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Endocrinology, doi:10.1210/en.2005-1208
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Endocrinology Vol. 147, No. 4 1725-1734
Copyright © 2006 by The Endocrine Society

Expression of Growth Differentiation Factor 9 in the Oocytes Is Essential for the Development of Primordial Follicles in the Hamster Ovary

Cheng Wang and Shyamal K. Roy

Departments of Obstetrics and Gynecology (C.W., S.K.R.) and Cellular and Integrated Physiology (S.K.R.), University of Nebraska Medical Center, Omaha, Nebraska 68198-4515

Address all correspondence and requests for reprints to: Shyamal K. Roy, Department of Obstetrics and Gynecology and Cellular and Integrative Physiology, University of Nebraska Medical Center, 984515 Nebraska Medical Center, Omaha, Nebraska 68198-4515. E-mail: skroy{at}unmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Postnatal growth differentiation factor 9 (GDF-9) expression in the hamster oocytes precedes the formation of primordial follicles. We examined the functional significance of GDF-9 in primordial folliculogenesis in the hamster ovary using RNA interference knockdown of GDF-9 mRNA and protein expression. Fifteen-day-old fetal ovaries were cultured for 9 d with or without 1 ng FSH, 1 µl Metafectane, 100 nM control nontargeting small interfering RNA (siRNA), GDF-9 siRNA, or GDF-9 siRNA + FSH, and the development of primordial follicles examined. The efficiency of siRNA transfecting ovarian cells in the organ culture was tested by culturing ovaries with siGlo, a nontargeting control siRNA labeled with Cy3. More than 90% of cells in the ovary were siGlo positive, and neither the Metafectane nor the siRNA-induced cellular apoptosis. Control siRNA did not affect the basal levels of GDF-9 mRNA, but GDF-9 siRNA slightly but significantly reduced the level. FSH markedly up-regulated the levels of GDF-9 mRNA and protein, and the effect was completely suppressed by GDF-9 siRNA. However, GDF-9 siRNA did not affect the levels of bone morphogenetic protein receptor IA or ß-actin mRNA. GDF-9 siRNA alone also reduced GDF-9 protein expression. Concurrent with GDF-9 expression, FSH significantly augmented primordial follicle formation, but the effect was abolished by GDF-9 siRNA. These results suggest that endogenous GDF-9 plays an important role in somatic cell differentiation and the formation of primordial follicles. Furthermore, FSH, by virtue of regulating GDF-9 expression, modulates oocyte regulation of primordial follicles formation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PRIMORDIAL FOLLICULOGENESIS requires a coordinated interaction of events, such as cell cycle progression, apoptosis, and differentiation of pluripotent somatic cells into granulosa cell lineage. Although the exact factors or mechanisms that signal somatic cell differentiation into the pregranulosa cells remain elusive, evidence has accumulated to suggest that growth differentiation factor 9 (GDF-9), one of the members of the TGFß superfamily of ligands, plays an important role in early folliculogenesis (1, 2, 3). GDF-9 protein expression in the oocytes of a variety of species first appears when the follicles reach the primary stage (1, 2, 4, 5, 6); however, in the hamster, it appears in the primordial oocytes long before any morphologically distinct primordial follicle can be identified in the postnatal ovary (7). The presence of GDF-9 mRNA in the rodent and human ovaries has been reported (6, 8). Nilsson and Skinner (9) have shown that a recombinant mouse GDF-9 promotes the growth of the primary follicles in neonatal rat ovaries in vitro, but it has no effect on the growth of primordial follicles. Furthermore, recent studies have shown that GDF-9 stimulates the in vitro growth of preantral follicles and thecal cell differentiation in the rat (10, 11) and primary and early secondary follicle formation in human ovarian slices in vitro (12). All these lines of evidence suggest that GDF-9 influences follicle formation beyond the primary stage.

Previously, we have shown that in the hamster ovary, GDF-9 immunoreactivity is present exclusively in the oocytes as early as d 4 of postnatal life (7), long before the first cohort of primordial follicles is visible (13). The administration of a recombinant rat GDF-9 to fetal hamster ovary cultures results in a marked enhancement of the formation of primordial follicles and their subsequent growth (7). Furthermore, FSH administration in vitro up-regulates GDF-9 expression in the oocytes of fetal hamster ovaries corresponding to increased formation primordial follicles (7). We have also demonstrated that elimination of FSH action during the fetal life results in marked decrease in the percentage of primordial follicles, and the effect can be reversed by exogenously added equine chorionic gonadotropin (13). The objective of the present study was to examine whether endogenous GDF-9 of oocyte origin was necessary for the formation of primordial follicles in cultured fetal ovaries. The objective was addressed by RNA interference (RNAi) knockdown of GDF-9 gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Male and female golden hamsters (90–100 g) were purchased from Charles River Laboratories (Charles River, MA) and maintained in a light-and climate-controlled room with free access to food and water according to the Institutional Animal Care and Use Committee (IACUC) and the United States Department of Agriculture guidelines. The use of hamsters for this study was approved by the IACUC. Females with at least three consecutive estrous cycles were mated with males on the evening of proestrous, and the presence of sperm in the vaginal smear the next morning was considered d 1 of pregnancy. Hamster gestation lasts for 16 d, and pups are born on d 16 of gestation.

DMEM was purchased from Invitrogen Life Technologies (Carlsbad, CA); human transferrin, selenium, and bovine insulin were from Collaborative Research (Bedford, MA); linoleic acid, BSA, and other fine chemicals were from Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific Co. (Pittsburgh, PA); Falcon tissue culture inserts and plates and solvents for histology were from Fisher Scientific Co. Plastic embedding medium was from Electron Microscopy Sciences (Hatfield, PA); GDF-9 antibody (catalog no. SC-7407) was from Santa Cruz Biotechnology (Santa Cruz, CA); second antibody-Alexa conjugate was from Molecular Probes (Eugene, OR); RT-PCR chemicals, Hot-start Taq polymerase and plasmid purification kit were from QIAGEN (Valencia, CA).

Cloning of hamster GDF-9 and designing GDF-9 small interfering RNA (siRNA)
An 1037-bp cDNA was amplified from the total RNA of 10-d-old hamster ovaries using RT-PCR protocol as described previously (14, 15). Briefly, after activating the enzyme for 15 min at 95 C, the reaction was continued for 30 cycles. The annealing temperature was 55 C. The PCR condition was: 1 min at 95 C, 2 min at 55 C and 1 min at 72 C. The cDNA was cloned into a PCR II-TOPO plasmid (Invitrogen), sequenced in the DNA sequencing core facility (University of Nebraska, Lincoln, NE), and blast-searched in the National Institutes of Health (NIH) GenBank. After confirmation, hamster GDF-9 sequence was compared with the rat, mouse, and human GDF-9 sequences using a Vector NTI (Invitrogen) gene analysis software (Durham Research Center Molecular Biology Core, University of Nebraska Medical Center). Forward and reverse primers (Table 1Go) for GDF-9 RT-PCR were designed from rat GDF-9 sequence (accession no. NM_0216672) using Amplify (Dr. Bill Engels, Genetics Department, University of Wisconsin, Madison, WI) and Vector NTI primer design software. The hamster GDF-9 cDNA and the deduced amino acid sequences were deposited in the GenBank (accession number: DQ237894). Using hamster GDF-9 cDNA sequence as a template, and rat GDF-9 cDNA sequence as the reference, 10 probable siRNA sense sequences were designed using siDesign software (Dharmacon, Inc., Lafayette, CO). The cRNA sequence of ACACCAUGGUCCAGAAUA was selected because it matched 100% with the hamster GDF-9 cDNA and fulfilled all eight essential criteria set by the software. The siRNA was synthesized by Dharmacon, Inc.


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  		TABLE 1. Primers and probes used for cloning and quantifying the levels of various transcripts present in the hamster ovary

 
Culture of prenatal hamster ovaries
Ovaries were collected from 15-d-old fetal golden hamsters in DMEM containing antibiotics (100 U/ml of penicillin G, 100 µg/ml of streptomycin sulfate, 0.25 µg/ml amphotericin B) and 0.5% BSA at room temperature, cleaned, rinsed three times with fresh DMEM and twice with DMEM containing 1% ITS+ (final concentration: 100 ng/ml insulin, 6.25 µg transferrin, 6.25 ng selenium, 5.35 µg of linoleic acid/ml, antibiotics and BSA (16) and cultured for 9 d at 37 C under 5% CO2 in air as described previously (7, 17). Medium was changed every 48 h, and after termination, ovaries were processed for morphometric evaluation of follicular development, detection of apoptotic DNA fragmentation, levels of mRNA, or immunofluorescence localization of protein.

Detection of transfection efficiency using siGLO-control siRNA
To validate the specificity of the RNAi approach, ovaries were cultured with 100 nM siGLO RISC-Free siRNA (Dharmacon) delivered in 2 µl Metafectane (BionTex, Munich, Germany), a transfection reagent, in DMEM without antibiotics. According to our fetal ovary culture procedure (17), the medium was replaced every 48 h with fresh medium without siRNA and the culture continued for an additional 7 d. siGLO was a cyanine 3-labeled stable, nontargeting control siRNA with enhanced fluorescence and had no similarity with any gene sequence in the human, rat, or mouse as verified by extensive genome database searching followed by microarray validation of the pattern of gene expression in control siRNA exposed cells of these species. Furthermore, siGLO modifications were nontoxic, had no detrimental effects on siRNA silencing activity, and could be visualized by fluorescence microscopy to assess the transfection efficiency. Ovaries were retrieved, and the formation of primordial follicles was examined by morphometry. Some ovaries were processed for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining to determine apoptotic DNA fragmentation.

Effect of GDF-9 siRNA on the formation of primordial follicles
In the first experiment, ovaries were cultured in the absence or presence of 100 nM nontargeting control or GDF-9 siRNA in 2 µl Transfectane for 48 h for siRNA. The medium was replaced every 48 h with fresh medium containing no siRNA, and ovaries were cultured for 9 d with medium changed every 48 h. In the next experiment, ovaries were cultured in the presence of 1 ng/ml of ovine-FSH-20 with or without 100 nM of GDF-9 siRNA. Ovaries were processed for morphometric assessment of the percentage of primordial follicles, real-time RT-PCR determination of the levels of GDF-9, bone morphogenetic protein receptor IB (BMPRIB) and ß-actin mRNA, and immunofluorescence quantification of GDF-9 protein expression.

TUNEL assay to determine DNA fragmentation
DNA fragmentation as an index of apoptosis was determined by ApopTag Fluorescein in situ apoptosis detection kit (Chemicon, Temecula, CA) according to the manufacturer’s protocol using frozen sections of cultured ovaries that were fixed in 1% paraformaldehyde in PBS (pH 7.4) at 4 C, and examined under a Leica DMR microscope (North Central Instruments, Minneapolis, MN) equipped with epifluorescence. Cells with fluorescence-labeled nuclei were apoptotic.

Real-time RT-PCR quantification of the levels of GDF-9, BMPRIB, and ß-actin mRNA
Ovaries were collected in RNA later solution (Ambion, Austin, TX), and total RNA isolated using a RNeasy mini kit (QIAGEN). The RNA was quantified using the Ribogreen kit (Molecular Probes) according to the manufacturer’s protocol. Real-time quantification of mRNA levels was done as described previously (15). Briefly, a series of mRNA standards for GDF-9, BMPRIB, and for ß-actin were used in parallel with the sample RNA to obtain true quantitative values. Real-time PCR primers and probes were designed from the hamster GDF-9, BMPRIA, and ß-actin cDNA sequences. The mRNA was synthesized using AmpliScribe T7-Flash transcription kit (Epicenter, Madison, WI), quantified using Ribogreen, the quality and the authenticity verified by routine RT-PCR using the forward and reverse primers designed specifically for real-time RT-PCR. A known amount of standard mRNA was reverse-transcribed parallel with 0.5 µg ovarian RNA samples in a total volume of 7 µl (14). A series of eight standards ranging from 0.1 fg to 1 ng in 10-fold increments was prepared from the cDNA. Aliquots of reverse-transcribed product of each ovarian sample were used for real-time quantification of GDF-9, BMPRIA, and ß-actin using the Opticon 2 thermocycler (MJ Research, Reno, NV). PCR was continued for 40 cycles after an initial denaturation at 95 C for 15 min to activate the HotStart Taq polymerase. Each cycle of PCR consisted of 30 sec of denaturation at 94 C, 45 sec of annealing at 55 C and 45 sec extension at 72 C, followed by a plate reading with a final extension at the end for 10 min at 72 C. Because PCR product size was kept around 100 bp, shorter annealing and extension time was adequate. The quantity of the mRNA was calculated from the linear portion of the amplification signal. The authenticity of the PCR signal was verified using tubes containing no RNA or RNA without reverse transcriptase. Furthermore, in preliminary experiments, the authenticity of the cDNA generated by real-time PCR was also verified by sequencing. The levels of mRNA were normalized against the total RNA, and the data presented as femtograms mRNA per microgram of total RNA. The levels of ß-actin mRNA for various groups were determined to check the specificity of GDF-9 and BMPRIA gene expression during in vitro development. The sequences of primers were presented in Table 1Go.

Morphometric evaluation of folliculogenesis
Cultured ovaries were embedded in JB-4 plastic, and 4-µm-thick hematoxylin and eosin-stained sections were examined under a Leica DMR microscope to identify primordial follicles. The morphometric evaluation was performed as described previously (13). Because primordial oocytes (i.e. clusters of oocytes without any definite somatic cell partners) dominated in the ovaries cultured up to 9 d (equal to 8 d of in vivo development) and primordial follicles were forming for the first time, the total number of oocytes with a nucleolus, regardless of their follicular association, was counted in a given optical field. Next, the number of primordial follicles corresponding to those oocytes was determined. The fields were chosen at random from the entire ovary, and 300 oocytes for each ovary were counted. The proportion of primordial follicles in an ovary was then expressed as percentage of oocytes. Based on the morphological definition, an oocyte surrounded by the cytoplasmic processes of at least one flattened parenchymal cell (pregranulosa cells) was considered a primordial follicle, whereas a primary follicle contained an oocyte surrounded by a layer of primarily cuboidal granulosa cells. Because the only class of follicles present in cultured ovaries was primordial and they were structurally distinct from primordial oocytes, no primordial oocyte could be counted as a primordial follicle. Furthermore, identification of primordial follicles was performed in a double-blind manner by two individuals. Therefore, errors in follicle counting were eliminated.

Detection and quantification of GDF-9 protein by fluorescence immunohistochemistry
Frozen sections at 6 µm were fixed in freshly prepared ice-cold 4% paraformaldehyde in PBS (pH 7.4), for 10 min and stained for GDF-9 protein as described previously (7). The specificity of the antibody was verified using sections of 3-d-old ovaries, in which GDF-9 protein was absent in the oocytes, and exposing sections of 8-d-old ovaries to nonimmune IgG or the GDF-9 antibody that was preneutralized with a recombinant rat GDF-9. The images were captured by a Qimaging digital camera (Burnaby, British Columbia, Canada) and Openlab image analysis software (Improvision, Lexington, MA). The exposure time of the camera was set for subtracting background fluorescence that was present in sections incubated with the nonimmune IgG of the host species. GDF-9-specific fluorescence signal (immunosignal) was merged with the nuclear signal to determine the cellular site of protein expression. The blue color of 4'-6-diamidino-2-phenylindole (DAPI) was converted to red for contrast, whereas the GDF-9 signal remained green. GDF-9 fluorescence intensity was measured using NIH Image 1.6 and Openlab image analysis software. Signal intensity (OD/pixel) was recorded randomly from at least 100 oocytes regardless of their association with follicles, and the average signal intensity for each ovary was calculated. Same sections were then examined for the percentage of oocytes with or without GDF-9 immunoreactivity by counting a total of at least 200 oocytes from different areas of each section. Sections from three ovaries were analyzed to get the mean ± SEM. The data were presented as average OD/pixel or percentage of oocyte with GDF-9 immunoreactivity.

Statistical analysis
For morphological evaluation of follicle development, three ovaries from different fetuses were cultured for each treatment group and the cultures were repeated at least twice. For all fluorescence localization, there was one ovary per group and cultures were repeated three times. For RNA preparation, three ovaries per group were cultured and then pooled to form one sample. The cultures were repeated three times to obtain three samples for statistical analysis. Representative fluorescence images were presented. Ovaries from untreated and siRNA-treated groups were cultured in parallel. GDF-9 immunofluorescence signal in the oocytes for each group reflected an average of at least 300 oocytes from three different ovaries. All quantitative data were analyzed using one-way ANOVA with Scheffé’s post hoc test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of the hamster GDF-9 and validation of siRNA
The rationale was to clone hamster GDF-9 cDNA to determine the partial nucleic acid and amino acid sequences for cross-species comparison and to design an effective siRNA. PCR amplification of hamster ovarian RNA generated a predicted 1037 bp cDNA, which was 90%, 89%, and 80% similar to the corresponding region of the rat (6), mouse (18), and human (19) GDF-9 cDNA, respectively (Fig. 1Go). A comparison of the deduced amino acid sequence revealed 97%, 89%, and 72% similarity with the rat, mouse, and human GDF-9 protein, respectively (Fig. 2Go). Interestingly, human GDF-9 had more nucleotide bases and corresponding amino acids compared with all three rodent species.


Figure 1
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  		FIG. 1. Partial nucleic acid sequence of hamster GDF-9 cDNA, and its comparison with that of rat, mouse, and human. F1 and R1 indicate the corresponding rat GDF-9 sequences selected to design the forward and reverse primers, respectively, for PCR amplification of the hamster GDF-9 mRNA. Nucleotides highlighted in bold reflect the sequence of the reverse primer used in real-time PCR. RF1 and probe indicate the sequences selected to design the forward primer and the probe, respectively.

 

Figure 2
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  		FIG. 2. Deduced amino acid sequence of hamster GDF-9 and its comparison with that of rat, mouse, and human. Human GDF-9 protein was longer than that of the rodents. The table summarizes the amino acid similarity between species.

 
To determine whether siRNA could efficiently transfect ovarian cells in culture, fluorescence-labeled nontargeting siRNA was used. Confocal microscopy revealed that more than 90% of ovarian cells were siGLO positive, and the siRNA remained in cells throughout the culture period despite the removal of the siRNA after 48 h (Fig. 3Go), indicating that siRNA-induced gene knockdown might continue for extended period with a relatively shorter exposure to the cRNA.


Figure 3
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  		FIG. 3. Detection of the efficiency of siRNA delivery into ovarian cells in vitro using siGLO, a nontarget Cy3-labeled control siRNA. Fifteen-day-old fetal ovaries were cultured for 9 d without (A) or with (B) 100 nM of siGLO that was delivered with 2 µl/ml Metafectane, a transfection agent. The fluorescence signal as an index of transfection was detected by confocal microscopy after 9 d of culture. Bar, 10 µm.

 
Because siRNA delivery into the cells required transportation agent, we selected Metafectane because of its less cytotoxic property compared with other commonly used transfection agents (personal conversation with the technical staff at Dharmacon), and superior transfection efficiency in prostate cancer cells (20) and ovarian cells (data not shown). TUNEL staining indicated that only few oocytes had DNA fragmentation; however, neither the 2 µl/ml dose of Metafectane nor the siRNA enhanced the number of TUNEL-positive oocytes compared with those present in untreated 9-d cultured ovaries or 8-d-old ovaries developed in vitro (Fig. 4Go). For the sake of brevity, quantitative data were not furnished.


Figure 4
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  		FIG. 4. Detection of the apoptotic effect of GDF-9 siRNA and Metafectane on cells of hamster ovaries in culture. DNA fragmentation (green fluorescence) as an index of apoptosis was determined by TUNEL, and the nuclei were counterstained with DAPI. The blue color of DAPI was converted to red color for contrast. Sections of an 8-d-old ovary grown in vivo (A), fetal ovaries cultured for 9 d (B–E), without siRNA (B), with 100 nM GDF-9 siRNA (C), with 1 ng/ml FSH (D) or with FSH + GDF-9 siRNA (E). Bar, 10 µm.

 
GDF-9 siRNA knockdown of GDF-9 mRNA and protein, and folliculogenesis
The rationale for the study was to examine whether endogenous GDF-9 was important for FSH induction of primordial follicle development during neonatal ovary development in the hamster. The specificity of the GDF-9 siRNA effect was verified by examining the levels of BMPRIB and ß-actin mRNA in ovaries exposed to GDF-9 siRNA. The rationale was that FSH up-regulated BMPRIB expression, whereas ß-actin mRNA expression remained unaltered regardless of the treatment. Although nontargeting control siRNA did not affect the basal transcript levels of any of the selected genes, GDF-9 siRNA specifically and appreciably knocked down the levels of GDF-9 mRNA in ovaries cultured without FSH (Fig. 5Go). Marked increases in the levels of GDF-9 and BMPRIB mRNA occurred in ovaries exposed to FSH; however, GDF-9 siRNA completely knocked down FSH-induced increase in GDF-9 mRNA without affecting the levels of BMPRIB mRNA (Fig. 5Go). The levels of ß-actin mRNA were not affected by any treatment (Fig. 5Go), thus suggesting the specificity of the GDF-9 siRNA effect.


Figure 5
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  		FIG. 5. Effect of GDF-9 siRNA without or with FSH on the levels of GDF-9, BMPRIB, and ß-actin mRNA in 15-d-old fetal ovaries cultured for 9 d. Marked reduction in the basal and FSH-induced increase in GDF-9 mRNA levels was evident in the presence of GDF-9 siRNA. Each bar represents nine ovaries. Bars with a same letter were not significantly (P < 0.05) different from each other. NT-Con, Nontargeting siRNA-control.

 
Varying degree of GDF-9 immunosignal was visible in prefollicular primordial oocytes in 6-d-old ovaries when no primordial follicle were visible (13), and primordial oocytes remained in clusters surrounded by undifferentiated somatic cells (Fig. 6AGo; area marked by dotted line). GDF-9 protein expression continued in the oocytes of 8-d-old ovaries developed in vivo (Fig. 6BGo). Although some clusters of primordial oocytes were still present, primordial follicles with an oocyte surrounded by flattened pregranulosa cells were readily visible (Fig. 6BGo). GDF-9 immunosignal in the oocytes remained unaffected when fetal ovaries were cultured for 9 d with control siRNA and 100% of the oocyte showed some degree of GDF-9 immunosignal (Figs. 6CGo and 7AGo), and the results were comparable to the ovaries cultured without RNAi (data not shown). However, the percentage of oocytes with GDF-9 protein declined significantly (P < 0.05) when the ovaries were cultured with GDF-9 siRNA (Figs. 6DGo and 7BGo), and the intensity of GDF-9 immunosignal in the remaining oocytes declined significantly (Figs. 6DGo and 7AGo). GDF-9 immunoreactivity increased markedly in ovaries cultured with FSH compared with the untreated or control siRNA-treated ovaries (Figs. 6EGo and 7AGo) with 100% oocytes showing GDF-9 immunosignal (Fig. 7BGo). Consistent with the results obtained with GDF-9 siRNA alone, FSH-induced increase in GDF-9 immunosignal was completely knocked down by GDF-9 siRNA (Figs. 6FGo and 7AGo) and a significantly lower percentage of GDF-9-positive oocytes was visible (Fig. 7BGo).


Figure 6
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  		FIG. 6. Effect of GDF-9 siRNA without or with FSH on the levels of GDF-9 protein in 15-d-old fetal ovaries cultured for 9 d. GDF-9 protein (green fluorescence) was present only in the oocyte. Nuclei were counterstained with DAPI, which was converted to red color for contrast. Sections of a 6-d-old in vivo grown ovary when no primordial follicle had developed (A), an 8-d-old ovary grown in vivo when primordial follicles first appear (B), fetal ovaries cultured for 9 d (C) without siRNA, with 100 nM GDF-9 siRNA (D), with 1 ng/ml FSH (E) or with FSH + GDF-9 siRNA (F). Note intense GDF-9 expression in prefollicular primordial oocytes (A). O, Oocyte, GC, granulosa cells, S, somatic cells, IC, interstitial cells, S0, primordial follicle, S1, primary follicle, OC, oocyte cluster, marked by dotted line. Bar, 10 µm.

 

Figure 7
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  		FIG. 7. Quantitative data of the GDF-9 immunosignal (A) and the percentage of oocytes expressing GDF-9 immunoreactivity (B) presented in panels C–F. The intensity of GDF-9 fluorescence was quantified using the NIH Image and Openlab image analysis software after appropriate background correction. The data were presented as OD/pixel. Each bar represented a mean of at least 100 oocytes from three ovaries. Same sections were examined for the percentage of oocytes with or without GDF-9 immunoreactivity. Each bar represented a mean of three ovaries. Bars with a same letter were not significantly (P < 0.05) different from each other. NT-Con, Nontargeting siRNA control.

 
Fetal ovaries cultured for 9 d without hormonal stimulus contained approximately 12% primordial follicles, and the percentage was not altered when the ovaries were exposed to nontargeting-siRNA (Fig. 8Go). Consistent with reduced GDF-9 mRNA expression and protein level, GDF-9 siRNA alone significantly retarded the formation of primordial follicles under basal condition (Fig. 8Go). Whereas FSH markedly stimulated the formation of the primordial follicles, the effect was completely abolished by GDF-9 siRNA (Fig. 8Go). Furthermore, the typical morphological association of the somatic cells with the oocytes was abrogated in ovaries treated with FSH and GDF-9 siRNA compared with ovaries developing under control culture, with FSH or in vivo; hence, those cells seemed not to form the pregranulosa cells and consequently, most of the primordial oocytes in the "egg-nests" or "oocyte cluster" failed to engage in folliculogenesis (data not shown). No GDF-9 immunosignal was observed in sections of 3-d-old ovary (7) or sections of 8-d-old ovary incubated with nonimmune IgG or the primary antibody preneutralized with a recombinant rat GDF-9, indicating the specificity of the antibody detection of GDF-9 protein (data not shown).


Figure 8
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  		FIG. 8. Effect of GDF-9 siRNA without or with FSH on the formation of primordial follicles in 15-d-old fetal ovaries cultured for 9 d. The percentage of primordial follicles was calculated with respect to the number of oocytes. Each bar represents at least three ovaries. Bars with a same letter were not significantly (P < 0.05) different from each other. NT-con, Nontargeting siRNA control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the study indicate that FSH up-regulates GDF-9 expression and endogenously produced GDF-9 appears to play an important role in the differentiation of somatic cells into pregranulosa cells, leading to the formation of primordial follicles. The results also suggest that a relatively shorter exposure of 48 h to siRNA is adequate to impose a long-term knockdown of gene expression in a multicellular structure, such as fetal ovaries. It is, however, equally possible that the knockdown of GDF-9 expression during the first 48 h is critical for the observed inhibitory effect on primordial folliculogenesis. In our culture method, fresh medium is replaced every 48 h. Hence, it is possible that an exposure to siRNA for few hours may be adequate for the observed effects. Because morphologically distinct primordial follicles in postnatal hamster ovaries cannot be identified until 8 d of postnatal life or 9 d of culture of fetal ovaries, it is necessary to culture ovaries for 9 d to allow the development of primordial follicles so that comparison between groups can be done properly. Previously, we have shown that modest GDF-9 expression in the oocytes occurs in vitro 48 h after the initiation of culture of 15-d-old fetal ovaries, and in vivo on d 4 of postnatal life (7). Compared with d 4 of postnatal life (7), appreciable but varying degree of GDF-9 protein expression is evident in prefollicular primordial oocytes at 6 d of age when no primordial follicle is visible, thus emphasizing the potential import role of GDF-9 in the differentiation of somatic cells to pregranulosa cells. The varying degree of GDF-9 expression may also explain why the siRNA knockdown is prominent for majority of the oocytes, whereas the remainders still have lower than basal levels of GDF-9. The failure of the oocytes to form primordial follicles despite a low GDF-9 expression suggests that when GDF-9 protein level falls below the threshold, its biological action is compromised. Future studies on mechanisms of action may shed light to this conjecture. It has been suggested that a complete knockout of gene expression by RNAi may not be expected because of the characteristic mechanisms of siRNA action (21, 22), and our data seem to corroborate the findings. Although siRNA knockdown of gene expression has been reported for cell cultures (23, 24, 25), the results of the present study provide the first evidence for a successful use of RNAi strategy to interfere cell functions during follicular development in an ovary organ culture system. The presence of siGLO signal in most of the ovarian cells suggests the efficiency of the siRNA transfection, whereas the lack of apoptosis in the presence of nontargeting control or GDF-9 siRNA indicates that the inhibition of folliculogenesis in the presence of the siRNA is not resulting from random cytotoxicity. Because GDF-9 is expressed exclusively in the oocytes of the hamster ovary, the effect was specific despite a possible entry of GDF-9 siRNA in somatic cells. The failure of GDF-9 siRNA to affect the levels of BMPRIB or ß-actin mRNA confirms this contention. Oocyte-specific expression of GDF-9 protein has been reported for the mouse (4), human (5), and rat (6). However, we cannot rule out the possibility that the resulting lack of GDF-9 action on the somatic cells may alter the ability of these cells to differentiate into pregranulosa cells. In fact, such findings will be consistent with the effect of GDF-9 on ovarian cells (10, 26). Future studies will address this contention.

Deletion of GDF-9 gene in mice results in the arrest in folliculogenesis beyond the primary stage (1), and exogenously added GDF-9 promotes FSH-induced growth of rat preantral follicles in vitro (11). Furthermore, GDF-9 has been shown to promote the growth and survival of human follicles in ovarian slices in vitro (12). Previously, we have demonstrated that administration of a recombinant rat GDF-9 (a kind gift from Dr. A. J. W. Hsueh, Stanford University Medical Center, Stanford, CA) in fetal hamster ovary organ culture results in the formation and subsequent growth of primordial follicles (7). We have also shown that FSH stimulates cAMP production by fetal hamster ovaries in vitro and FSH action during fetal ovary development is important for the formation of primordial follicles during postnatal life (13). Furthermore, exogenously added FSH stimulates the formation of primordial follicles and induces GDF-9 expression in the oocytes in vitro (7). Full-length FSH mRNA is expressed in fetal hamster ovaries (Roy, S. K., unpublished observation). The results of the present study not only corroborate FSH-induction of GDF-9 expression in the hamster oocytes but also provide novel information that FSH action in follicular morphogenesis, at least partly, is mediated by GDF-9 produced in the oocytes. Using an organ culture of newborn rat ovaries, Nilsson and Skinner (9) have suggested that recombinant mouse GDF-9 does not promote the development of primordial follicles. However, Vitt et al. (10) have shown that treatment of immature rats with a recombinant rat GDF-9 results in a decrease in the number of primordial follicles with a corresponding increase in the number of primary follicles, suggesting a primordial to primary follicle transition. Because a recombinant mouse GDF-9 is not available to us, its efficacy in hamster folliculogenesis cannot be tested. Nevertheless, it is noteworthy that unlike the present findings, no effect of GDF-9 on the formation of primordial follicles in the rat or mouse has been demonstrated. What is the possible explanation of such discrepancy in findings? Immunohistochemical studies have shown unequivocally that GDF-9 protein expression in the mouse or rat oocytes first occurs when follicles reach the primary stage (1, 6). Therefore, consistent with the genetic findings (1), it is unlikely that GDF-9 will be needed in vivo for folliculogenesis earlier than the primary to secondary stage transition. In contrast, despite the expression of GDF-9 mRNA from the fetal life (Roy, S. K., unpublished observation), in vivo GDF-9 protein expression in the hamster oocytes occurs on fourth postnatal day when the oocytes are still in the prefollicular primordial stage and 4 d away to become primordial follicles (7). Secondly, newborn ovaries of rats and mice contain the full complement of primordial follicles and some primary follicles; therefore, follicular development in vitro can be expected to occur only beyond the primordial class. Some primordial follicles may form during in vitro culture, but a significant increase in the percentage of primordial follicles may not be observed because GDF-9 also promotes the transition of primordial follicles to primary and primary to secondary stage in vitro (7). In light of these lines of evidence, the present findings are not surprising. These findings further suggest a very important species difference in the requirement of factors influencing primordial follicle formation.

The formation of primordial follicles, albeit significantly lower than the control group, despite the knockdown of GDF-9 expression suggests that endogenous stimulus, perhaps FSH, has initiated the process during the fetal life. Because the formation of the oocytes from oogonia occurs in succession, the first batch of oocytes is expected to interact with the surrounding somatic cells to form the first cohort of primordial follicles (27) and so on. Furthermore, the levels of GDF-9 receptor, such as TGFß-receptor type I and BMPRII (28, 29), which are expressed in the hamster ovarian somatic cells from the fetal life are not affected by the GDF-9 siRNA (data not shown). Therefore, it is likely that there is not enough time for the GDF-9 siRNA to be effective for the first batch of oocytes, and consequently, a low percentage of primordial follicles develops. A similar trend in primordial follicle development has been observed when endogenous FSH levels are neutralized at different days of fetal development (13). Formation of some primordial follicles has been reported for the women carrying an inactivating mutation of the FSH receptor (30). It is noteworthy that ovaries of these women can produce a small amount of cAMP in vitro in response to FSH (31). These lines of evidence allow us to speculate that even a low intensity of appropriate stimulus can trigger the differentiation of somatic cells to pregranulosa cells leading to the formation of some primordial follicles, but the full complement of primordial follicle pool does not develop unless the stimulus is sustained. Conversely, it can be argued that even though more than 90% of ovarian cells are transfected with the siRNA, the effective amount of siRNA present in the oocytes may vary due to inherently limited efficiency of lipophylic transporter-mediated transfection of nucleic acids. Such inefficiency may lead to suboptimal knockdown of GDF-9 in a small number of oocytes, and consequently, a low percentage of primordial follicles develops. A modest level of GDF-9 protein expression in vitro occurs after 2 d of culture of fetal hamster ovaries (7). The in vivo and in vitro difference in GDF-9 protein expression suggests that some putative factors may induce a translational block in vivo, the effect of which is partially removed during in vitro culture. This is consistent with the finding that GDF-9 mRNA is present in the hamster ovary from the fetal life (Roy, S. K., unpublished observation).

GDF-9 has been shown to affect many functions in the ovary, including the induction of LH receptor, thecal androgen synthesis, inhibin production by the rat granulosa cells, and cumulus expansion in the mouse (3, 32, 33). Gui and Joyce (34) have shown that oocytes regulate cumulus expansion in the mouse via GDF-9. In this context, it is important to note that when FSH-induced folliculogenesis is compromised by GDF-9 siRNA, ovarian somatic cells surrounding the egg nests or primordial oocyte clusters undergo morphological changes that are quite distinct from that occur in untreated ovaries. It has been demonstrated that granulosa cells lose their follicle forming potential when the oocytes is surgically removed (35, 36). Conversely, oocytes from secondary follicles when transferred to primordial follicles, accelerate the growth of the pregranulosa cells in primordial follicles (37). Based on these lines of evidence, it can be conjectured that somatic cells in neonatal hamster ovaries lose their cue to form follicles when the oocyte-derived GDF-9 signal is withdrawn or severely reduced; however, stimuli for the chronological growth of somatic cells remain unperturbed. Therefore, somatic cells assume a dense and slender appearance resembling fibroblast-like cells. Whether such unique development eventually causes severe ovarian deformation is the subject of future studies.

In summary, the results of the present study provide the first and direct evidence that GDF-9 production in the oocytes is critical for the formation of primordial follicles, at least in the hamster. Furthermore, FSH action during neonatal ovary development is important for the expression of GDF-9 in the oocytes.


   Footnotes
 
This work was supported by Grant HD38468 from the National Institute of Child Health and Human Development, National Institutes of Health (to S.K.R.)

First Published Online December 29, 2005

Abbreviations: BMPR, Bone morphogenetic protein receptor; DAPI, 4'-6-diamidino-2-phenylindole; GDF-9, growth differentiation factor 9; RNAi, RNA interference; siRNA, small interfering RNA; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling.

Received September 20, 2005.

Accepted for publication December 19, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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