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Ovary |
Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 277 21 Libechov, Czech Republic
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cumulus cells, follicle, granulosa cells, growth factors, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The first oocyte-specific factor influencing the function of granulosa cells was identified and characterized in mice with targeted deletion of the growth differentiation factor 9 (GDF9) gene. In these mice, primordial and primary one-layer follicles could be formed, but follicular development beyond the one-layer follicle stage was blocked [10, 11]. In wild-type mouse, GDF9 mRNA was synthesized only in oocytes from the primary stage until after ovulation and not in somatic follicular cells [10, 12]. Thus, it appears that the GDF9, a member of the transforming growth factor (TGF) ß family, is specifically expressed in oocytes and is essential for the normal progression of folliculogenesis in the mouse. The extended expression of GDF9 throughout the oocyte development suggests that GDF9 affects processes in later stages of follicular development. Further data supporting such an idea were obtained after the preparation of a recombinant mouse GDF9 using a Chinese hamster ovary cell as an expression system [12, 13]. Those authors found that recombinant GDF9 stimulates synthesis of hyaluronan synthase 2 (HAS-2) and cyclooxygenase 2 (COX-2) but suppresses synthesis of the urokinase plasminogen activator (uPA) and LH-receptor mRNA. Because the induction of HAS-2 and suppression of protease uPA in cumulus cells are key events that regulate production of the hyaluronic acid-rich extracellular matrix during cumulus expansion, those authors tested a hypothesis that GDF9 could mimic this process. They found that GDF9 enabled FSH-stimulated expansion even in complexes in which oocyte had been removed. These data suggest that GDF9 is involved in the regulation of cumulus expansion and, perhaps, is identical with the CEEF [12]. Next, GDF9-stimulated expression of COX-2 and steroidogenic acute regulator protein (StAR) in mouse granulosa cells [12] indicates that GDF9 may be involved in the regulation of progesterone synthesis by cumulus cells in preovulatory follicles, which in vivo is required for optimal cumulus expansion, ovulation, and fertilization [14, 15]. These studies demonstrate that GDF9 plays multifunctional roles in the regulation of follicular development. To our knowledge, however, the expression and function of GDF9 in ovarian tissues has not yet been studied in the pig.
Significant differences have been shown in the regulation of cumulus oophorus expansion between mice and large animal species. In contrast to the mouse, porcine and bovine cumulus cells are able to undergo FSH-stimulated expansion without factors secreted by the oocyte [16–19]. This may result from autocrine production of a factor that chemically appears to be similar, if not identical, to the CEEF produced by mouse oocytes [20]. Moreover, the factor produced by porcine cumulus enables expansion of mouse oocytectomized complexes in an interspecies testing system [17, 20]. Because the CEEF is probably identical to GDF9 [12, 21], in the present study we tested the hypothesis that GDF9 may be expressed not only in oocytes but also in cumulus and mural granulosa cells of porcine growing and preovulatory follicles. In addition, we questioned whether the relative abundance (RA) of GDF9 mRNA changes in oocytes and somatic follicular cells during maturation of OCCs in vivo and in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of OCCs, Denuded Oocytes, Cumulus, and Mural Granulosa Cells of Immature Gilts
Ovaries of slaughtered gilts were collected at a local abattoir, kept in a thermos in prewarmed (37°C) PBS, and transported to the laboratory within 1 h. The contents of the growing follicles (diameter, 3–5 mm) were aspirated by a syringe connected with a 20-G needle, pooled in a 15-ml test tube, allowed to settle, and washed three times by PBS with 3 mg/ml of polyvinylpyrrolidone. The OCCs and clumps of mural granulosa cells (diameter, 
300 µm) were picked from the aspirate. In addition, clumps of mural granulosa cells were isolated from halves of dissected follicles that lacked cumulus oophorus to avoid contamination by cumulus cells. Cumulus cells were obtained by repeated pipetting of OCCs through a fine-bore pipette. Special attention was paid to avoid rupture of any oocyte during the removal of cumulus, which might result in the contamination of cumulus cells by oocyte mRNA. Denuded oocytes were exposed to a 0.1% trypsin solution in PBS to ensure complete removal of cumulus cells. To prepare samples for extraction of the total RNA, various numbers of OCCs (up to 50), oocytes (up to 1000), as well as cumulus cells removed from 150 to 300 OCCs and 150–300 pieces of mural granulosa cells (both representing approximately 1.5 to 3 x 105 cells) were lysed in 350 µl of lysis buffer (Qiagen Sciences, Germantown, MD) and stored frozen at –70°C.
Isolation of OCCs and Mural Granulosa Cells of Cycling Gilts
To assess in vivo the expression of GDF9 mRNA during the periovulatory period, the samples were prepared from follicular cells of 10- to 14-mo-old cycling gilts crossbred between Minnesota and Göttingen strains of miniature pigs. The gilts were examined daily for estrus and stimulated on Day 15 of the reproductive cycle with 500 IU of eCG (Folligon; Intervet, The Netherlands) and, to initiate expansion of the OCCs, with 500 IU of hCG (Pregnyl; Organon, Oss, The Netherlands) given 72 h after the eCG. The gilts were slaughtered either 72 h after eCG administration or at different intervals (8, 16, 24, and 32 h) after hCG administration. The ovaries were excised immediately after slaughter, and the OCCs and mural granulosa cells were isolated from large preovulatory follicles (diameter, 6 mm). In addition, the tissues were isolated from follicles of nonstimulated gilts slaughtered on Day 15 of the cycle. The samples for extraction of RNA from the collected tissues were prepared as described above.
Culture of OCCs In Vitro and Assessment of Cumulus Expansion
To assess expression of GDF9 mRNA in OCCs expanding in vitro, the OCCs isolated from the ovaries of immature gilts were washed and cultured in M-199 (Sevapharma, Prague, Czech Republic) supplemented with 6.25 mM Hepes, 20 mM sodium bicarbonate, 0.91 mM sodium pyruvate, 1.62 mM calcium lactate, penicillin G (50 mg/L) and streptomycin (50 mg/L). (all from Sigma, Prague, Czech Republic). Only OCCs surrounded by compact, multilayered cumulus were selected for the experiments. Twenty OCCs were cultured in 1 ml of M-199 with 5% fetal calf serum (Sigma) in four-well dishes (Nunclon, Roskilde, Denmark) at 38.5°C in an atmosphere of 5% CO2 in the air. Expansion of the cumulus cells was stimulated by the addition of 100 ng/ml of FSH (Puregon; Organon) into the culture medium. The degree of cumulus expansion was assessed at different intervals (4, 8, 16, 20, and 24 h) following the onset of culture using a subjective scoring method [7]. Briefly, the scoring method was as follows: 0, no response; 1, minimum observable response (i.e., cells in the outermost layer of the cumulus become round and glistening); 2, expansion of outer OCC layers; 3, expansion of all OCC layers except the corona radiata; and 4, expansion of all OCC layers. The aim of the present experiment was to determine when the cultured OCCs begin to expand (i.e., undergo transition from degree 1 to degree 2). For this reason, the proportions of OCCs with expansion scores of 1–4 and 2–4 were assessed at the indicated times of culture.
Detection of GDF9 mRNA by Reverse Transcription-Polymerase Chain Reaction
The total RNA from denuded oocytes, OCCs, cumulus, and mural granulosa cells was extracted with the use of the RNeasy Mini Kit (Qiagen) following the manufacturer's instructions. To exclude contamination of the samples by genomic DNA, an on-column DNase digestion was carried out during isolation of the RNA from all tested cell types using RNase-free DNase set (Qiagen). The concentration of the RNA in the samples was assessed by a spectrophotometer (Helios 2; Spectronic Unicam, Cambridge, UK). The reverse transcription-polymerase chain reaction (RT-PCR) was carried out by One-Step RT-PCR Kit (Qiagen) using oligonucleotide primers directed against homologous sequences of human and mouse GDF9 (5'-TAGTCAGCTGAAGTGGGACA-3' and 5'-ACGACAGGTGCACTTTGTAG-3') [22]. These primers were expected to generate a 277-base pair (bp) cDNA fragment. For glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene, the RT-PCR was performed using primers (5'-GTTCCAGTATGATTCCACCCACGGCAAGTT-3' and 5'-TGCCAGCCCCAGCATCAAAGGTAGAAGAGT-3') that generated a 763-bp fragment.
The total RNA of the samples was reverse-transcribed and subsequently amplified in a reaction mixture (total volume, 25 µl) containing 5 µl of 5x reaction buffer, 1 µl of dNTP mix (10 mM stock of each), 0.5 µl of both reverse and forward primer (0.1 mM stock), 0.15 µl of RNasine (20 U/µl stock; Promega, Madison, WI), 1 µl of enzyme mix, and RNA. For each sample, the amplification of each gene was run in a separate tube. The reaction conditions were as follows: cDNA synthesis at 50°C for 30 min, predenaturation at 95°C for 5 min, and then various numbers of PCR cycles consisting of denaturation (95°C for 30 sec), annealing (58°C or 65°C for 30 sec for GDF9 and GAPDH, respectively), extension (72°C for 45 sec), and final extension (72°C for 5 min). For semiquantitative RT-PCR, the number of cycles was optimized for each set of samples by a gradient method over the range of 20–40 cycles to ensure that amplification of cDNA for both primer sets was terminated in the exponential phase of the PCR. Products of the RT-PCR were separated by electrophoresis on 1.5% agarose gel and visualized by ethidium bromide staining. The intensity of the objective bands was determined by scanning densitometry using Image J Version 1.29 free software (National Institute of Mental Health, Bethesda, MD). The RA of GDF9 mRNA was expressed as the ratio of GDF9 to GAPDH.
Quantification of GDF9 mRNA Expression by Real-Time RT-PCR
In some experiments, real-time RT-PCR was carried out to confirm significant differences in the GDF9 mRNA expression indicated by the scanning densitometry. The reaction mixture was the same as described above except for a lower concentration of the primers (0.02 mM stock). In addition, 0.5 µl of SYBR Green I of 1.000x stock solution (Molecular Probes, Eugene, OR) was added in each reaction. The amplification was performed on the RotorGene 2000 cycler (Corbett Research, Sydney, Australia) under the same reaction conditions as described above. Fluorescence data were acquired during an additional step at approximately 3°C below the product's melting temperature (Tm). For quantification, an endogenous standard curve was generated for each transcript (GDF9 and GAPDH) by amplifying serial dilutions of RNA from control (a serial 10-fold dilution series spanning three orders of magnitude). After the cycling, the melting curve was generated to verify the amplification of one specific target (one peak at a specific melting temperature demonstrates the specificity). No primer-dimers were generated during the 35 amplification cycles of real-time PCR. In addition, specificity of RT-PCR products was assessed by gel electrophoresis and staining as described above.
Nucleotide Sequencing of the RT-PCR Product
To confirm the identity of the RT-PCR products with GDF9, DNA was isolated from gel using the MinElute Gel Extraction Kit (Qiagen) according to the manufacturer's instruction, reamplified by 40 cycles of PCR under the conditions described above, and analyzed by an ABI PRISM sequencer (Applied Biosystems, Foster City, CA).
Statistical Analysis
One-way ANOVA followed by the Tukey posttest was used to compare the results of densitometry, real-time RT-PCR quantification, and proportions of expanding OCCs. The experiments were carried out in at least three replicates. The results quantifying RA of GDF9 mRNA are expressed as the mean of the GDF9:GAPDH ratio. The differences were considered to be significant at P < 0.05. The software program GraphPad Prism version 4.0 (GraphPad Software, San Diego, CA) was used for the statistical calculations.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The RT-PCR resulted in amplification of a product of the expected size (277 bp) in all samples prepared from freshly isolated oocytes, cumulus, and mural granulosa cells but not in control samples of porcine fibroblast cells (Fig. 1). The RA of GDF9 mRNA was approximately fourfold higher in oocytes than in cumulus and mural granulosa cells (Fig. 2). Running the PCR without RT yielded no detectable product (Fig. 2). In addition, treatment of the samples with DNase did not disturb the production of the specific band by RT-PCR. These control treatments thus excluded the possibility of sample contamination by genomic DNA. The identity of the PCR products obtained by amplification of oocyte, cumulus, and the mural granulosa cell cDNA were assessed by analysis of their nucleotide sequence (submitted to EMBL/GenBank/DDBJ databases under the accession no. AJ620358). The analysis showed that the sequence of the 277-bp fragments was 88%, 90%, and 91% homologous to human, bovine, and ovine GDF9, respectively, based on published sequences (GenBank accession no. NM 005260, NM 174681, and AF 078545, respectively). The sequences of the fragments relevant to oocyte, cumulus, and mural granulosa GDF9 were identical. The sequence of 92 amino acids encoded by the 277-bp fragment in the pig was 97% and 99% identical to human and bovine sequences, respectively. No significant similarity was found between the 277-bp fragment and the published sequences of the most relative members of the TGFß superfamily: bone morphogenetic protein 15 (GenBank no. NM 009757, NM005448, and AJ 534391) and TGFß1 (GenBank no. AF461808).
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Expression of GDF9 mRNA in Expanding OCCs During Culture In Vitro
In the cultured mouse granulosa cells, GDF9 increases expression of HAS-2, which regulates production of hyaluronic acid by the expanding cumulus cells [12]. The aim of the present experiment was to determine whether FSH-stimulated expansion of porcine OCCs cells in vitro is preceded by an increase in RA of GDF9 mRNA. For this reason, proportions of expanding OCCs and RA of GDF9 mRNA were measured in OCCs cultured for 0–24 h. Expansion 1 was observed after 8 h, and expansion 2 occurred in this experiment between 8 and 16 h in a great majority of the cultured OCCs (Fig. 3C). Therefore, the RA of GDF9 mRNA should have increased by 16 h of culture. However, we found that it did not change during the first 16 h of culture and decreased significantly during later stages of the culture period (Fig. 3). This decrease occurred in the cumulus cells, because the RA of GDF9 mRNA remained stable in oocytes throughout the culture period (Fig. 4).
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Expression of GDF9 mRNA in OCCs and Mural Granulosa Cells During Periovulatory Period
In these experiments, we assessed whether expansion of cumulus cells under in vivo conditions is accompanied by an increase in the expression of GDF9 mRNA. The RA of this transcript was examined in OCCs and mural granulosa cells isolated from large preovulatory follicles of gilts stimulated by eCG and by eCG plus hCG. In vivo, the cumuli still remained compact at 8 h after hCG administration, and expansion of the outer layers of cumulus cells was observable at 16 h after hCG administration. Extensive expansion (degrees 3 and 4) of the cumulus and adjacent part of the mural granulosa was observed at 24 h post-hCG. The RA of GDF9 mRNA in OCCs isolated 8–32 h after hCG administration was significantly lower than in compact OCCs isolated from nonstimulated gilts at Day 15 of the cycle and from eCG-stimulated gilts (Fig. 5). Similarly, no increase in the RA of GDF9 mRNA was observed in mural granulosa cells isolated from eCG+hCG-stimulated follicles when compared to nonstimulated or eCG-stimulated follicles (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of GDF9 was oocyte specific in mouse ovarian tissue as determined by immunohistochemistry [12] and in situ hybridization [23, 24]. In the rat, as with mice, GDF9 expression was consistently found exclusively in oocytes from the primary follicle stage onward [25]. In ovine and bovine ovaries, GDF9 mRNA was also expressed exclusively in oocytes [26, 27]; however, in contrast to the mouse, the positive signal was already found at the primordial follicle stage [22]. This indicates that GDF9 may be involved not only in the maintenance of folliculogenesis beyond the primary stage in these species but also in the process of recruiting primordial follicles to the growth phase. Localization of GDF9 mRNA and protein in human and primate ovarian tissues is a matter of debate. In some studies, expression of GDF9 was detected only in oocytes by immunohistochemistry and in situ hybridization [28– 30]. Recently, however, the GDF9 protein and mRNA were also detected in human and primate granulosa and cumulus cells by Western blot analysis, immunohistochemistry, and RT-PCR [31–33].
The present data support the idea that GDF9 mRNA may not be expressed exclusively in oocytes within the ovary in some species. Using primers directed against highly conserved regions of the GDF9 gene, we were able to detect GDF9 mRNA in samples of either oocytes or cumulus and mural granulosa cells. The identity of the RT-PCR products with GDF9 was confirmed by nucleotide sequencing. This approach also completely excluded the possibility that the primers designed against the GDF9 gene interact with another member of the TGFß family. Contamination of the samples by genomic DNA was excluded by running the PCR without RT with a negative result and also by treatment of the samples from cumulus and granulosa cells by DNase, which did not disturb the positive outcome of the RT-PCR. These results support our hypothesis about expression of GDF9 in pig cumulus and mural granulosa cells raised on the basis of our previous study, in which we reported that media conditioned by pig cumulus and mural granulosa cells isolated from growing and preovulatory follicles exhibit the CEEF activity [20]. Because the GDF9 is the most probable candidate for the CEEF [12, 21], it appeared reasonable to speculate about its production by pig somatic follicular cells. It is now conceivable that at least part of the CEEF activity produced by pig cumulus and mural granulosa cells during their culture in vitro is mediated by expression of GDF9 mRNA and the synthesis of an active GDF9 protein.
Like other members of the TGFß family, GDF9 is produced as a glycosylated preprotein and is activated by an unknown mechanism [12]. Regulation of the amount of active GDF9 in follicular tissues and follicular fluid is not clear. It has been suggested that small amounts of active GDF9 are produced throughout follicular development and that a bolus of active GDF9 is released by the oocyte only after the LH surge [34]. A concentration-dependent effect would explain why GDF9 does not stimulate enzymes regulating cumulus expansion (HAS-2 and COX-2) prematurely in growing follicles. Under this scheme, a low concentration of GDF9 would stimulate proliferation and prevent differentiation of granulosa (cumulus) cells via suppression of Kit-ligand and LH-receptor expression. On the other hand, large amounts of GDF9 would promote expression of HAS-2 and COX-2 and, therefore, expansion of the cumulus cells. We have tested the possibility that the large amounts of active GDF9 result from an increased transcription of the GDF9 mRNA in OCCs and/or mural granulosa cells. However, we found that no significant increase in the RA of GDF9 mRNA occurred both in vivo, during the periovulatory period, and in vitro, during FSH-stimulated expansion of the OCCs. Instead, we found a significant decrease in the RA of GDF9 mRNA in cumulus cells during expansion of OCCs. This decrease may reflect a down-regulation of GDF9 mRNA synthesis following activation of processes leading to increased production of hyaluronic acid and its incorporation in the extracellular matrix. To our knowledge, involvement of GDF9 in these processes has yet to be confirmed in the pig.
It follows that further experiments are necessary to elucidate changes in concentration of premature GDF9 protein in porcine preovulatory follicles and mechanisms of its activation. An activation of a GDF9 preprotein-specific protease after the LH surge is certainly one of the mechanisms to be considered in this respect. In addition, the recent discovery of different susceptibilities of homo- and heterodimers of GDF9 and BMP-15, an oocyte-specific member of the TGFß superfamily, to proteolytic degradation offers a further mechanism that might be involved in regulation of the GDF9 activity [35]. It is interesting to note here that no significant change in concentration of both premature and mature GDF9 protein has been found in follicular fluid of rhesus monkeys during the periovulatory period [33].
In the present study, we show that expression of GDF9 mRNA is essentially higher in oocytes than in cumulus/ granulosa cells. Thus, it could be expected that the concentration of an active GDF9 protein is higher in the oocyte surroundings than in other compartments of the follicle. In this sense, the present results do not oppose a view of GDF9 as an oocyte growth factor that affects surrounding cells on the basis of a concentration gradient [36]. Nevertheless, the findings of the present and other studies [31– 33] about a likely production of GDF9 by somatic follicular cells suggest the possibility that GDF9, in interactions with other members of the TGFß family and gonadotropins, participates in regulation of complex follicular environment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Radek Prochazka, Academy of Sciences of the Czech Republic, Institute of Animal Physiology and Genetics, Rumburská 89, 277 21 Libechov, Czech Republic. FAX: 420 315 639 510; prochazka{at}iapg.cas.cz
Received: 30 January 2004.
First decision: 16 February 2004.
Accepted: 27 May 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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