investigate the possibility that oocytes cultured in the presence of KITL-deficient fibroblasts were undergoing premature GVBD due to an early onset of meiotic competence, oocytes were cultured in the presence of dbcAMP or PMA to inhibit spontaneous GVBD, as previously reported [39, 40]. In the absence of KITL, 67% of oocytes underwent GVBD (Fig. 4). This was decreased by 17% in the presence of dbcAMP, which was not statistically significant. Similarly, PMA failed to maintain the oocytes in meiotic arrest, with the percentage undergoing GVBD not significantly different from the KITL-deficient group, indicating that the meiotic resumption could not be controlled by these known meiosis inhibitors. In contrast, GVBD was significantly decreased by 48% in oocytes cultured in the presence of KITL2 (P < 0.05), compared with the KITL-deficient controls.


Figure 04
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  		FIG. 4 Percentage of oocytes undergoing germinal vesicle breakdown (% GVBD) after a 2-day culture on KITL-deficient fibroblasts treated with dbcAMP (n = 30; 0.3 mM) or phorbol 12-myristate 12-acetate, PMA (n = 29; 0.1 nM). Oocytes cultured on KITL2-expressing fibroblasts were included as a comparison. Bars indicated with different letters are significantly different (P < 0.05).

Role of PI3K in Oocyte Growth on KITL2-Producing Fibroblasts

Experiments initially designed to determine the role of PI3K in KITL2-mediated oocyte growth showed that Wortmannin, an inhibitor of PI3K, dramatically reduced the expression of Kitl2 in the transfected fibroblasts (Fig. 5C). This reduction in Kitl2 expression was associated with an impairment of oocyte growth (Fig. 5A) and an increase in the proportion undergoing spontaneous GVBD (Fig. 5B), further implicating KITL2 in promoting oocyte growth.


Figure 05
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  		FIG. 5 Wortmannin (100 nM), an inhibitor of PI3K, was added to the oocytes (n = 25) cultured in the presence of KITL2-producing fibroblasts. Results are presented as the mean oocyte diameter ± SEM (A) and the percentage of oocytes that had undergone GVBD (B) after the 2-day culture. Bars indicated with different letters are significantly different (P < 0.05). C) Kitl2 mRNA abundance in Sl4 fibroblasts (no Kitl), and fibroblasts transfected with Kitl1 and Kitl2, cultured with mouse zona-free oocytes for 48 h with (+) or without (–) Wortmannin. Bars indicated with different letters are significantly different (P < 0.01, n = 3 or 4), and error bars represent mean ± SEM.

KIT Expression in Oocytes after Culture on KITL-Deficient and KITL-Producing Fibroblasts

Immunofluorescence was performed to determine KIT receptor expression in oocytes cultured with KITL-deficient, KITL1-producing, and KITL2-producing fibroblasts. KIT receptors were present on the oocyte membrane (Fig. 6A). Expression on the oocyte surface was low in the absence of KITL (Fig. 6A, a), but the intensity of the fluorescent signal was increased in the presence of KITL2 (Fig. 6A, b). When exogenous KITL1 was added to cultures containing KITL2-producing fibroblasts, KIT expression was less than in the presence of KITL2 alone and was comparable to the oocytes cultured with the KITL-deficient fibroblasts (Fig. 6A, c). Processing of control oocytes where the primary antibody was omitted showed no background fluorescence (Fig. 6A, d).


Figure 06
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  		FIG. 6 A) Detection of expression of KIT receptors by immunocytochemistry on mouse oocytes cultured for 48 h in the presence of KITL-deficient fibroblasts (a), KITL2-producing fibroblasts (b), or KITL2-producing fibroblasts with the addition of exogenous KITL1 (c) by immunofluorescence. A control oocyte where the primary antibody was omitted showed no background fluorescence (d). Bar = 8.5 µm. B) Real-time PCR analysis comparing Kit expression in oocytes cultured for 48 h in the presence of KITL-deficient (no KITL) or KITL1- or KITL2-producing fibroblasts. Bars indicated with different letters are significantly different (P < 0.05, n = 3–4), and error bars represent mean ± SEM.

RT-PCR analysis was performed to confirm Kit expression in the cultured oocytes and showed that oocytes cultured with KITL2-producing fibroblasts expressed markedly higher Kit than those cultured with KITL-deficient and KITL1-producing cells (P < 0.05; Fig. 6B).

DISCUSSION

These studies demonstrate that Kitl mRNA is not only dramatically reduced in cultured rat granulosa cells, but also that these cells shift from producing more Kitl2 to expressing predominantly Kitl1 mRNA. To determine the relative biopotency of membrane-bound versus soluble KITL in promoting oocyte growth, it was first necessary to develop a culture system in which KITL2 expression was sustained. With murine oocytes cocultured with fibroblasts stably expressing either KITL1 or KITL2, the results demonstrate that KITL2 is the principal KITL isoform associated with oocyte growth in vitro. KITL2 also increased expression of the KIT receptor on the oocyte surface, whereas KIT expression was lower in the absence of KITL or when soluble KITL1 was added to oocytes grown on KITL2-producing fibroblasts. Three lines of evidence support a KITL2-mediated mechanism in the modulation of oocyte growth. First, both Gleevec, a KIT inhibitor, and ACK2, a specific neutralizing anti-KIT antibody, suppressed oocyte growth induced in the presence of KITL2-producing fibroblasts. Moreover, Gleevec attenuated the KITL2-mediated suppression of GVBD. Second, when the KITL1:KITL2 ratio was increased by the addition of soluble KITL1 to cultures in the presence of KITL2, oocyte growth was suppressed. This result is in agreement with our previous data showing that the correct balance of KITL1:KITL2 is necessary for optimum oocyte growth in vitro [30]. Third, Wortmannin-induced suppression of Kitl2 expression simultaneously inhibited oocyte growth. These results provide the first direct evidence that KITL2 is the isoform that promotes oocyte growth.

In vivo, KITL expression is low during early preantral development (in 7- and 8-day-old mice) and increases during later preantral stages (12- and 13-day-old mice) before declining in early antral follicles of 15-day-old mice [11, 42]. The timing of expression of KITL2 during follicle development is consistent with a role for this ligand in stimulating oocyte growth [6, 11, 42]. Although a role for soluble KITL1 in promoting early oocyte growth (in 8-day-old mice) in vitro has previously been demonstrated [31], growth was limited, and the relative contributions of membrane-bound and soluble KITL during oocyte development were not elucidated. In a previous study, we showed an increase in the Kitl1:Kitl2 ratio in oocyte-granulosa cell complexes containing fully grown oocytes isolated from 19-day-old mice, compared with the oocyte-granulosa cell complexes from 15-day-old mice, which contain growing oocytes [30]. This pattern of expression is in agreement with our in vitro data from the same study, where a low steady-state Kitl1:Kitl2 mRNA ratio was associated with growing oocytes on Day 3 of culture, with a subsequent increase in the Kitl1:Kitl2 ratio between Days 3 and 7, when no further increase in oocyte diameter was observed [30]. While these studies have all correlated KITL2 expression with oocyte growth, the results of this study provide the first direct evidence that KITL2 stimulates oocyte growth.

Evidence of the physiological importance of membrane-bound KITL2 has been shown in several cell types. A study of mast cell attachment to fibroblasts derived from KitlSl/KitlSl-d mice showed that the extracellular domain of the membrane-associated form of KITL was required to mediate this attachment [43]. KITL2 is also a more potent stimulator of primordial germ cell survival and proliferation than is soluble KITL1 [44–46]. In testes, membrane-bound KITL is more mitogenic to KIT-bearing germ cells [47, 48] and facilitates adhesion of germ cells to Sertoli cells [49], a process that is imperative for germ cell survival and development. Mice homozygous for the KitlSl-d allele, which produce only KITL1, are sterile because of a deficiency in germ cells [13], whereas mice that exclusively produce KITL2 are fertile [50]. Based on this evidence, as well as the results of the present study, it is clear that membrane-bound KITL2 has a quality of activity that cannot be replaced by soluble KITL1.

The mechanisms that regulate KIT expression in oocytes are poorly understood. In this study, KITL2 increased and maintained KIT receptor expression on the surface of the oocyte during the culture period, whereas KIT expression was reduced in the absence of KITL, as well as when exogenous KITL1 was added to oocytes cultured on the KITL2-producing fibroblasts. As KITL2 is membrane anchored, it may prevent subsequent down-regulation and internalization of activated KIT, as has been shown in mast cells [15]. In a stromal cell line, KITL2 has been previously reported to induce a more persistent activation of KIT receptor kinase than the soluble form of KITL [16, 17]. KITL2 may thus be the more potent isoform for promoting oocyte growth by its stabilization of KIT receptor expression and persistent activation.

Oocyte meiotic competence, defined as the ability of the oocyte to resume and complete meiosis, is acquired as the oocyte approaches its full size. A number of follicular factors have been shown to delay or inhibit the resumption of meiosis, of which cAMP is the best characterized. Oocytes cultured with membrane-permeable analogues of cAMP inhibit meiosis in a dose-dependent and reversible manner [51]. Inactivation of protein kinase C also appears to be involved in the process of FSH-mediated oocyte meiotic maturation in the mouse [40]. In this study, oocyte growth was limited in the absence of KITL, and yet spontaneous GVBD was observed in these oocytes, suggesting the acquisition of meiotic competence. However, the inability of both dbcAMP and PMA to suppress this GVBD suggests that this breakdown of the nuclear envelope is not a physiologic, albeit premature, resumption of meiosis. Although a role for KITL2 in the maintenance of meiotic arrest cannot be ruled out, the evidence that KITL promotes the survival of several cell types [52–54], including oocytes [26], and that nuclear envelope breakdown is an indicator of oocyte degeneration leads to the possibility that KITL2 promotes oocyte survival and, in its absence, the apparent GVBD denotes the early stages of oocyte degeneration, rather than the resumption of meiosis.

This hypothesis is strengthened by previous studies that have investigated the spontaneous resumption of meiosis of mouse oocytes cultured with fibroblasts or in somatic cell-conditioned medium [55, 56]. Because fibroblasts are known to express KITL [57], our experimental design differs from that of Canipari et al. [55], as we have used KITL-deficient fibroblasts, as well as fibroblasts expressing either KITL1 or KITL2. By employing this method, we have been able to show the specific importance of KITL2 in oocyte growth and survival, as well as pinpoint functional differences between the two isoforms. There are at least three studies showing that mouse oocytes do not undergo GVBD at least until Day 15 of overall age, i.e., inclusive of days spent both in vivo (postpartum) and in vitro [55, 56, 58]. In the present study, oocytes from 12-day-old mice were cultured for 2 days on KITL-deficient fibroblasts, with almost 70% of oocytes undergoing GVBD. Given the previous work and the failure to inhibit meiotic resumption with known meiosis inhibitors, it seems unlikely that such a high proportion of oocytes at this stage attained the competence to resume meiosis, which strengthens the hypothesis that KITL2 promotes oocyte survival.

The importance of intraovarian paracrine factors in ovarian function has been established but is not well understood. In this study, we have provided insight into the relative contribution of the specific KITL isoforms during murine oocyte development and have identified KITL2 as the principal isoform regulating oocyte growth and perhaps survival in vitro. The culture system established in this study may also be helpful in future studies to examine the interactions with other paracrine factors that regulate oocyte-granulosa cell interactions during folliculogenesis.

ACKNOWLEDGMENTS

The authors thank Kerri Courville and Elizabeth Macdonald for their assistance with oocyte collection and maintenance of the cell lines and David Thomas for advice on the statistical analyses.

FOOTNOTES

1This study is part of the Program on Oocyte Health funded under the Healthy Gametes and Great Embryos Strategic Initiative of the CIHR, Institute of Human Development, Child and Youth Health, grant HGG62293 (to B.C.V.). F.H.T. is a Canadian Institutes of Health Research (CIHR) Strategic Initiative Fellow, funded by a STIRRHS bursary. Back

Correspondence: 2Barbara C. Vanderhyden. FAX: 613 247 3524; e-mail: bvanderhyden{at}ohri.ca

Received: 31 October 2006.

First decision: 6 December 2006.

Accepted: 20 September 2007.

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