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RESEARCH |
Follicle Biology Laboratory, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, 1090 Brussels, Belgium
Correspondence should be addressed to J C Sadeu; Email: vsadeu{at}yahoo.co.uk
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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Over the past years, the growth of rodent (Eppig & O'Brien 1996, Parrott & Skinner 1999, Klinger & de Felici 2002, Obata et al. 2002, Kezele & Skinner 2003, O'Brien et al. 2003, Lee et al. 2004, Nilsson & Skinner 2004, Shen et al. 2006a, 2006b, 2007), cattle (Wandji et al. 1996, Braw-Tal & Yossefi 1997, Fortune et al. 1998), and primate (Wandji et al. 1997), including human (Hovatta et al. 1999, Wright et al. 1999, Hreinsson et al. 2002, Sadeu et al. 2006) primordial follicles has been investigated in vitro. In these ovarian culture experiments, primordial follicles within newborn or neonatal mouse ovaries, ovarian cortex of fetal or adult bovine ovaries as well as fetal baboon ovaries, or fetal or adult human ovaries were able to initiate growth and to develop to primary or secondary follicles. But generally, only a few growing primary follicles progressed to the secondary stage in cattle, primates, and humans. Meiotically competent oocytes have been mainly obtained after isolation and in vitro growth of mouse primary or secondary follicles as single functional units (Spears et al. 1994, Cortvrindt et al. 1996, Eppig & O'Brien 1996, O'Brien et al. 2003, Lenie et al. 2004, Kreeger et al. 2005, Shen et al. 2006b, 2007). Regarding the production of mature oocytes in vitro, there is scarce proof of concept in the non-rodent species (Hirao et al. 1994).
It is known that the primary to secondary transition is induced by an autocrine/paracrine regulatory process that involves growth factors produced by the oocyte and GC. Growth differentiation factor-9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific proteins secreted by growing oocytes in rodents, sheep, and humans (McGrath et al. 1995, Dube et al. 1998, Fitzpatrick et al. 1998, Aaltonen et al. 1999, Bodensteiner et al. 1999, Otsuka et al. 2000). They are known to control folliculogenesis by acting on GC in developing follicles. Studies in genetic mutations have elucidated the role of these proteins in regulating the primary to secondary follicle transition. Mutations in GDF9 (Dong et al. 1996) and BMP15 (Galloway et al. 2000, Hanrahan et al. 2004) result in growth arrest at the primary stage. Furthermore, consistent with its role in controlling follicular growth, GDF9 has been shown to enhance the development and growth of human (Hreinsson et al. 2002), and rodent (Hayashi et al. 1999, Nilsson & Skinner 2002) early-stage follicles when added to culture media.
BMP15 and GDF9 play a synergistic role in folliculogenesis. Mice lacking both copies of the Bmp15 gene and one copy of the Gdf9 gene show increased oocyte loss and decreased late-stage follicles, whereas those lacking both copies of Gdf9 and Bmp15 genes show follicle growth arrests at the primary stage similar to the Gdf9 knockout mice (Yan et al. 2001).
Anti-Müllerian hormone (AMH) is expressed postnatally by the GC of developing follicles from the early primary stage (oocyte surrounded by a mixture of flattened and cuboidal GC) to the early antral stage (Durlinger et al. 2002a, 2002b). Increased levels of AMH secreted by growing follicles have been associated with the decreased activation of the pool of primordial follicles (Behringer et al. 1990). It is proposed that AMH inhibits the primordial to primary transition. In addition, a homozygous mouse knockout model for AMH shows an early depletion of the primordial follicle pool that is consistent with the role of AMH in regulating the activation of primordial follicles (Durlinger et al. 1999).
GDF9, BMP15, and AMH are major regulators for the growth and function of ovarian follicles, and it was questioned whether in the in vitro culture situation the actions of these factors might be altered in comparison with their observed roles in vivo, where they decrease the number of secondary follicles. Therefore, to investigate whether the decrease in the number of growing follicles was related to any abnormal level of expression of mRNAs for GDF9, BMP15, and AMH, primary follicles were isolated from cultured intact neonatal mouse ovaries and used to address the question. The aims of the study were: (1) to compare the ovarian follicular population between cultured neonatal mouse ovaries and freshly isolated age-matched ovaries; (2) to quantify mRNA of key growth factors in activated follicles in relation to the stage they have reached in vitro; and (3) to immunolocalize GDF9 and AMH in cultured ovaries.
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Results |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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Localization of GDF9 protein in cultured mouse ovaries
The ovaries cultured in the absence of FSH for 10 days were further used for immunohistochemical analysis. A weak to strong immunostaining for GDF9 was detected in the oocytes. The immunostaining was weak in primary follicles (Fig. 4A and C) and absent in primordial (pr) follicles (Fig. 4E) and some primary follicles (Fig. 4C; Table 2). No staining was found in the negative control sections (Fig. 4G).
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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Because communications between oocytes and follicular cells all contribute to follicular development, any alterations in growth-supporting factors may disrupt these processes. In this study, the primordial to primary transition in vitro proceeded similar to the in vivo situation. However, the deficiency in secondary follicle development in vitro might suggest an altered hormone processing, a lack of some stage-specific growth factors in the in vitro environment, or an alteration of response of GC to oocyte-secreted factors. Given that the null mutation of the Gdf9 and Bmp15 genes (Dong et al. 1996, Galloway et al. 2000, Hanrahan et al. 2004) results in growth arrest at the primary stage in vivo, it can be postulated that low expression of Gdf9 and/or Bmp15 transcripts in vitro may contribute to the decrease in the number of small and medium secondary stage follicles observed in ovarian culture experiments. However, there is no evidence for or against this idea in the literature.
Freshly isolated ovaries at PND 3 contained mostly primordial follicles. A low number of these follicles activated in vitro during whole-ovary organ culture, similar to previously described observations (Eppig & O'Brien 1996). No significant change in the rate of development of primary and early secondary follicles was found between in vivo control and cultured ovaries although there was a trend towards increased primary follicle development in vitro. Follicle development in vivo was different from in vitro conditions in that the number of small secondary follicles was significantly increased in vivo. In addition, the development of medium secondary follicles did not occur in vitro. These findings show that most of the primordial follicles that initiated growth in vitro underwent developmental arrest at the primary stage. The number of atretic follicles was comparable between the in vivo and in vitro conditions, suggesting that atresia was not affected by the conditions of culture. Similar to the results with rodents, human, bovine, and baboon ovarian cortical tissue cultures showed that only a few growing primary follicles progress to the secondary stage (Wandji et al. 1996, 1997, Braw-Tal & Yossefi 1997, Fortune et al. 1998, Hovatta et al. 1999, Wright et al. 1999, Hreinsson et al. 2002, Sadeu et al. 2006).
Gene expression in ovarian organ culture experiments is regularly performed with mRNA extracted from whole ovaries. The limitation in such a methodological approach is that different follicle stages exist in the cultured ovaries and therefore it is impossible to indicate which follicle stage is showing a change in expression of any particular gene. In that respect, in contrast to other studies, the present analysis on mRNA expression in cultured ovaries was performed with RNA from a well-defined follicle class. After 10 days of culture of PND 3 mouse ovaries, the Gdf9 and Bmp15 mRNA levels in primary follicles were similar to the control levels in in vivo follicles. This observation indicates a normal expression of growth-supporting factors in vitro. On the basis of the data from in vivo studies, one would expect that normal Gdf9 and Bmp15 mRNA expression in vitro would correlate with normal primary follicle progression into the secondary stage. This was not the case in our study. Nevertheless, the decreased progression in vitro might still depend on GDF9 and BMP15 since these growth factors act as homo- or heterodimers. Their prohormones must first undergo proteolytic cleavage before they can form the bioactive dimers or it is possible that expressions of GDF9 and BMP15 receptors did not occur in present ovarian organ culture experiment.
Early-stage follicles contain growing oocytes and this may explain the increased Gdf9 and Bmp15 mRNA levels from day 7 to day 10 of culture. Interestingly, both oocyte-secreted factors showed similar mRNA expression patterns. The observation that Gdf9 and Bmp15 mRNA levels are slightly higher in FSH conditions might suggest that FSH could positively regulate Gdf9 and Bmp15. Using total RNA from immature mouse ovaries, Guéripel et al. (2006) have observed that Bmp15 but not Gdf9 mRNA levels increased in the gonadotropin-treated immature mice. This was an in vivo study and RNA was extracted from whole ovaries.
A negative regulator of early follicular development is AMH, which has been shown to inhibit the transition from primordial to primary follicles. An increased number of developing follicles was found in mice with null mutation of the Amh gene (Durlinger et al. 1999). The Amh mRNA level was significantly lower after 10 days of culture without FSH. However, this decrease was not reflected in the number of growing follicles, which may suggest that AMH expression in vitro could be high enough to keep control over follicle recruitment. By contrast, the Amh mRNA level of follicles cultured in the FSH conditions was similar with the control level in vivo, suggesting a possible direct or indirect relationship between FSH and AMH. It was shown that FSH-stimulated preantral follicle growth in vitro was attenuated in the presence of AMH and, in addition, that more follicles start to grow in vivo under the influence of exogenous FSH in AMH-deficient mice compared with the wild types (Durlinger et al. 2001). Given that Amh mRNA levels were not significantly different between the follicles cultured in the FSH and those cultured in the no FSH conditions, direct evidence to support the role of FSH in regulating AMH expression cannot be provided.
Consistent with Gdf9 and Amh mRNA expressions detected by real-time PCR, we showed by immunohistochemical analysis that GDF9 protein was first detected in oocytes of primary follicles, and AMH, in cuboidal GC of primordial (oocyte surrounded by a mixture of flattened and cuboidal GC) follicles, suggesting that these proteins were present at these stages of follicle development as well as in the more advanced stages where positive immunoreactions were further detected. This provided evidence for the maintenance of the stage-specific specialized GC and oocyte functions during follicle growth in vitro. These findings are in agreement with previously described observations from in vivo studies where Gdf9 mRNA expression and protein were demonstrated in oocytes of primary and advanced follicle stages in mice, rats, and humans (McGrath et al. 1995, Aaltonen et al. 1999, Elvin et al. 1999, Hayashi et al. 1999, Jaatinen et al. 1999), and AMH in cuboidal GC of primordial and advanced follicle stages in mice and humans (Durlinger et al. 2002a, 2002b, Weenen et al. 2004).
In summary, primary follicles isolated from cultured PND 3 mouse ovaries were used to determine whether any alteration in the intrinsic expressions of GDF9, BMP15, and AMH could explain the block in follicle transition from the primary to secondary stage in ovary organ culture. It was found that no significant change in expression levels of Gdf9 and Bmp15 occurred in primary follicles grown in vitro compared with control age-matched follicles grown in vivo, whereas significantly lower Amh expression was found after in vitro growth in the absence of FSH. In addition, GDF9 and AMH protein expressions were detected at similar stages of follicle development as described in vivo. The present observations indicated that the disruption of the primary to secondary transition in vitro might not result from defective Gdf9, Bmp15, and/or AMH transcription. Additional investigations of other genes playing a significant role in the oocyte, GC, and theca cell interactions would provide more insight into the abnormal process of follicle development beyond the primary stage in vitro.
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Materials and Methods |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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Postnatal 3-day-old (PND 3) mice were killed by decapitation. Ovaries were aseptically removed and collected in 1.5 ml Leibovitz-L15 medium supplemented with 10% fetal bovine serum (FBS), 100 µg/ml streptomycin, and 100 IU/ml penicillin. Under a light microscope, the bursa and surrounding tissues were carefully dissected away from the ovaries. The procedure was carried out at 37 °C. Unless indicated, otherwise all chemicals used were purchased from Invitrogen.
Ovary organ culture
Ovaries were cultured as described in Parrot & Skinner (1999) with some modifications. Each well of 4-well culture plates (Nunc, VWR, Leuven, Belgium) was filled with 500 µl Dulbecco's modified Eagle's medium–Ham's F-12 (1:1, v/v) supplemented with 10% FBS, 10 µg/ml insulin, 10 µg/ml transferrin, and without or with 50 mIU/ml FSH. A floating filter (0.4 µm isopore membrane filter, Millipore, Brussels, Belgium) was laid over the medium and the plates were pre-equilibrated for 
4 h. Ovaries (2–4/well) collected at PND 3 were placed onto the floating filters, covered with drops of medium, and cultured for 7 (D7) or 10 (D10) days at 37 °C in a humidified incubator with 5% CO2 in air. Three-day-old ovaries cultured for 10 days should be comparable with ovaries of similar age (i.e., ovaries of 13-day-old mice). Consequently, a normal number of small secondary follicles (similar to the development after 13 days in vivo) should be obtained. Half the volume of culture medium was replaced with fresh pre-equilibrated medium every 3 days. At the end of cultures, the ovaries were fixed in 4% formaldehyde (Sigma) for morphological or immunohistochemical analysis. Alternatively, they were used for an isolation of primary follicles. Each experiment was performed at least three times. One group of ovaries (n=5) was collected from age-matched 13-day-old (PND 13) mice for in vivo comparisons of follicular populations and another group (n=6) for isolation of primary follicles for in vivo comparisons of expressions of Gdf9, Bmp15, and Amh mRNAs.
Tissue processing
The ovaries were fixed for 4 h at room temperature. Subsequently, they were transferred into 70% ethanol at 4 °C until transported to the histology laboratory for processing and embedding into paraffin blocks. The ovaries were completely serial sectioned at 5 µm thickness on a sliding microtome (Bromma, Stockholm, Sweden). Every other set of five consecutive sections was mounted onto a microscope slide.
Quantification of primordial and primary follicles
Thirteen cultured ovaries and five ovaries of PND 13 immature animals were used for the analysis. The sections were deparaffinized, hydrated, and stained with hematoxylin, eosin, and safran. Every tenth section was examined under a light microscope for the presence of follicles. Primordial and primary follicles were counted in the entire sections. To avoid counting the same follicle more than once, only follicles with visible oocyte nucleolus were counted.
Early-stage follicles were classified as: (a) primordial follicle, oocyte surrounded by flattened or a mixture of flattened and cuboidal GC; (b) primary follicle, oocyte surrounded by one layer of cuboidal GC; (c) early secondary follicle, oocyte surrounded by one and a half layer of GC; (d) small secondary follicle, oocyte surrounded by two layers of GC; and (e) medium secondary follicle, oocyte surrounded by two and a half to three layers of GC. Follicles were considered atretic based on the following criteria: pyknotic GC or theca cells, eosinophilia of the ooplasm, a contracted chromatin material, or loss of basement membrane integrity. The size of the ovaries did not change significantly between treatments, and an estimation of the total follicle numbers per ovary was obtained by multiplying the number of primordial or primary follicles present in every tenth section analyzed by 10 to account for the ovarian sections not included in the analysis (Flaws et al. 2001).
Quantification of early, small, and medium secondary follicles
The number of healthy early, small, and medium secondary follicles per ovary was determined by exact counts from an examination of each of the entire consecutive 5 µm sections throughout the whole ovary. Attention was paid to avoid double counting in adjacent sections.
Isolation of primary follicles
Primary follicles were isolated from ovaries after 7 or 10 days of culture and from in vivo D13 ovaries (of immature mice) by mechanical isolation with fine 25
gauge needles (Becton Dickinson, Erembodegem, Belgium). The diameter of the follicles was measured with a caliper in the eyepiece of an inverted microscope. A total of 185 primary follicles were isolated and immediately frozen (2–12 follicles/cryovial) in liquid nitrogen and stored until analyzed for gene expression as described below.
Gdf9, Bmp15, and Amh mRNA expressions in primary follicles
RNA extraction and RT
Total RNA was extracted from pooled follicles using the RNeasy Micro kit (Qiagen) according to the manufacturer's instructions. An exogenous control 10 pg of luciferase mRNA (Promega) was added and samples were eluted in 14 µl RNase-free water and stored at –80 °C. Subsequently, RT of 10 µl total RNA was carried out using the iScript cDNA Synthesis Kit (Bio-Rad laboratories) according to the manufacturer's instructions using the blend of oligo(dT) and random hexamers in a total volume of 20 µl. Negative controls were generated by omitting the RNA or the RT enzyme. Reverse transcribed cDNA was diluted 2:5 with DEPC-treated water and stored at –80 °C until real-time PCR was performed.
Real-time RT-PCR
Quantitative PCR was performed on the LightCycler 480 (Roche Diagnostics). PCR primer sequences are indicated in Table 3. Amplification reactions were carried out in a total volume of 15 µl containing 2 µl cDNAs, 7.5 µl SYBR Green PCR Master Mix 2x (Roche Diagnostics), and 0.6 µM forward and reverse gene-specific primers. Cycle amplification protocol was as follows: 95 °C for 10 min followed by 55 cycles of 95 °C for 10 s. and 60 °C for 30 s. For the quantification, standard curves were generated by amplifying serial dilutions of each amplicon. The specificity of the PCR products was checked by the melting curve analysis performed after the amplification cycles, and was further confirmed by sequencing the amplicons. The amount of gene of interest was normalized with the amount of luciferase in the same sample and with the number of follicles in the sample. Luciferase has been used as an appropriate exogenous control gene for quantitative PCR studies (Pennetier et al. 2006). Each sample was tested in triplicate and the normalized expression level of each gene was then related to the average amount as found in the in vivo control samples, PND 13.
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Statistical analysis
All data are expressed as mean±S.E.M. Statistical analysis of follicle number was performed by Student's t-test and the differences in Gdf9, Bmp15, and Amh mRNA levels between control and cultured groups were determined by one-way ANOVA followed by Tukey's post hoc test. The GraphPad Prism 4.01 statistical analysis software (GraphPad Software Inc., San Diego, CA, USA) was used for all analysis. P<0.05 was considered significant.
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Declaration of interest |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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Funding |
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Acknowledgements |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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Received February 11, 2008
First decision March 14, 2008
Revised manuscript received May 2, 2008
Accepted May 8, 2008
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References |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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