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Gene Expression Profiles of Cumulus Cell Oocyte Complexes during Ovulation Reveal Cumulus Cells Express Neuronal and Immune-Related Genes: Does this Expand Their Role in the Ovulation Process?

Department of Molecular and Cellular Biology (I.H.G., I.G.R., M.S., C.M.W., J.S.R.), Department of Molecular and Human Genetics (L.W.), and Huffington Center on Aging (S.A.O.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: JoAnne S. Richards, Ph.D., Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: joanner{at}bcm.tmc.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Ovulation is a complex process initiated by the preovulatory LH surge, characterized by cumulus oocyte complex (COC) expansion and completed by the release of a mature oocyte. Although many ovarian genes that impact ovulation have been identified, we hypothesized that genes selectively expressed in COCs would be overlooked by approaches using whole ovary or granulosa cell samples. RNA isolated from COCs collected from preovulatory follicles of equine chorionic gonadotropin (CG) primed mice and at selected times after human CG treatment was subjected to microarray analyses and results confirmed by RT-PCR analyses, Western blotting, and immunofluorescent studies. A remarkable number of genes were up-regulated in COCs including Areg, Ereg, and Btc. Several genes selectively expressed in cumulus cells compared with granulosa cells were related to neuronal (Mbp, Tnc, Nts) or immune (Alcam, Pdcd1, Cd34, Cd52, and Cxcr4) cell function. In addition to Sfrp2, other members of the Wnt/Fzd family (Sfrp4, Fdz1 and Fdz2) were expressed in COCs. Thus, there is a cumulus cell-specific, terminal differentiation process. Furthermore, immunofluorescent analyses documented that cumulus cells are highly mitotic for 4–8 h after human CG and then cease dividing in association with reduced levels of Ccnd2 mRNA. Other down-regulated genes included: Cyp19a1, Fshr, Inhb, and the oocyte factors Zp1–3 and Gja4. In summary, the vast number of matrix, neuronal, and especially immune cell-related genes identified by the gene- profiling data of COCs constitutes strong and novel evidence that cumulus cells possess a repertoire of immune functions that could be far greater than simply mediating an inflammatory-like response.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
OVULATION IS INITIATED by the LH surge, requires marked changes in the function and structure of preovulatory (PO) follicles, and culminates in the release of a mature metaphase II oocyte. The ovulated oocyte is surrounded by cumulus cells, a structure known as the cumulus oocyte complex (COC). Before ovulation COCs produce a specialized extracellular, hyaluronan-rich matrix during a process referred to as "COC expansion." Based on the high levels of prostaglandins and other factors produced by ovulatory follicles, ovulation has been likened to an inflammatory response (1). Recent studies have identified numerous genes induced in PO follicles in response to the ovulatory LH surge that support this concept (2, 3). These include prostaglandin synthase-2 (Ptgs2; also known as Cox2), which encodes the rate-limiting enzyme in prostaglandin biosynthesis (4, 5) and genes associated with COC matrix formation: hyaluronan (HA) synthase-2 (Has2) (6), the HA-binding proteins TNF-induced protein-6 (Tnfaip6) (6, 7, 8), the proteoglycan veriscan (Cspg2) (9), pentraxin 3 (Ptx3) (10), and the cell surface molecule Cd44 (11). In addition, numerous proteases such as a disintegrin and metalloproteinase with thrombospondin-like repeats (Adamts1, Adamts4) (12, 13, 14) and cathepsin L (Ctsl) (12, 15) are induced. Importantly, mice null for Ptgs2 (16, 17, 18), the prostaglandin E2 receptor subtype, EP2 (Ptger2) (19), and Tnfaip6 (20) exhibit impaired ovulation associated with defective COC expansion. In addition, mice null for bikunin (1 microglobulin; Ambp), a component of inter--trypsin inhibitor (21, 22), which binds HA and TNFAIP6 as well as mice null for Ptx3 (8, 10, 23) and the type I bone morphogenetic protein (BMP) receptor (BpmrIB) (24), are infertile and present impaired matrix stability.

These observations have indicated that changes in the function of cumulus cells, as well as granulosa cells, are critical for matrix formation and stabilization. Although many hypotheses have been proposed for the function of the cumulus mass (25, 26), including its role as a structural shield for the oocyte (3), relatively little is known about the specific functions of cumulus cells before and during ovulation. For example, cumulus cells exhibit some morphological and functional features similar to granulosa cells (25); however, the COC represents a unique microenvironment within the follicle (27). Specialized cumulus cells within the corona radiata make direct contact with the oocyte via the gap junction protein connexin 37 (GJA4) (28, 29). This communication channel is critical because mice null for Gja4 are infertile due to impaired follicular growth and ovulation (30). Factors released by the oocyte such as growth differentiation factor 9 and BMP15 clearly impact cumulus cell function whereas the cumulus cells factors, such as kit ligand, impact oocyte function (31, 32). The number of cumulus cells also appears to be important because mice null for the cell cycle regulator cyclin D2 (Ccnd2) are infertile, fail to ovulate, and have entrapped oocytes probably because there is an insufficient number of cumulus cells to form a stable matrix (33, 34). The reduced number of cumulus cells in the Ccnd2 null mice also indicates that in wild-type mice these cells express Ccnd2 mRNA and proliferate, a concept that has only recently been supported by experimental data (35, 36). Although cumulus cells, like granulosa cells, acquire steroidogenic potential (37, 38), they do not become vascularized to form a corpus luteum in response to the LH surge. Rather, they are released as part of the expanded COC and remain in a relatively hypoxic environment within the oviduct. Thus, we hypothesized that the unique interactions of cumulus cells with the oocyte and the altered cell fate of the cumulus cells compared with granulosa cells might be associated with specific gene expression profiles within cumulus cells.

Several approaches have been taken to identify genes induced in the whole ovary after the LH surge and have provided a wealth of information (39, 40, 41, 42). However, to our knowledge, no systematic study of genes expressed in cumulus cells or COCs has been done. Therefore, because cumulus cells directly communicate with the oocyte and appear to play critical roles before, during, and after ovulation, we sought to identify genes selectively expressed in COCs that might impact the ovulation process. Accordingly, immature mice were treated with equine (e) choriogonadotropin (CG) to stimulate follicular growth and human (h)CG to stimulate ovulation. At selected time intervals, COCs were isolated from ovaries before ovulation or from the oviduct after ovulation. RNA was prepared and subjected to microarrary analyses. In addition, the mitotic activity of cumulus cells during this time course was analyzed by immunofluorescent detection of phosphohistone H3, a marker of prophase, as well as tubulin, a marker of the mitotic spindle.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Gene Profiling of COCs during Ovulation: Microarray Analyses
Microarray analyses were done to identify genes that are selectively expressed in cumulus cells and that may impact COC matrix formation and/or function. For these studies, COCs were isolated from ovaries of immature mice treated with eCG to stimulate growth of PO follicles (PO; 0 h) and from mice treated with eCG followed by hCG (8 h) to stimulate COC expansion and meiotic maturation of cumulus cell-enclosed oocytyes. Ovulated COCs were isolated from the oviducts of eCG-hCG-treated mice at 16 h (16 h). As shown schematically in Fig. 1, a remarkable number of genes were induced in COCs in response to hCG stimulation in vivo and have been divided in several categories: those induced before ovulation (panel A, 0–8 h), those that continued to increase after ovulation (panel B, 8–16 h), and those that remained constant (panel C, 8–16 h) or declined (panel D, 8–16 h) after ovulation. Many of the induced genes encoded factors related to matrix formation, inflammation, innate immunity cell migration, and neuronal activity (Table 1). An impressive number of genes were also turned off in response to hCG (Fig. 1E, 0–16 h). Many of these genes encoded factors associated with specific cell-signaling cascades, proliferation, and the oocyte. Thus, the processes of COC expansion and meiotic maturation of the cumulus cell-enclosed oocytes are associated with extensive genetic reprogramming of these cells.

View larger version (45K): [in this window] [in a new window]   Fig. 1. Schematic of Microarray Analyses Showing the Number of Genes Induced or Repressed in COCs at Selected Time Intervals after hCG Administration in Vivo RNA was prepared from each treatment group (two biological samples per time interval), and expression analysis was performed in duplicate at each time interval with the Affymetrix Mouse Genome 430 2.0 array. Expression analysis was performed using the open sourced BioConductor bioinformatic software [version 1.5, http://www.bioconductor.org/ (111 )]. Raw expression numbers (probe level data) were corrected, normalized, and modeled using the RMA function (112 ) with the Affy package (version 1.5.8) for BioConductor. Comparison of expression values across the time courses was performed using the Affy (version 1.5.8) and Limma (version 1.8.6) packages. Genes were classified as those induced greater than 4-fold (P < 0.05) at 8 h (A), those induced greater than 4-fold (P < 0.051) from 8–16 h (B), those induced at 8 h but unchanged at 16 h (C), those induced at 8 h but reduced greater than 4-fold (P < 0.05) at 16 h (D), and those down-regulated more than 4-fold (P < 0.05) between 0–16 h (E). The 56 most highly induced genes in category A, all nine induced genes in category B, and the 40 most highly reduced genes in category E are listed. In addition, selected genes from category A that show no change or decrease are listed in C and D. Genes that were selected for further analyses or are discussed in the text are listed by functional categories in Table 1. (For complete microarray analyses, see supplemental data published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org.) (Figure continues on next page.)

View larger version (35K): [in this window] [in a new window]   Fig. 1A. Continued

Steroidogenic Enzymes.
Steroidogenic enzymes were also expressed and induced (Table 1 and data not shown), confirming that COCs synthesize cholesterol from acetate (Hmgcl:3-hydroxy-3-methylglutaryl-coenzyme A reductase and Dhcr-7: 7-decholesterol reductase) (44, 45) and progesterone from endogenous cholesterol: (Star, Cyp11a1, Hsd3b) (46).

Induction of Genes Not Previously Identified in COCs
The expression in COCs of mRNAs encoding factors that were unexpected fell into several specific categories: 1) epidermal growth factor (EGF) signaling, 2) WNT/Frizzled signaling, 3) neuronal activity and cell migration, and 4) immune cell function. The expression of mRNAs of selected genes in each of these categories was confirmed by RT-PCR and analyzed in more detail. To determine the cell-specific and temporal expression of some selected genes, COCs and granulosa cells were collected from ovaries of eCG-primed mice at multiple time intervals before and after hCG stimulation. RNA was prepared and analyzed by semiquantitative RT-PCR using specific primer pairs and L19 as the control.

The EGF Superfamily.
The first group of genes analyzed encodes the EGF-like family of ligands, amphiregulin (Areg), epiregulin (Ereg) and betacellulin (Btc). Previous studies by Conti and associates (47, 48, 49) indicated that these genes were induced by LH in granulosa cells and then, via a paracrine mechanism, activate EGF receptor (EGFR) present on cumulus cells. Herein we show that mRNA encoding each of these factors was induced in cumulus cells (Fig. 1A and Table 1). RT-PCR analyses of COCs collected at 0, 4, 8, 12, 16, and 24 h shows that induction of mRNA encoding these factors was highest at 4 h and similar to that observed in granulosa cells isolated from the same ovarian samples. Immunohistochemical, immunofluorescent, and Western blot data confirm the presence of the cellular (60 kDa) form of AREG in cumulus cells. FSH, prostaglandin E2, and AREG induce Areg and Ptgs2 mRNA in COCs in culture. Inhibitor studies showed that Ptgs2 expression is down-stream of AREG and EGFR activation. Our results establish an autocrine as well as a paracrine regulatory loop within the COCs and granulosa cells that mediates induction of these EGF-like factors (50). The expression of mRNA encoding these genes in isolated COCs is a novel observation and extends those of Conti and associates (47, 48, 49).

WNT/FZD Signaling Cascades.
The second group of genes analyzed belongs to the WNT/Frizzled signaling cascade. In particular, we selected Sfpr2 and Sfrp4 because mRNA encoding these genes was markedly increased at 8 and 16 h on the microarray (Fig. 1, A and B, and Table 1) and because previous studies in our laboratory have focused on the expression and function of Wnt/Fzd factors in ovarian cells (51, 52, 53, 54). By RT-PCR we show that Sfrp2 mRNA was selectively and significantly induced in COCs (P < 0.0001) compared with granulosa cells with the most dramatic increases occurring after ovulation (Fig. 2). In contrast, Sfrp4 mRNA was induced significantly in both cell types (Fig. 2). Furthermore, whereas peak levels of Sfrp2 mRNA were observed in ovulated COCs (16–24 h post hCG), Sfrp4 mRNA levels in COCs and granulosa cells were maximal by 8 h and maintained thereafter. Sfpr2 mRNA was also induced dramatically in COCs cultured in the presence of FSH (inset). Western blots confirmed the marked induction of SFRP2 protein in COCs at 8 and 16 h compared with granulosa cells as well as the high expression of SFRP4 in both cell types (Fig. 2). Immunolocalization of SFRP2 and SFRP4 in isolated COCs revealed that these proteins were present and localized to specific structures within the cytoplasm (secretory vesicles) of cumulus cells (Fig. 2).

View larger version (49K): [in this window] [in a new window]   Fig. 2. Regulators of Wnt/Fzd Signaling Are Induced in COCs Semiquantitative RT-PCR was used to verify gene induction profiles observed by microarray analyses. These include Sfrp2, Sfrp4, Fzd1, and Fdz2. Expression of SFRP2 and SFRP4 were confirmed by Western blot analyses. Immunofluorescence localized SFRP2 and SFRP4 protein to the cumulus cells of ovulated COCs, 16 h post hCG. Insets depict immunostaining of SFPR2 and SFRP4 with DAPI to illustrate the cytoplasmic (particulate) localization of these proteins. Immunostaining of the oocyte is also evident, but whether or not this is specific for SFPR2 or SFRP4 remains to be verified by additional analyses. Two separate sets of RNA were analyzed for each COC and granulosa cell treatment group. Values of the gene product relative to the internal standard L19 were calculated and expressed as fold-induction relative to COC PMSG (0 h). The mean ± SD for each treatment group is presented for each gene analyzed in this and all subsequent figures. ANOVA and Newman-Keuls multiple comparison test were used to determine statistical significance. Values were significant if P < 0.05 or lower and designated by asterisks: *, P < 0.05; **, P < 0.01; ***, P < 0.001. GC, Granulosa cell.

Neuronal Genes.
A large number of transcripts induced in COCs before ovulation encode genes previously associated with neuronal cells (Fig. 1, A and B, and Table 1). These neuronal-associated genes include: myelin basic protein (Mbp) (55), the extracellular glycoprotein Tenascin C (Tnc) (56), the tridecapeptide neurotensin (Nts) (57), Sfrp2 (58), the heparin-binding growth factor pleiotrophin (Ptn) (59), and cGKII (Prkg2) (60). As shown in Fig. 3, levels of mRNA encoding Mbp increased progressively between 4–16 h, with peak levels observed at 16 h (P < 0.001 compared with 0 h) in the ovulated complexes. In contrast levels of Mbp, mRNA in granulosa cells appeared to decrease at 4 h after hCG and then increased significantly at 8–12 h (P < 0.05). Western blot analyses confirmed increased expression of MBP in COCs compared with constant levels of this protein in granulosa cells at 0 and 8 h. Immunofluorescent analyses showed that MBP was low in COCs at 0 h, increased markedly by 8 h where it was localized to the surface of all cumulus cells (not a subset of cells) and to extensions between cumulus cells and the oocyte (Fig. 3, arrowhead), and remained elevated in ovulated COCs at 16 h. Thus, MBP is uniformly expressed in cumulus cells of COCs before ovulation.

View larger version (55K): [in this window] [in a new window]   Fig. 3. Neuronal-Like Genes Are Expressed in COCs RT-PCR analyses of Mbp, Tnc, and Nts mRNA show selective expression of Mbp and Tnc in cumulus cells, whereas Nts was also highly expressed in granulosa cells. Expression of MBP was verified by Western blot and immunofluorescent localized MBP to the cumulus cell membrane. The apparent increase in MBP staining of the oocyte at 8 h (5x) compared with 0 h (5x) is related to the intense membrane staining of cumulus cells that surround the oocyte as well as to projections between the cumulus cells and oocyte as shown. Because granulosa cells also express MBP mRNA and protein, the somatic cells are the most likely source of this protein in COCS. TNC was localized to selected regions of the cumulus cells, whereas NTS was localized to the membrane. Data were analyzed and presented as described in the legend of Fig. 2. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Nts mRNA was induced rapidly in both COCs and granulosa cells after hCG (P < 0.01 and P < 0.001, respectively) with maximal levels of about 80-fold at 4–8 h post hCG and declined significantly thereafter (Fig. 3). Induction of mRNA encoding Ptn, as revealed by the microarray, was also confirmed by RT-PCR (data not shown). cGMP-dependent protein kinase II (cGKII; PRKG2) is highly induced in COCs as well as in granulosa cells where it is a PR target gene (60).

Immune Cell-Related Genes.
In addition to the expression of "neuronal" genes, many genes thought to be specifically associated with immune cell function and innate immunity were induced in COCs before and/or after ovulation (Fig. 1 and Table 1; microarray data not shown). These include Adam8 (61), CD28: Pdcd1 (62), Cd34 (63, 64), Cd52 (65), Cd81 (66), Cd97:Emr1 (67, 68), Cd97a (Ig- or mb-1) (69), Cd147:Embigin (70), Cd166:Alcam (71, 72), Cxcr4 (73, 74), Il6 (75), Nurp1:P8 (76), Ptx3 (10, 23), Runx1 (69, 77, 78), and Saa3 (79). Expression of Ptx3, Cd147, Runx1, and Saa3 mRNA in COCs extends previous observations that these genes are expressed in ovarian cells (10, 23, 39, 70, 79). However, with the exception of Ptx3, these genes have not been reported previously to be expressed in cumulus cells. Herein we show that induction of two additional genes, programmed cell death 1 (Pdcd1;Cd28) associated with autoimmune responses, and Cd52, a B-cell marker, occurs selectively in cumulus cells compared with granulosa cells (Fig. 4). Levels of Pdcd1 mRNA increased dramatically in ovulated COCs (P < 0.001) whereas Cd52 mRNA increased progressively in COCs before ovulation and peaked at 70-fold after ovulation (P < 0.001). Immunofluorescent analyses localized PDCD1 and CD52 protein to the cumulus cell surface (Fig. 4). Expression of CD52 appeared to be enhanced in mitotic cells of COCs collected at 8 h as revealed by intense membrane staining of CD52 on cells immunopositive for phosphohistone H3 (Fig. 4; see also Fig. 7B). Based on the cloverleaf-like pattern of phosphohistone H3 staining of the chromatin, these cells appear to be in early prophase (80). By 16 h CD52 was uniformly present on all cumulus cells. mRNA encoding RUNX1 and RUNX2, putative transcriptional regulators of the Pdcd1 gene (78), were also highly induced in COCs by 8–12 h and maintained in ovulated COCs at 16 h (P < 0.001; Fig. 4), confirming microarray analyses (Figs. 1 and 4 and Table 1). Unlike Pdcd1, however, Runx1 and Runx2 mRNA was also induced significantly in granulosa cells at 8 h (P < 0.01) but declined by 24 h (P < 0.05 and P < 0.01, respectively). Western blot analyses confirmed induction of RUNX2 protein in cumulus cells and granulosa cells.

View larger version (39K): [in this window] [in a new window]   Fig. 4. Immune-Like Genes Are Expressed in COCs A, RT-PCR analyses of Pdcd1 and Cd52 show selective expression of Pdcd1 and Cd52 mRNA in cumulus cells compared with granulosa cells. B, Immunofluorescence analyses localized PDCD1 and CD52 to the surface of cumulus cells. The progressive appearance of PDCD1 is depicted by its localization to a few cumulus cells at 4 h and to essentially all cumulus cells at 16 h. At 8 h, immunostaining of CD52 was enhanced in mitotic cumulus cells as shown by costaining with phosphohistone H3, a marker of mitotic prophase. C, RT-PCR analyses showed induced expression of Runx1 and Runx2 mRNA in COCs isolated at 4 and 8 h, respectively, that was maintained at 16 h. Increased expression of RUNX2 protein in COCs as well as granulosa cells at 8 h was shown by Western blot analyses. D, When nonexpanded COCs were isolated from PO follicles and cultured in the presence of FSH (100 ng/ml), Runx1 and Runx2 mRNA increased progressively at 4 and 8 h, a pattern similar to that observed in vivo, but declined at 16 h. Data were analyzed and presented as described in the legend of Fig. 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001. GC, Granulosa cell.

View larger version (48K): [in this window] [in a new window]   Fig. 7. Cumulus Cells Are Highly Proliferative A, RT-PCR analyses confirm that Ccnd2 mRNA is high in COCs at 0 h but declines by 16–24 h. B, COCs at 4 h were immunostained using antibodies to phosphohistone H3 (green) and acetylated tubulin (red). DAPI was used to stain nuclei (blue, DNA). Cumulus cells in early prophase exhibit a cloverleaf pattern of condensed chromatin that was decorated with phosphohistone H3. The condensing chromatin was organized around the spindle fibers as shown by the localization of tubulin. Cumulus cells in metaphase were also observed. In these cells the DNA (blue) was clearly at the metaphase plate, tubulin (red) was within the spindle fibers, and phospho-p38MAPK/MAPK14 (green) was localized to the centriolar region of the mitotic spindle. (Figure continues on next page.)

View larger version (70K): [in this window] [in a new window]   Fig. 7A. Continued In cumulus cells completing cytokinesis, tubulin was restricted to the centriolar region of the cells with phospho-p38MAPK/MAPK14 in the center of the centriole. C, Oocytes isolated from PO follicles are arrested in prophase of meiosis and have not undergone GVBD (0 h; upper panels). Oocytes enter meiotic metaphase I by 4 h post hCG and exhibit a meiotic spindle (tubulin, red), phospho-p38MAPK/MAPK14 in the centrosomal protein complex (green), and condensed nuclear DNA (DAPI/blue) (middle panels). The colocalization of tubulin and DNA, tubulin and phospho-p38MAPK/MAPK14, and tubulin, DNA, and phospho-p38MAPk/MAPK14 are merged in the lower panels.

View larger version (59K): [in this window] [in a new window]   Fig. 5. COCs Express Additional Immune Related Genes Alcam, Cxcr4 and Cd34 Alcam mRNA is selectively induced in COCs compared with granulosa cells at 12 h post hCG. Immunofluorescent analyses showed increased immunostaining of ALCAM protein by 8 h and 16 h where it localized to the cumulus cell surface. mRNA encoding the GPCR Cxcr4 is induced in COCs and granulosa cells with protein also localized to the cell membrane. Cd34 was induced most dramatically in COCs before ovulation. At 8 h the CD34 protein was clearly present on the membrane of cumulus cells. Data were analyzed and presented as described in the legend of Fig. 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Induction of Genes in Culture
To determine whether the genes induced in COCs during ovulation could be induced in culture and what signaling cascades might be operative, COCs were collected from PO follicles of eCG-primed mice and cultured for specific time intervals in the presence of FSH with or without inhibitors of specific signaling cascades: protein kinase A (KT5720; 10 µM), MAPK kinase 1 (MEK1) (PD98059; 20 µM), p38MAPK/MAPK14 (SB203580; 20 µM) (6), and EGFR activation via tyrosine phosphorylation (AG1478; 10 µM) a dose used successfully in ovarian cells (47, 83, 84) as well as PTGS2 (NS398; 10 µM) (85). As shown in Fig. 6, MBP was induced by FSH in cultured COCs, and this induction was reduced significantly (P < 0.05) by protein kinase A (PKA) and MEK1 inhibitors. More dramatic, however, was the time-dependent induction of Tnc, Pdcd1, Cd52, and Alcam mRNA by FSH (Fig. 6) that closely mimicked the induction of each gene in vivo (Figs. 3–5). Furthermore, induction of Tnc, Pdcd1, and Alcam mRNA by FSH was reduced dramatically by inhibitors of MEK1, p38MAPK/MAPK14, and EGFR signaling but not by inhibitors of protein kinase A or PTGS2/COX2. Expression of Cd52 mRNA was reduced significantly by all inhibitors including those of PKA and PTGS2. Runx1 and Runx 2 mRNAs were also induced by FSH (Fig. 5) via MEK1-, P38MAPK/MAPK14-, and EGFR-responsive mechanisms (data not shown). Collectively, these data indicate that PKA is one, but not the only, signaling factor controlling the induction of genes selectively expressed in COCs before and after ovulation. Rather, the MAPKs and EGF receptor pathways appear to predominate and support the emerging concept that the EGF-like factors impact gene expression during COC expansion (48, 49, 83, 84).

View larger version (29K): [in this window] [in a new window]   Fig. 6. Induction of Genes in COCs in Culture COCs isolated from PO follicles of eCG-primed mice were cultured in the presence of FSH (100 ng/ml) for 0–16 h (panel A) or with FSH for 8 h without or with inhibitors of PKA (KT5720; 10 µM), MEK1 (PD98059; 20 µM), p38MAPK/MAPK14 (SB203580; 20 µM), PTGS2 (NS398; 10 µM), and EGFR tyrosine kinase (AG1478; 10 µM) as described in Materials and Methods. Data were analyzed and presented as described in the legend of Fig. 3 with the exception that the mean ± SD was calculated based on three separate samples in each treatment group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

When oocytes were examined with these same markers, tubulin was clearly evident in the meiotic spindles of oocytes that had undergone germinal vesicle breakdown and entered meiotic metaphase I (4 h) and metaphase II (16 h) but was not present in oocytes collected from PO follicles that still retained the germinal vesicle (0 h). In the metaphase I oocytes (Fig. 7C) as in metaphase II oocytes (data not shown), phospho-p38MAPK/MAPK14 was localized to a diffuse region at the pole of the meiotic spindle that contains centrosomal proteins. Collectively, these data document that cumulus cells collected from PO follicles are highly mitotic and that mitosis persists up to about 8 h but is reduced markedly by 16–24 h. During this same time period oocytes have entered meiotic metaphase II.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Ovulation is a complex process that involves marked changes in follicular cell function (2, 3, 41, 95). Previous studies have focused primarily on altered gene expression patterns obtained by analyzing RNA prepared from whole ovarian tissue (39, 40, 41). Herein we document that COCs isolated from PO follicles at specific times after hCG-induced ovulation undergo extensive genetic reprogramming. The molecular mechanisms by which LH/hCG mediates these dramatic changes in gene expression in COCs appear to involve, at least in part, the induction of the EGF-like ligands AREG, EREG, and BTC, as proposed recently by Conti and colleagues (48, 49, 83). According to their paracrine hypothesis, the EGF-like ligands induced by LH/hCG in granulosa cells are released and activate EGF receptors in cumulus cells. Our data extend this hypothesis by showing that these EGF-like factors are expressed not only in granulosa cells but also in cumulus cells where they regulate the expression of Ptgs2 and other genes, establishing that these EGF-like ligands exert autocrine actions to regulate cumulus cell function before ovulation (50). Another major signaling cascade that is highly induced in the ovulating COCs is made up of members of the WNT/FZD family. Although the function of WNT/FZD signaling in the ovary has not yet been well defined, many members are present and hormonally regulated (51, 52, 53). Misregulated expression of ß-catenin, a down-stream mediator of the canonical pathway, induces granulosa cell tumors (54). That Fzd2 mRNA is expressed at high levels in cumulus cells and that induction of Fzd1 occurs in association with that of Sfrp2 and Sfrp4 suggests that these secreted negative regulators of WNT may act to provide a brake on WNT signaling events occurring via FZD1 and/or FZD2. Because induction of Sfrp2 has been linked to neuronal and osteoblast cell differentiation (58, 96) whereas induction of Sfrp4 is related to terminal differentiation of granulosa cells (52), these factors may dictate cumulus cell-specific terminal differentiation associated with ovulation.

Although cumulus cells express some genes in a manner similar to granulosa cells, they also express a distinct set of genes, indicating that cumulus cell differentiation has some unique features. Microarray analyses confirmed that cumulus cells express mRNAs encoding steroidogenetic enzymes (Star, Cyp11a1) and regulators of inflammation (Ptgs2, Tnfaip6 and Ptx3), as well as matrix and matrix-related molecules, some of which are typically expressed in the formation of cartilage (Has2, Cspg2, Adamts1 and Adamts4) and bone (Runx2). It is important to note, however, that although cumulus cells express many genes (Cyp11a1, Sfrp4, Prlr) associated with the luteinization process in granulosa cells (52, 97), cumulus cells do not luteinize. Although cumulus cells do not specifically make cartilage or bone, the matrix molecules that are important components of these tissues during development are expressed in COCs (9, 98). Microarray analyses revealed further that genes commonly associated with neuronal cell activity (Mbp, Sfrp2, and Tnc) and immune cell functions (Alcam, Cd34, Cd52, Cxcr4, Pdcd1, and Runx1) are induced and highly expressed in expanded COCs compared with granulosa cells. Although their specific functions in COC expansion, ovulation, and oocyte maturation remain to be defined, collectively, the data indicate that cumulus cells are multipotential and appear to function uniquely to ensure that the oocyte is released within a stable protective environment.

That many genes normally associated with neuronal and immune cell function are induced in cumulus cells was not expected because neither neuronal nor immune cell types appear to be present in COCs. However, two of the most highly induced genes on the microarray were myelin basic protein (Mbp) and Nts. MBP is a major component of the myelin sheath of oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system. Recent studies have shown that MBP is also present in immune cells and hematopoietic cells, most specifically CD34+ dendritic cells (55), and in these cells the major product is derived from mRNA transcribed from an alternate region upstream of the classical Mbp exons. This variant mRNA encodes a protein referred to as Golli-MBP. Based on RT-PCR analyses and specific primer pairs, we conclude that this variant transcript encoding golli-Mbp is expressed in ovarian cells. Thus, the induction of Mbp mRNA and protein as well as Cd34 mRNA in cumulus cells of ovulating follicles provides the first evidence that these proteins are present in cells other than neuronal, immune, or hematopoietic cells and that cumulus cells (and granulosa cells) have immune cell-like features. The even distribution of MBP on all cumulus cells indicates that there is no obvious subset of cells within the COCs. The mechanism by which Mbp mRNA is induced remains unclear because induction by FSH in culture did not mimic that observed in vivo; nor was Mbp mRNA markedly down-regulated by any signaling inhibitors used. Although the function of MBP in cumulus cells has not been determined, it is known to stabilize regions of lipid rafts in other cells and thereby stabilize membranes or provide unique microdomains within the cell membrane (99).

The other neuronal-related genes have diverse functions. Nts has recently been shown to be expressed in neuroendocrine cells in prostate cancer that are presumed to be derived from nonneuroendocrine cancer cells (100). Thus, the expression of elevated levels of neurotensin in cumulus cells indicates that these cells may be capable of assuming neuroendocrine functions possibly related to immunoprotection. Tenascin C is a hexameric extracellular matrix glycoprotein that binds versican and HA. Although TNC is most prominent during embryonic development of the nervous system, where it is made predominantly by oligodendrocyte precursor cells (56), it is also present in the embryonic and adult tissues that are undergoing remodeling events. The induced expression of Tnc mRNA selectively in cumulus cells compared with granulosa cells provides evidence that cumulus cells exhibit some functions that are distinct from granulosa cells. However, Tnc null mice are fertile, indicating that it is not essential for the ovulation process (101).

The remarkable number of hematopoietic and immune cell-related genes that are expressed in cumulus cells suggests that cumulus cells may have a unique plasticity and may be pluripotential. Hematopoietic and immune-related genes that are highly induced in COCs of ovulatory follicles include Alcam, Cd34, Cd52, Cd147, Cxcr4, Ptx3, Pdcd1, and Runx1. Of these, Ptx3 and Cd147 (EMBIGIN; BASIGIN) have already been shown to be expressed in the ovary and play key roles in fertility (10, 23, 70, 102). PTX3 binds to TNFAIP6, thereby stabilizing the matrix of ovulated COCs (8) whereas CD147 appears to be critical for both ovulation and implantation (70). ALCAM, which is a marker of hematopoeitic stem cells, is also expressed on other cells (71) and is induced and localized to endometrial epithelial cells and blastocysts (72). Because ALCAM has been shown to promote cell adhesion and because it is uniformly distributed on the surface of all cumulus cells, the induction of Alcam in COCs compared with granulosa cells suggests that it may promote cell-cell adhesion or enhance cell adhesion to the matrix. The uniform distribution of ALCAM on the surface of all cumulus cells provides additional evidence that it is not associated with a specific subset of immune cells within the COCs.

As noted above, some CD34+ dendritic cells express Mbp defining one subset of immune stem cells (55). In addition, CD34 has been linked to hematopoietic progenitor cells, mast cells, and vascular endothelial cells and is thought to play a role in cell migration by blocking adhesion. CXCR4, the G protein-coupled receptor involved in the homing of hematopoietic cells, is expressed in some CD34+ stem cells (73, 74). The marked coinduction of Cd34, Cxcr4, and Mbp in COCs indicates that cumulus cells may acquire specific functions of a subset of immune-like cells. Although the factors that induce Cd34 expression in cumulus cells are not yet known, Cxcr4 is induced during hypoxia by HIFa as well as EGFR signaling events (103). Thus, transcription of the Cxcr4 gene in cumulus cells may be mediated by the EGF-like factors such as AREG. In addition, CD34+ cells that also express the tetraspanin CD81 (CD34+/CD81+) have been proposed to delineate a specific subset of lymphohematopoietic stem cells (104). In lymphocytes, CD81 interacts with the Ig superfamily member prostaglandin (PG) regulatory-like protein (PGRL) to mediate prostaglandin induced T cell differentiation and motility (82). That Cd81 and Pgrl mRNAs are expressed at high levels in COCs (microarray data not shown) provides additional evidence for immune cell-like markers in cumulus cells. One might postulate that cumulus cells that are PGRL+ and CD81+ in COCs of PO follicles and acquire CD34 as well as PTGS2 within 8 h of hCG have alternative ways to mediate prostaglandin action during ovulation. It is tempting to speculate that CD34+ cells that also express PGRL, CD81, CXCR4, and MBP may acquire unique functional properties.

CD52 is a small glycosylphosphatidylinositol-anchored glycopeptide that is expressed in mature T lymphocytes, monocytes, and monocyte-derived dendritic cells and in B cells and B cell chronic lymphocytic leukemia cells (65). A specific antibody (CAMPATH1) is used in vivo to facilitate transplantation and reduce graft-vs.-host disease. This antibody is also used to alleviate B cell leukemia. CD52 is also expressed in epithelial cells of the epididymus and ductus deferens, secreted and transferred to the surface of sperm. However, the lipid and carbohydrate moieties of CD52 present in lymphocytes differ from those present in sperm. CD52 protein was clearly associated with the membrane of all cumulus cells, including those in mitosis, but its functions remain to be determined because Cd52 null mice have not been reported.

Perhaps most curious is the induction of Pdcd1 selectively in COCs compared with granulosa cells. PDCD1 is a member of the IgG CD28 superfamily originally isolated from an apoptosis-induced T cell hybridoma (62). PDCD1 is expressed in activated CD4–/CD8– T cells but is not present in CD4+/Cd8+ unstimulated T cells. PDCD1 in humans is linked to autoimmune diseases such as systemic lupus erythromatosus. Pdcd1 receptor null mice exhibit splenomegaly with increased myeloid cells, progressive arthritis, and lupus-like glomerulonephritis (105). Because PDCD1 protein was localized to the surface of all cumulus cells, it may be involved in preventing autoimmune-like responses once the COCs are released. However, Pdcd1 null mice are fertile (H. Nishimura, personal communication) and thus the specific role for this factor remains unclear. Of particular interest, however, the Pdcd1 (Pd1) gene in humans is regulated by the transcription factor RUNX1, and mutations in the Runx1 gene lead to autoimmune diseases (78). That Runx1 and Runx2 mRNAs are highly up-regulated in COCs (Fig. 5 and Table 2) indicates that both of these factors may impact Pdcd1 mRNA expression in cumulus cells. RUNX1 is also known to impact expression of Cd97a, the Ig-signaling component of the B cell receptor (69) that is also expressed at high levels in COCs. Expression of Runx2 mRNA is most highly expressed and obligatory for osteoblast differentiation (106) and in these cells appears to be regulated, at least in part, by WNT-signaling cascades (107). Thus, a similar regulatory network may operate in cumulus cells of ovulating COCs where members of the WNT signaling cascade (Sfrp2, Sfrp4, Wnt 4, and Fzd1) are induced.

Microarray analyses revealed that many genes were down-regulated during COC expansion. These include genes essential for PO follicular function such as Fshr, Cyp19a, and Ccnd2 and oocyte function such as Zp1–3, Natch5/Mater, and Gja4. Although it has been generally assumed that cumulus cells are not mitotic during COC expansion, the array data indicated that mRNA encoding the cell cycle regulator Ccnd2 was expressed. This was confirmed by RT-PCR analyses that also showed that Ccnd2 mRNA declined but more slowly in COCs than in granulosa cells, supporting previous studies (34, 35). Unequivocal evidence that cumulus cells are proliferative was shown by immunofluorescent detection and localization of phosphohistone H3 to condensing chromatin and tubulin decorating the mitotic spindle of cells in prophase (Fig. 8A). Cumulus cells at metaphase were also observed in COCs isolated at 0–4 h. However, by 8 h many cells were undergoing cytokinesis as shown by the localization of tubulin and phospho-p38MAPK/MAPK14 to a single discrete centriolar region (80, 108). This proliferative activity appears critical for ovulation, because Ccnd2 null mice that have few cumulus cells surrounding the oocyte are infertile and present entrapped oocytes within luteinized follicles. Furthermore, inhibition of p38MAPK/MAPK14 blocks COC expansion suggesting that there may be a functional link between proliferation, germinal vesicle breakdown, and alterations in gap junctions (117). As in cumulus cells, phosphohistone H3 localized to condensed chromatin, tubulin decorated the meiotic spindles, and phospho-p38MAPK/MAPK14 localized to the diffuse centrosomal region within oocytes at metaphase I (4 h post hCG) and metaphase II (24 h post hCG). That phospho-p38MAPK/MAPK14 was present in the centrosomal region of oocytes as well as the centriolar region of cumulus cells indicates that this kinase impacts both the meiotic and mitotic processes. These results support recent studies by others that cumulus cells are mitotic (35, 36) and provide novel information on a possible role for p38MAPK/MAPK14 in centrioles of proliferating cells.

In summary, LH/hCG induced ovulation is associated with an amazingly complex series of events within the COCs: matrix formation, reduced proliferation, altered interactions of the somatic cells with the germ cell, and the resumption of meiosis. Although changes in gene expression patterns were expected, the vast number of genes up-regulated and down-regulated was somewhat surprising. Importantly, cumulus cells, unlike mural granulosa cells, do not become vascular and form corpora lutea. Rather they appear to undergo a cumulus cell-specific terminal differentiation process during which they express specific genes not observed in high abundance in granulosa cells (Alcam, Cd34, Cd52, Cd147, Cxcr4, Pdcd1, Sfrp2, Tnc). Many of these are related to immune cell-like functions. Even as early as postnatal d 1, neonatal ovaries express immune cell-related genes (109) including some that also appear on the COC array. Although immune cells have been reported present in the ovulated COCs (26, 110), the number of these cells (based on immunostaining) relative to the abundance of cumulus cells is remarkably small. Based on the vast number of immune cell-like genes expressed in essentially all cumulus cells within expanded complex (our immunofluorescent data), we propose that this constitutes strong evidence that cumulus cells have a repertoire of potential immune-like functions that far exceeds that of mediating an inflammatory response. In additional to providing a structural barrier, the matrix and cumulus cells may also provide factors that have the potential to immunoprotect the oocyte and thereby prevent/control innate immune responses.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Animals
Immature female C57BL/6 mice were obtained from Harlan, Inc. (Indianapolis, IN). On d 23 of age, female mice were injected ip with 4 IU of eCG (Pregnyl; Organon, West Orange, NJ) to stimulate follicular growth followed 48 h later with 5 IU hCG (Gestyl; Diosynth, Oss, The Netherlands) to stimulate ovulation and luteinization (12). Animals were housed under a 16-h light/8-h dark schedule in the Center for Comparative Medicine at Baylor College of Medicine and provided food and water ad libitum. Animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals, as approved by the Animal Care and Use Committee at Baylor College of Medicine.

COC Isolation and Expansion in Culture
Ovaries of immature mice primed with eCG for 48 h contain multiple PO follicles. COCs cells were isolated from these follicles by needle puncture, collected by pipette, pooled, frozen, and stored at –80 C for RNA or protein extraction (6). Granulosa cells that were released by needle puncture of the follicles were also pooled, collected by centrifugation, and frozen at –80 C. COCs and granulosa cells were also isolated from ovaries of eCG-primed mice exposed to hCG for 2, 4, 8, or 12 h. During this time, COCs expand but have not yet ovulated. Ovulated (fully expanded) COCs were collected by needle puncture of the oviducts of mice 16 h or 24 h after hCG. Each pool of COCs and granulosa cells was obtained from 15 mice. Each experiment generating a separate pool of COCs and granulosa cells was repeated twice.

In other experiments nonexpanded COCs (20–30) were collected from the ovaries of eCG-treated mice, plated in separate wells of a Falcon 24-well plate (Becton Dickinson, Franklin Lakes, NJ) in 0.2 ml of defined medium (6) containing 1% fetal bovine serum without or with FSH (100 ng/ml) and various protein kinase inhibitors: PKA (KT5720; 10 µM), MEK1 (PD98059; 20 µM), p38MAPK/MAPK14 (SB203580;20 µM) (6), and EGFR activation via tyrosine phosphorylation (AG1478; 10 µM) (47, 84) as well as a selective inhibitor of PTGS2/COX2 (NS398; 10 µM) (85). After 16 h, the COCs were extracted for RNA (see below). Each pool represents COCs from 15 mice. Each experiment generating a separate pool was repeated three times.

RNA Isolation Microarry Analyses
For microarray gene expression analysis, total RNA was obtained from COCs (200) collected from the ovaries of 15 eCG-primed mice (0 h), 15 eCG-primed mice at 8 h post hCG (8 h), and from the oviducts of 15 eCG-primed mice at 16 h post hCG (16 h) to generate one pool of COCs for each time point and repeated once to provide two independent COC pools for all three time points. Pooling COCs was not only necessary to obtain a sufficient amount of RNA for the microarray analyses but also provided one means to reduce variation among mice, treatments, and individual COCs. RNA was isolated from these six COC pools using the RNeasy mini kit (QIAGEN Sciences, Germantown, MD) according to the manufacturer’s instructions. RNA quality was confirmed using the Agilent Bioanalyzer 2100 and the NanoDrop ND-1000 Spectrophotometer. Total RNA was labeled and hybridized to Affymetrix Mouse Genome 430 2.0 Array gene chips by the Microarray Core Facility at Baylor College of Medicine (Lisa White, Ph.D., Director.) Affymetrix .cel files for each chip can be found at GEO (<http://www.ncbi.nlm.nih.gov/geo/). Expression analysis was performed using the open sourced BioConductor bioinformatic software [version 1.5, http://www.bioconductor.org/ (111)]. Raw expression numbers (probe level data) were corrected, normalized, and modeled using the RMA function (112) with the Affy package (version 1.5.8) for BioConductor. Comparison of expression values between the time courses (0 h vs. 8 h, 0 h vs. 16 h, and 8 h vs. 16 h) was performed using the Affy (version 1.5.8) and Limma (version 1.8.6) packages. Limma uses an empirical Bayes method to moderate standard errors of the estimated log-fold changes between samples and provides more stable inference and improved power, especially for experiments with a small number of arrays (113). The processed data were exported to a Filemaker Pro 7 database, and data were filtered and parsed with Filemaker Pro 7. Genes of interest were called if three criteria were met based on the Limma analysis: BioConductor. Comparison of expression values across the time courses was performed using the Affy (version 1.5.8) and Limma (version 1.8.6) packages. The processed data were exported to a Filemaker Pro 7 database, and data were filtered and parsed with Filemaker Pro 7. Genes of interest were called if the t test P value (P 0.05), difference probability (>90%), and fold change at least 4-fold (up-regulated) or not exceeding 4-fold (down-regulated) were true for the comparison between the two time points of interest.

RT-PCR Analyses
To verify changes in the expression of specific genes identified on the microarray, semiquantitative RT-PCR analyses were performed. Total RNA was reverse transcribed using 500 ng poly-dT (Amersham Pharmacia Biotech, Piscataway, NJ) and 0.25 U avian myeloblastosis virus-reverse transcriptase (Promega Corp., Madison, WI) at 42 C for 75 min and 95 C for 5 min. For the amplification of the cDNA products, specific primers pairs were selected and analyzed as indicated in Table 2. All PCRs contained [³²P]dCTP (ICN, Los Angeles,CA), Taq Polymerase, and Thermocycle buffer (Promega Corp.) as in previous studies (14, 114). cDNA products were resolved on 5% polyacrylamide gels that were dried and exposed to film. The radioactive PCR product bands were quantified using a Storm 860 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). All RT-PCR reactions were run within the linear range, and the authenticity of the PCR products was verified by sequencing.

Western Blot Analyses
To confirm that altered expression of specific mRNAs was associated with changes in protein levels, Western blots were done. For these analyses, protein samples from COCs and isolated granulosa cells were prepared either by homogenization in whole-cell extract buffer [100 mM NaCl, 100 mM Na₄P₂O₇, 50 mM NaF, 0.1 mM NaV0₄, 1% Triton X100, 2.5 mM HEPES (pH 7.5), 10% glycerol, 5 mM EDTA, and 5 mM EGTA] (115) or boiling SDS sample buffer (116) as indicated in the text and as described previously. Extracts were resolved by SDS-PAGE (12%) and transferred to Immobilon-P nylon membranes (Millipore Corp, Bedford, MA). Membranes were blocked in Tris-buffered saline and Tween 20 [TBST; 10 mM Tris (pH 7.5), 150 mM NaCl, and 0.05% Tween 20) containing 3% nonfat Carnation instant milk (Nestle Co., Solon, OH). Blots were incubated with specific antibodies (MBP, SFRP2, SFRP4, and RUNX2) (Table 3) overnight at 4 C. After washing in TBST, enhanced chemiluminescence detection was performed by using Pierce Super Signal according to the manufacturer’s specifications (Pierce) and appropriate exposure of the blots to Kodak x-ray film (Eastman Kodak, Rochester, NY). Specific bands were quantified by densitometric analyses using a Molecular Dynamics Personal Densitometer.

Statistics
The semiquantitative RT-PCR data are represented as means + SD. Data were analyzed by using GraphPad Prism Programs (ANOVA or t test; GraphPad Prism, San Diego, CA) to determine significance. Values were considered significantly different if P < 0.05 or P < 0.01.

	ACKNOWLEDGMENTS

 
We thank Derek Boerboom (Department of Molecular and Cellular Biology) and Dorothy Lewis (Department of Immunology) at Baylor College of Medicine (BCM) for their helpful suggestions and comments. We thank the Integrated Microscopy Core for their assistance, expertise and equipment.

	FOOTNOTES

 
This work was supported, in part, by the Spanish Ministry of Education, Culture and Sport and Autonomous Community of Canary Islands (Consejeria de education, Cultura y Deportes) (Grants IH-G and IG-R) as well as National Institutes of Health Grants HD-16229 and HD-07495-SCPRR (to J.S.R.).

I.H.G., I.G.R., M.S., C.M.W., S.A.O., and L.W. have nothing to declare; J.S.R. consults for Schering, AG.

First Published Online February 2, 2006

Abbreviations: AREG, Amphiregulin; BMP, bone morphogenetic protein; BTC, betacellulin; CG, choriogonadotropin; COC, cumulus oocyte complex; DAPI, 4',6-diamidino-2-phenylindole; EGF, epidermal growth factor; EGFR, EGF receptor; EREG, epiregulin; HA, hyaluronan; MEK, MAPK kinase; NTS, neurotensin; PGRL, regulatory-like protein; PKA, protein kinase A; PO, prevovulatory; TLR, Toll-like receptor; TNC, Tenascin C.

Received for publication October 21, 2005. Accepted for publication January 23, 2006.

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TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

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