Induction of Ovarian Primordial Follicle Assembly by Connective Tissue Growth Factor CTGF

Primordial follicle assembly is a process that occurs when oocyte nests break down to form individual primordial follicles. The size of this initial pool of primordial follicles in part determines the reproductive lifespan of the female. Connective tissue growth factor (CTGF) was identified as a potential regulatory candidate for this process in a previous microarray analysis of follicle development. The current study examines the effects of CTGF and associated transforming growth factor beta 1 (TGFβ-1) on follicle assembly. Ovaries were removed from newborn rat pups and placed in an organ culture system. The ovaries treated with CTGF for two days were found to have an increased proportion of assembled follicles. CTGF was found to regulate the ovarian transcriptome during primordial follicle assembly and an integrative network of genes was identified. TGFβ-1 had no effect on primordial follicle assembly and in combination with CTGF decreased oocyte number in the ovary after two days of culture. Over ten days of treatment only the combined treatment of CTGF and TGFβ-1 was found to cause an increase in the proportion of assembled follicles. Interestingly, treatment with TGFβ-1 alone resulted in fewer total oocytes in the ovary and decreased the primordial follicle pool size after ten days of culture. Observations indicate that CTGF alone or in combination with TGFβ-1 stimulates primordial follicle assembly and TGFβ-1 can decrease the primordial follicle pool size. These observations suggest the possibility of manipulating primordial follicle pool size and influencing female reproductive lifespan.


Introduction
Oocytes in newborn rodents are present in 'nests' that are composed of many adjacent oocytes with no intervening cells. Follicle assembly occurs in the first few days after birth in rodents. In humans, follicle assembly begins during mid-gestation near week 18 and continues into the third trimester [1,2]. During the developmental process of follicle assembly primordial follicles are formed from oocyte nests [3,4,5,6,7]. The reproductive lifespan of a female is determined in part by the size of the primordial follicle pool generated [8]. Initially in the embryonic ovary oogonia undergo mitotic proliferation and then enter the first meiotic division to become oocytes. These unassembled oocytes are present directly adjacent to one another in nest structures which are surrounded by somatic cells (i.e. precursor granulosa cells) [3,4,5,6,7,9]. The process of follicle assembly results in the breakdown of oocyte nests, due partially to apoptotic cell death of many of the oocytes. Somatic cells move into the nests and intersperse between the remaining oocytes [10,11,12]. An oocyte must be surrounded by an adequate number of pregranulosa cells to form a primordial follicle [13,14,15]. The primordial follicles formed constitute a pool of follicles having oocytes arrested in prophase I of meiosis [3,4,5,6]. Primordial follicles gradually leave the arrested pool by undergoing the primordial to primary follicle transition. After transition follicles as they grow either undergo apoptosis/atresia or the follicles ovulate. In humans, when the pool of follicles is depleted reproduction ceases and women enter menopause [16,17,18,19,20]. Some studies have suggested the possibility that new follicles with oocytes may form in adulthood [21,22,23], but the preponderance of literature suggests that a defined and finite pool of primordial follicles exists [24]. It is possible that if the size of the follicle pool could be manipulated the reproductive capacity and fertile lifespan of the organism may change.
Although several signal transduction and transcription factors have been shown to influence the primordial follicle pool [25,26,27,28,29], few extracellular signaling factors have been shown to have an effect on ovarian follicle assembly. Treatment of neonatal mice with activin resulted in an increase in the initial pool size [30]. It has been shown that both estrogen and progesterone slow the rate of follicle assembly [31]. This study also demonstrated that progesterone acts at least in part through an inhibition of oocyte apoptosis. Previous studies have demonstrated that apoptosis of oocytes is part of follicle assembly [9,32]. The actions of progesterone were later found to be inhibited by tumour necrosis factor-alpha (TNFa) [33]. TNFa did not have an effect on the rate or percentage of assembled follicles, but promoted the apoptosis of oocytes. TNFa was found to block the inhibitory actions of progesterone and promote normal primordial follicle assembly [33]. Analysis of the inhibitory actions of progesterone on follicle assembly using a microarray analysis of the ovarian transcriptome demonstrated that progesterone promoted a dramatic up-regulation of connective tissue growth factor (CTGF), as well as an increase in the TGFb family member TGFb-3 [33].
The current study was designed to investigate the role of CTGF in primordial follicle assembly due to its altered gene expression during this process [33,34]. Connective tissue growth factor (CTGF) has been shown to be expressed by multiple cell types and to have numerous functions. CTGF is well known for its roles in tissue remodeling [35] and extra-cellular-matrix formation [36]. Tissue remodeling and extracellular matrix are important for follicular development [37]. CTGF is the most studied member of the CCN family of proteins, which is composed of cysteine-rich protein 61 (CYR61, CCN1), CTGF (CCN2), nephroblastoma overexpressed gene (NOV, CCN3), and Wnt-inducible secreted proteins 1-3 (WISP-1, WISP-2, WISP-3), which are also known as CCN4 through CCN6 [38]. CTGF mRNA has been shown to be present in ovaries in pre-antral to preovulatory follicles in both rats and pigs [39,40]. CTGF and transforming growth factor-beta (TGFb-1) have been shown to modify the functions of each other. For instance, CTGF regulates connective tissue fibroblasts [41] and vascular smooth muscle cells [42] through a modulation of TGFb-1 actions.
Transforming growth factor beta-one (TGFb-1) has been shown to be present in both rat and human ovaries during follicle assembly [43,44]. TGFb-1, TGFb-2, and TGFb-3 are known to be expressed throughout adulthood in ovarian follicles. TGFbs have been shown to stimulate oocyte meiotic maturation in pubertal 23-25 day old rats [45]. In humans, TGFb-1 has been localized primarily to oocytes during the period from 18-22 weeks of gestation [44]. In contrast, in 1-3 day old rats, TGFb-1 has been localized primarily to granulosa cells [43]. The role of TGFb-1 in neonatal ovary development has not been determined.
The present study is designed to determine if CTGF and/or TGFb-1 affect the process of follicle assembly or alter primordial pool size. Since the expression of CTGF changes during follicle assembly, as shown in recent microarray studies [33,34], the functional role of CTGF was investigated. Interestingly, CTGF was found to induce primordial follicle assembly while TGFb reduced primordial follicle pool size.

Histology and Organ Cultures
Harlan Sprague-Dawley rats from Washington State University breeder colonies were used in this study. Female rat pups (,8 hours old) were euthanized and their ovaries were dissected. All procedures were approved by the Washington State University Animal Use and Care Committee (IACUC approval # 02568-014). Whole ovaries were cultured as previously described [46] on floating filters (.4 mm Millicell-CM, Millipore, Bedford, MD, USA) in .5 ml Dulbecco's modified Eagle's medium (DMEM)-Ham's F-12 medium (1:1, vol/vol) containing 0.1% BSA (Sigma), 0.1% Albumax (Gibco BRL, Gaithersburg, MD, USA), 27.5 mg/ml transferrin, and 0.05 mg/ml L-ascorbic acid (Sigma) in a four-well culture plate (Nunc plate, Applied Scientific, South San Francisco, CA, USA) for two or ten days. The medium was supplemented with penicillin and streptomycin to prevent bacterial contamination. Ovaries were randomly assigned to treatment groups, with 1-3 ovaries per floating filter. Wells were treated every two days with recombinant connective tissue growth factor (CTGF) (Peprotech, Rocky Hill, NJ, USA) at 500 ng/ml, transforming growth factor beta 1 (TGFb-1) (R&D Systems, Minneapolis, MN, USA) at 50 ng/ml, a combination of both CTGF and TGFb-1, or progesterone (P 4 ) (Sigma) at 10 26 M. After culture, ovaries were fixed in Bouin's fixative (Sigma) for two hours. Ovaries were then embedded in paraffin, sectioned at 3 mm and stained with hematoxylin/eosin for use in morphological analysis.

Morphological Analysis
The number of oocytes at each developmental stage was counted and the number for each developmental stage was averaged across two consecutive sections that had the largest ovarian cross section. The oocyte nucleus had to be visible for an oocyte to be counted. Normally, between 100 and 300 oocytes were present in each cross-section. Oocytes were microscopically examined and were classified as being not yet assembled into follicles (i.e. the oocyte was part of an unassembled oocyte nest), as primordial (stage 0) or as one of the developing pre-antral stages (stages 1-4) as described previously [31,47]. Unassembled oocytes are recognized from being grouped together without any intervening stromal cells. As assembly occurs, the surrounding pre-granulosa cells and the developing basement membrane around the follicle provide a more distinct and visible division of one oocyte from another. Primordial follicles consist of an oocyte partially or completely surrounded by a single layer of squamous pre-granulosa cells. Developing (stages 1-4) follicles contain successively more cuboidal granulosa cells in layers around the oocyte [4,5,46,47]. Total oocyte numbers per ovarian cross-section were divided by the area (mm 2 ) of the cross-section to account for any differences in ovary size between individuals.

Apoptosis Assay
The degree of oocyte atresia (i.e. apoptosis) was measured with the in situ cell death detection kit (Roche Applied Science, Indianapolis, IN). Briefly, neonatal ovaries were cultured for 2 days, at which time follicular assembly is readily observed. Then cultured ovaries were fixed, embedded in paraffin, and sectioned. Paraffin embedded slides were then treated as per instructions in the apoptosis detection kit. Use of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) in situ caused polymerization of fluorescein-labeled nucleotides to the free 39 ends of DNA that had been cleaved during apoptosis. Therefore, fluorescein-stained cellular nuclei had multiple breaks in their DNA and were undergoing apoptosis at the time of fixation.

Immunohistochemistry
Ovary sections from cultured postnatal day 0 (P0) ovaries (cultured for 2 days) were immunostained as described previously [48], for the presence of CTGF using anti-CTGF primary antibody (anti-CTGF rabbit IgG, 1 mg/ml; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Briefly, 3 mm sections were deparaffinized, rehydrated through a graded ethanol series, boiled in 10 mM sodium citrate buffer, quenched in 3% hydrogen peroxide/20% methanol and 0.1% Triton-X solution, and then blocked with 10% goat serum (normal goat serum; Vector Laboratories Inc., Burlingame, CA, USA) for 20 min prior to incubation with 1 mg/ml CTGF antibody (R&D Systems) for 12 h at 4uC. The sections were then washed with PBS and incubated with 1:300 diluted biotinylated secondary antibody for 45 min (goat anti-rabbit IgG; Vector Laboratories Inc.), washed again, and incubated with streptavidin peroxidase (Zymed, San Francisco, CA, USA) prior to color development with a DAB peroxidase substrate kit (Vector Laboratories Inc). Following development, the sections were dehydrated, coverslips mounted with xylene-based medium (Cytoseal-XYL; Richard Allan Scientific, Kalamazoo, MI, USA), and analyzed at 2006, 4006, and 10006 magnification using light microscope. Negative control experiments were performed using a non-specific primary antibody at 1 mg/ml (rabbit IgG; Sigma) and a negative control of an irrelevant anti-SRY antibody at 1 mg/ml (anti-SRY rabbit IgG, 1 mg/ml; Santa Cruz Biotechnology, Santa Cruz, USA).

RNA isolation and purification
Samples of 2-3 pooled control on treated ovaries were stored in TRIZOL (Invitrogen) at -80uC until RNA extraction following the manufacturer's protocol. High quality RNA samples were assessed with gel electrophoresis and required a minimum OD 260/280 ratio of 1.8. Three samples each of control and treated ovaries were applied to microarrays.

Microarray Analysis
mRNA processing and hybridization were performed at Genomics Core Laboratory, Center for Reproductive Biology, Washington State University, Pullman, WA using standard Affymetrix reagents and protocol. Briefly, mRNA was transcribed into cDNA with random primers, from the later, cRNA was transcribed, and from that, single-stranded sense DNA was synthesized which was fragmented and labeled with biotin. Biotin-labeled fragmented ssDNA was then hybridized to the Rat Gene 1.0 ST microarrays containing more than 27,000 transcripts (Affymetrix, Santa Clara, CA, USA). Hybridized chips were scanned on Affymetrix Scanner 3000. CEL files containing raw data were then pre-processed and analyzed with Partek Genomic Suite 6.5 beta software (Partek Incorporated, St. Louis, MO) using RMA, GC-content adjusted algorithm. The signals from 11 probe sets for each transcript were combined to give a single value. Lists of differentially expressed genes treatment were generated using following cut off criteria: signal ratio Control/ Treatment .1.20 or ,0.83, mean difference for un-logged signals between control and treatment .10, t-test p-values ,0.05. pvalues were generated in 2-way ANOVA, blocking for organ culture date batch effects.
CEL files (MIAME compliant raw data) from this study have been deposited with the NCBI gene expression and hybridization array data repository (GEO, http://www.ncbi.nlm.nih.gov/geo, #GSE22096) and can be also accessed through www.skinner. wsu.edu. For genes annotation, Affymetrix annotation file RaGene1_0stv1.na30.rn4.transcript.csv was used unless otherwise specified. Generation of affected KEGG pathways (Kyoto Encyclopedia for Genes and Genome, Kyoto University, Japan) used Pathway-Express, a web-based tool freely available as part of the Onto-Tools (http://vortex.cs.wayne.edu) [49].
Previous studies have demonstrated that microarray data are validated with quantitative PCR data [34,50]. Due to the presence of 11 different oligonucleotide sets for each specific gene being used on the microarray versus only a single primer set for a gene in a quantitative PCR, the microarray is more effective at eliminating false positive or negative data and provides a more robust quantification of changes in gene expression.

Statistical Analysis
Treatment groups are compared using analysis of variance (ANOVA) followed by comparative t-tests where appropriate. Groups were considered statistically significant with P#0.05. All statistics were calculated using GraphPad Prism version 5.0b for Macintosh, GraphPad Software, San Diego, CA, USA.

Results
Data from a previous microarray and ovarian transcriptome analysis demonstrated the relative expression of TGFb-1, 2, and 3 were highest in unassembled oocytes [34]. The inhibitory actions of progesterone on primordial follicle assembly was found to coincide with a dramatic increase in CTGF gene expression [33]. CTGF and TGFb-3 are secreted growth factors that showed a stimulation by progesterone during follicle assembly. TGFb-3 and TGFb-1 bind to the same receptor, but only TGFb-1 was commercially available. Therefore, CTGF and TGFb-1 were selected as candidates to investigate experimentally as being involved in the regulation of primordial follicle assembly.
The 0-day old rat ovary is composed almost entirely of nests of unassembled oocytes ( Figure 1A). In the rodent by 4 days of age the follicle assembly process is in large part completed, and the oocytes have assembled into primordial follicles ( Figure 1B, 1C). Therefore, the ovaries can be caught in an intermediate stage of assembly by using 0 day ovaries and culturing for 2 days. Ovaries from newborn 0-day-old rat pups were placed onto a floating filter organ culture system and treated with either 50 ng/ml transforming growth factor beta one (TGFb-1), or 500ng/ml connective tissue growth factor (CTGF), or a combination of TGFb-1 and CTGF, or 10 26 M progesterone (P 4 ), or were left untreated as controls. After two days of culture, the ovaries were fixed, sectioned and stained for morphological analysis. The relative proportion of assembled follicles was compared across treatment groups (Figure 2A). Treatment with CTGF was found to significantly increase the percentage of assembled follicles. As seen in previous studies [31], progesterone decreased the proportion of assembled follicles over 2 days. Response to progesterone was used as a positive control for these experiments. TGFb-1 had no effect alone. The combined treatment of CTGF and TGFb-1 was not different from CTGF alone. Observations indicate the presence of TGFb-1 did not have a direct effect on the percentage of assembled follicles (Figure 2A).
For each ovary section counted the cross sectional area was determined using ImageJ [51]. The total number of oocytes in each ovary was normalized by its area (mm 2 ) and the oocyte density for each ovary compared between treatment groups ( Figure 2B). There was no difference in ovarian cross-sectional area between treatment groups (data not shown). Interestingly, the combined CTGF and TGFb-1 treatment significantly decreased the oocyte density compared to controls. No other treatment groups had any significant effect. Therefore, the combination of CTGF and TGFb-1 appeared to decrease the initial oocyte cohort/pool in the ovary after two days of culture.
Oocyte apoptosis was investigated since the primordial follicle assembly process involves programmed cell death of the oocytes. The level of apoptosis was measured with TUNEL assays of DNA fragmentation. The number of apoptotic oocytes per section for each treatment group were compared ( Figure 3). No significant alteration in oocyte apoptosis was observed in any treatment group. Therefore, the actions of CTGF did not appear to involve altered oocyte apoptosis at 2 days of culture.
The effects of the growth factors on the primordial follicle pool size used a 10-day ovary culture system. The combined treatment of CTGF and TGFb-1 was found to increase the proportion of assembled primordial follicles and decrease the proportion of unassembled follicles ( Figure 4B and 4C). Progesterone (P 4 ) was found to decrease the percentage of assembled and developed follicles ( Figure 4A and 4B) and increase the percentage of unassembled follicles compared to controls ( Figure 4C). The unassembled follicles present were small clusters of 2-3 oocytes as shown in Figure 1D. The oocyte density and ovarian crosssectional area in each ovary were compared across treatments ( Figure 5). The TGFb-1 treatment group showed a significant decrease in the number of oocytes ( Figure 5A). In contrast, the total ovary section area (i.e. size) was not affected by any treatment ( Figure 5B). The effects of P 4 and CTGF on the 10-day cultures appeared to be due to altered unassembled follicle populations and not a change in pool size. However, the effects of TGFb1 alone appears to be due to a reduced follicle pool size.
Immunohistochemistry was performed on untreated P0 ovaries that had been cultured for two days to determine where CTGF was localized ( Figure 6). CTGF was localized to the basement membranes of both nests and more developed follicles. As ovaries in culture undergo growth and development it is expected that the cell type distribution of ligand and receptor will be similar to that in vivo [39,40]. Therefore CTGF appears to be localized to basement membranes and expressed throughout the ovary. Neither use of non-specific IgG as a primary antibody nor an irrelevant anti-SRY antibody resulted in any specific staining in the ovary.
The molecular actions of CTGF on primordial follicle assembly were investigated with a microarray analysis of the ovarian transcriptome. Ovaries from newborn 0-day-old rat pups were cultured for 1 day of treatment prior to collection for analysis. Morphological analysis of these ovaries demonstrated negligible effects on follicle morphology and assembly after only 1 day of treatment (data not shown). This allows an analysis of CTGF actions on the ovarian transcriptome independent of morphological changes to the ovary. CTGF was found to alter the expression of 76 genes with 33 having an increase in expression and 43 having a decrease in expression. A list of these known genes in their appropriate functional categories is presented in Table 1. The presence of Cxcl2 suggests this chemokine may also be involved in follicle assembly. Interestingly, several olfactory receptors were found to be regulated in the ovary by CTGF treatment, Table 1 and Supplemental Table S1. In addition, an integrated gene network analysis demonstrated 11 regulated genes from this list formed a gene network with appropriate interactions with critical cellular processes (Figure 7). The most abundant functional classes of regulated genes were cell differentiation, cell cycle and apoptosis. Therefore, insights into the molecular actions of how CTGF promotes follicle assembly are provided.

Discussion
Previous ovarian transcriptome analysis demonstrated that TGFb-1 is highly expressed during the follicle assembly process when compared to ovaries containing primordial or primary follicles [34]. In addition, the ability of progesterone to inhibit follicle assembly resulted in a dramatic increase in expression of CTGF [33]. CTGF and TGFb-3 were among the few growth factors shown to be regulated. TGFb-1 and TGFb-3 act on the same receptor complex to stimulate the same signaling cascade. CTGF and TGFb-1 are also known to regulate each others' functions [41,42]. Therefore, CTGF and TGFb-1 were investigated as potential regulators of primordial follicle assembly utilizing whole ovary organ cultures.
Observations from the two-day ovary culture experiments demonstrates that CTGF promotes primordial follicle assembly. TGFb-1 treatment was not found to have an effect alone. The combined treatment of CTGF and TGFb-1 stimulated follicular assembly to the same degree as CTGF alone. Therefore, TGFb-1 had no effect on the proportion of oocytes assembled into follicles after two days of culture. Treatment with progesterone (P 4 ) inhibited follicle assembly as previously described [31], and was used as a positive control in these experiments. Analysis of oocyte density demonstrated that the combined treatment of CTGF with TGFb-1 induced a significant decrease in oocyte number. This suggests that the two factors synergize to modify oocyte number and demonstrates that initially it is possible to manipulate follicle pool size. One possible way to stimulate follicular or ovarian development and decrease pool size is by increasing apoptosis of the oocytes. This was tested with a cellular apoptosis assay, but no significant effect of any treatment was demonstrated. Therefore, CTGF and the combined CTGF & TGFb-1 treatment groups appear not to act through altering apoptosis. However, it is possible that a difference in apoptosis rates may not be detectable at two days of culture. Further studies with shorter or longer culture periods are needed to clarify if there is a period at which CTGF and TGFb-1 affect apoptosis. Alternatively, CTGF may modify the extra-cellular matrix (ECM) of either pre-granulosa cells or the oocytes. In summary, after two days of ovary culture CTGF was found to induce primordial follicle assembly while a combined CTGF and TGFb-1 treatment initially reduced the pool of oocytes in the ovary.
Morphologically, the basement membranes of the nests are degraded and new membranes around primordial follicles are formed during follicle assembly. This ECM formation and breakdown is essential for follicular development [37]. CTGF has previously been shown to affect ECM structure and stability in other tissues [36]. CTGF was primarily localized to the ECM of nest basement membranes and to the cell membranes of individual oocytes. These results are consistent with the general sub-cellular localization of CTGF in other tissues [36]. So one mechanism of action for CTGF may be for CTGF to affect ECM during follicle assembly, possibly promoting the cell migration that is a part of primordial follicle formation [37].
Ovaries were also treated for ten days in culture to investigate the possibility of manipulating the primordial follicle pool size at a time near the end of follicle assembly. In the event the number of assembled follicles could be affected with a growth factor treatment, observations would indicate the possibility of affecting the primordial follicle pool size and thus reproductive lifespan of a female. The nests found after 10 days of treatment were markedly smaller than nests after two days ( Figure 2D). In vivo no unassembled nests would be observed after 10 days of postnatal development, but the ovary organ culture system has the capacity to retain small nests of unassembled oocytes. After 10 days of  culture, CTGF was not found to increase the proportion of assembled follicles as was observed after two days of treatment. The likely explanation for this is that CTGF only increases the rate of follicle assembly. The control untreated ovaries appear to have ''caught up'' developmentally over the longer 10-day culture time period.
Interestingly, the combined treatment of both CTGF and TGFb-1 was observed to increase the proportion of assembled follicles over ten days. This suggests that CTGF and TGFb-1 act synergistically to promote follicle development since the CTGF treatment group alone did not have an effect after ten days of culture. This also indicates this treatment may affect follicle transition which takes place immediately after assembly. In contrast, progesterone was seen to inhibit follicle assembly and development over ten days [31]. Progesterone was found to increase the proportion of unassembled follicles, and the combined CTGF and TGFb-1 treatment decreased the proportion of unassembled follicles. Therefore, the inhibitory effects of progesterone and stimulatory effects of the combined CTGF and TGFb-1 treatment on primordial follicle assembly can be explained by their respective actions on the unassembled follicle pool, even  without their having effects on the total number of oocytes. The separate actions of TGFb-1 and CTGF on follicle assembly appear different than their combined effects. For example, TGFb-1 alone decreased total oocyte number in 10-day ovary cultures, while TGFb-1 plus CTGF did not. This suggests that their actions combined are not simply the additive result of their separate actions. It is likely that the synergism of signaling pathways from both CTGF and TGFb regulate specific and novel cellular functions.
Observations suggest the expression of progesterone, CTGF and TGFb expression are coordinated with each other. Previous studies of rat follicle assembly indicates progesterone treatment of ovaries resulted in a 2.7-fold change in CTGF mRNA expression, and a 1.6-fold change in TGFb-3 expression [33]. In the current study microarray analysis revealed that CTGF treatment resulted in a small (1.1-fold) but statistically significant (p,0.05) increase in TGFb-3 expression. In other organ systems it has been shown that CTGF and TGFb-1 modulate each other's expression and actions [41,42]. The signaling pathways that link the expression suggest the assembly-inhibiting actions of progesterone stimulates the expression of CTGF and TGFb's that in turn can promote follicle assembly. Observations suggest the inhibition of follicle assembly may prime the factors that in turn stimulate assembly.
The total number of oocytes per ovarian cross-section was measured in the ten-day culture experiments. Interestingly, none of the treatments that affected the percentage of assembled follicles (i.e. CTGF and TGFb-1, or P 4 ) resulted in a significantly different number of oocytes when compared to controls. Therefore, the altered percentage of primordial follicles was due to corresponding alterations in the assembly of unassembled oocytes into primordial follicles and not from altered oocyte number. However, TGFb-1 treatment was found to significantly reduce the total number of oocytes per cross section compared to controls. Observations indicate that TGFb-1 reduced the total oocyte number, such that the actions of TGFb may reduce the future capacity for follicular development. Although the postnatal alteration in oocyte number suggests the possibility of manipulating the primordial follicle pool and reproductive lifespan for a female, previous studies have suggested that at puberty the primordial follicle pool may normalize. For example, an increased number of assembled follicles caused by activin treatment prepubertally was reduced to normal levels during puberty [30]. Further studies are needed to determine if the mechanisms involved during puberty compensate for any earlier prepubertal primordial follicle pool size manipulation.
Investigation of the molecular events involved in CTGF induction of primordial follicle assembly used a microarray  analysis of the ovarian transcriptome. The 76 genes regulated by CTGF impact numerous cellular processes with cell differentiation and cell cycle being prominent. The gene network identified suggested an extracellular growth factor (CXCL2), receptor signaling (GPR132 and CTNNB1), numerous signaling systems and transcriptional factors are all involved in primordial follicle assembly, Figure 7. Numerous potential signal transduction processes and families of transcription factors are regulated by CTGF, Supplemental Tables 1 and S1. Clearly the cytokine CXCL2 is a potential regulator of primordial follicle assembly that needs to be investigated. Further information is needed before specific pathways or transcription processes could be considered for therapeutic targets. However, the molecular actions of CTGF on primordial follicle assembly provide insights into this critical developmental process.
In summary, experiments using postnatal day 0 cultured rat ovaries were performed to determine the effects of CTGF and TGFb-1 on primordial follicle assembly. CTGF alone was found to stimulate primordial follicle assembly. Treatment with CTGF and TGFb-1 together was found to stimulate follicle assembly. Neither of these treatments were found to act through an apoptotic pathway or alteration in oocyte number. Interestingly, TGFb-1 alone was found to decrease the number of oocytes and follicles over ten days suggesting the possibility that primordial follicle pool size may be manipulated. This treatment will need to be tested in vivo to determine if the reproductive lifespan of females can be manipulated. Understanding the processes of follicle assembly and follicle transition could lead to therapies to prevent premature ovarian failure or to the ability to manipulate the onset of menopause. The current study identifies CTGF as one of the few  Red color represents up-regulated genes, blue color -down-regulated genes, grey color means that gene/protein is not from the list of 76. Arrows with plus sign show positive regulation/activation, arrows with minus sign -negative regulation/inhibition; grey arrows represent regulation, lilac -expression, and green -promoter binding. Node shapes code: oval and circle -protein; crescent -protein kinase and kinase; diamond -ligand; irregular polygon -phosphatase; circle/oval on tripod platform -transcription factor; ice cream cone -receptor, rectanglecell processes. doi:10.1371/journal.pone.0012979.g007 growth factors known to be involved in primordial follicle assembly, and TGFb-1 as a factor that can reduce prepubertal follicle pool size.