Knockout of Pentraxin 3, a Downstream Target of Growth Differentiation Factor-9, Causes Female Subfertility
Simona Varani,
Julia A. Elvin,
Changning Yan,
Janet DeMayo,
Francesco J. DeMayo,
Heidi F. Horton,
Michael C. Byrne and
Martin M. Matzuk
Department of Pathology (S.V., J.A.E., C.Y., M.M.M.), Department of Molecular and Cellular Biology (J.D., F.J.D., M.M.M.), Department of Molecular and Human Genetics (M.M.M.), Baylor College of Medicine, Houston, Texas 77030; and Wyeth Research (H.F.H., M.C.B.), Cambridge, Massachusetts 02140
Address all correspondence and requests for reprints to: Martin M. Matzuk, M.D., Ph.D., The Stuart A. Wallace Chair and Professor, Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: mmatzuk{at}bcm.tmc.edu.
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ABSTRACT
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The ovulatory process is tightly regulated by endocrine as well as paracrine factors. In the periovulatory period, extensive remodeling of the follicle wall occurs to allow the extrusion of the oocyte and accompanying cumulus granulosa cells. Growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) are secreted members of the TGFß superfamily that are expressed beginning in the oocyte of small primary follicles and through ovulation. Besides its critical role as a growth and differentiation factor during early folliculogenesis, GDF-9 also acts as a paracrine factor to regulate several key events in preovulatory follicles. By analyzing GDF-9-regulated expression profiles using gene chip technology, we identified TNF-induced protein 6 (Tnfip6) and pentraxin 3 (Ptx3 or PTX3) as novel factors induced by GDF-9 in granulosa cells of preovulatory follicles. Whereas Tnfip6 is induced in all granulosa cells by the LH surge, Ptx3 expression in the ovary is specifically observed after the LH surge in the cumulus granulosa cells adjacent to the oocyte. PTX3 is a member of the pentraxin family of secreted proteins, induced in several tissues by inflammatory signals. To define PTX3 function during ovulation, we generated knockout mice lacking the Ptx3 gene. Homozygous null (Ptx3-/-) mice develop normally and do not show any gross abnormalities. Whereas Ptx3-/- males are fertile, Ptx3-/- females are subfertile due to defects in the integrity of the cumulus cell-oocyte complex that are reminiscent of Bmp15-/-Gdf9+/- double mutant and BMP type IB receptor mutant mice. These studies demonstrate that PTX3 plays important roles in cumulus cell-oocyte interaction in the periovulatory period as a downstream protein in the GDF-9 signal transduction cascade.
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INTRODUCTION
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OVULATION, THE PROCESS in which a mature follicle under LH stimulation ruptures and releases the oocyte into the oviduct, is often compared with an inflammatory response (1). Follicular hyperemia and edema occur within a few hours of the gonadotropin surge and are mediated by vasoactive agents such as histamine, kinins, and PGs. Before the ovulatory stimulus, the cumulus granulosa cells (adjacent to the oocyte) are connected to each other and the oocyte through gap junctions that allow the oocyte to be exposed to factors that regulate its growth and meiotic maturation. After exposure to LH, the oocyte resumes meiosis, and the cumulus cells secrete an extensive extracellular matrix (a process known as cumulus expansion) (2, 3). This expanded matrix is critical for normal and efficient reproduction because it binds the oocyte and the cumulus cells together, protects the oocyte from the proteolytic and mechanical stresses during extrusion, and allows sperm binding, penetration, and fertilization.
There is increasing evidence that the oocyte plays an active role in cumulus expansion (for review see Ref. 4); a factor secreted from the oocyte has been shown to induce cumulus expansion through increased hyaluronic acid matrix synthesis. Growth differentiation factor-9 (GDF-9 or Gdf9), an oocyte-secreted member of the TGF-ß superfamily, is expressed in growing oocytes throughout all stages of folliculogenesis and in cumulus cell-oocyte complexes after ovulation (5, 6, 7). Knockout of the Gdf9 gene leads to infertility due to a block at the primary follicle stage, absence of thecal layer formation, and defects in oocyte meiotic competence (8, 9, 10). In vitro, recombinant GDF-9 can substitute for the oocyte in inducing cumulus expansion and regulates the expression of several genes important for ovulation in preovulatory granulosa cells (6). Recombinant GDF-9 induces hyaluronic acid synthase 2 (6), the cumulus granulosa cell enzyme responsible for hyaluronic acid deposition (11), cyclooxygenase 2 (COX2 or prostaglandin endoperoxide synthase 2), the prostaglandin pathway enzyme expressed in granulosa cells of the preovulatory follicle after the LH surge (10, 12), and the PGE2 receptor, EP2 [PTGEREP2; (13)]. Cox2 knockout mice are infertile and demonstrate reduced ovulation and fertilization rates. Similarly, EP2 receptor knockout mice show reduced female fertility due to a decreased rate of in vivo fertilization and defects in cumulus expansion (14, 15, 16). Consistent with the recombinant GDF-9 studies, Bmp15-/- Gdf9+/- double mutant female mice are subfertile due to defects in ovulation and the integrity of the cumulus-oocyte complexes (i.e. the cumulus cells are not adherent to the oocyte after ovulation) (17). Thus, in vitro and in vivo, GDF-9 regulates the expression of cumulus cell proteins that play key functions in the periovulatory period.
To identify additional genes that are regulated by GDF-9 in preovulatory granulosa cells and play roles at this stage of folliculogenesis, we used Affymetrix (Santa Clara, CA) gene chip technology. In addition to confirming Cox2 and Has2 (hyaluron synthase 2) as genes downstream of GDF-9, we identified TNF-induced protein 6 (Tnif6) and pentraxin 3 (Ptx3 or PTX3) as novel genes induced by GDF-9 in the periovulatory granulosa cells of the ovary. PTX3 is a secreted protein belonging to the long pentraxin family of inflammatory proteins, named after the characteristic discoid arrangement of the five noncovalently bound subunits (18). Because the physiological roles of PTX3 in cumulus cell physiology and ovulation were unknown, we generated a Ptx3 knockout mouse model. Ptx3 knockout mice are subfertile secondary to ovulatory and cumulus cell-oocyte defects.
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RESULTS
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Ptx3 and Tnif6 Are Induced by GDF-9 in Vitro and after Superovulation Treatment
The Affymetrix expression chip system was used to identify genes differentially regulated by recombinant GDF-9 in preovulatory granulosa cells. We isolated mural granulosa cells from large antral follicles, cultured them with and without recombinant GDF-9, and isolated total RNA for oligonucleotide array hybridization. Several generations of Affymetrix gene expression oligonucleotide array chips were used to monitor the expression of 30,000 genes and expressed sequence tags. The results of this analysis (Table 1
) confirmed our previous data (6, 13) that GDF-9 regulates the synthesis of several important ovarian gene products in the preovulatory follicle including Cox2, which encodes the enzyme responsible for prostaglandin synthesis in cumulus granulosa cells, and Has2, expressed in cumulus cell-oocyte complexes and involved in cumulus expansion. Ovaries from Gdf9 knockout females demonstrate a decrease in the levels of follistatin, Igf1, and inhibin/activin ßB mRNAs but show no difference in the levels of inhibin
mRNA (10). In the gene chip experiment, GDF-9 appears to positively regulate the synthesis of follistatin, IGF-I, and inhibin/activin ßB mRNAs. As a control, expression of inhibin
, one of the most abundant ovarian mRNAs, is unchanged in the presence or absence of GDF-9. These findings were confirmed by Northern blot analysis (fold change presented in Table 1
).
Our GeneChip studies also identified the hyaluronan binding protein, Tnfip6 (also called TNF-stimulated gene 6) and Ptx3 as novel genes that were not previously known to be positively regulated by GDF-9 in the periovulatory period. An average of two different GeneChip hybridization experiments showed that Tnfip6 was 6.2-fold induced by GDF-9 relative to control-treated granulosa cells, although the relative mRNA copies in each experiment were somewhat divergent (Table 1
). Our Northern blot results of Tnfip6 were more consistent with the 11K ChipB results because Tnfip6 was significantly increased in the absence of GDF-9 (compared with freshly isolated granulosa cells), and the presence of GDF-9 resulted in a 5.6-fold up-regulation of Tnfip6 mRNA (Fig. 1A
). In addition, Tnfip6 was absent in Gdf9 knockout ovaries, detectable at low levels in adult wild-type ovaries, and up-regulated by PMSG (48 h) and human CG (hCG) (5 h) treatment of immature mice (Fig. 1B
). These findings suggest that GDF-9 and other factors may regulate Tnfip6 synthesis in the mammalian ovary. Similar analyses of Ptx3 expression were performed. In two independent series of experiments, Ptx3 was assigned "absent" (4 and 5 mRNA copies/100,000 total mRNA copies) in cells incubated in the absence of GDF-9 but "present" (206 and 146 mRNA copies/100,000 total mRNA copies) in cells treated with GDF-9, thereby showing an average 35.2-fold induction by GDF-9 (Table 1
). Northern blot analysis confirmed these findings, demonstrating that Ptx3 is up-regulated 34.8-fold in RNA samples derived from GDF-9-treated granulosa cells (Fig. 1C
) compared with RNA from granulosa cells incubated in the absence of GDF-9 (control) or freshly isolated granulosa cells (at time 0). Similar to control granulosa cells, Ptx3 mRNA is undetectable in adult ovaries from Gdf9 knockout mice and barely detectable in adult ovaries from wild-type mice. Similar to Tnfip6, Ptx3 is dramatically up-regulated in ovaries from mice superovulated with PMSG for 48 h and hCG for 5 h (Fig. 1D
).

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Figure 1. Northern Blot Analysis of Tnfip6 (A, B) and Ptx3 (C, D) Expression in in Vitro Cultured Granulosa Cells and in Ovaries
A and C, RNA samples are: t=0, granulosa cells immediately after isolation; Control, granulosa cells after 5 h culture in control media; GDF-9, granulosa cells after 5 h culture in the presence of 50 ng/ml GDF-9. B, D, Ovarian RNA samples are: WT, control; -/-, GDF9 -/-; S.O. WT, 3-wk-old CD1 females superovulated (S.O.) with 5 IU of PMSG for 48 h then 5 IU of hCG for 5 h. Each lane contains 15 µg total RNA, Gapdh is shown as a control for RNA loading.
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To determine the cellular site of expression of Ptx3 and Tnfip6, we performed in situ hybridization. Ovaries from wild-type mice, stimulated for 48 h with hCG, do not show any detectable Tnfip6 or Ptx3 signal (data not shown). However, mice treated with PMSG for 48 h and subsequently hCG for 1 h, begin to express both Ptx3 and Tnfip6 mRNAs. Ptx3 localizes to the granulosa cells closest to the oocyte, whereas Tnfip6 is present both in cumulus and mural granulosa cells (Fig. 2
, A and B, and Fig. 3
, A and B). By 5 h after hCG injection, an intense Ptx3 mRNA signal in the cumulus granulosa cells was found, and a few mural granulosa cells that line the antral cavity also express Ptx3 (Fig. 2
, CF). At this time point, Tnfip6 mRNA was detected in all granulosa cells in antral follicles (Fig. 3
, CF). By 12 h after hCG injection, expression of both Ptx3 and Tnfip6 decreased. Ptx3 was no longer seen in mural granulosa cells, but persisted in the cumulus granulosa cells of some cumulus-oocyte complexes (Fig. 2
, G and H). Tnfip6 was localized to expanded cumulus cells, with lower levels of expression detected in mural granulosa cells (Fig. 3
, G and H). Interestingly, the spatiotemporal expression of Ptx3 is nearly identical to the pattern of Cox2 (10, 12) and Has2 (11), which are also induced in cumulus cells after the LH surge and are up-regulated by GDF-9 in preovulatory granulosa cells in vitro (6, 13). Thus, the findings that Ptx3 is expressed after the LH surge in granulosa cells in closest proximity to the oocyte and also induced in granulosa cells in vitro in response to GDF-9 suggest that the Ptx3 gene is a physiological downstream target of GDF-9.

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Figure 2. Localization of Ptx3 mRNA in the Ovary after Superovulation
Ovaries from CD1 female mice given PMSG for 48 h and hCG for 1 h (A, B), for 5 h (CF), and for 12 h (G, H), were analyzed by in situ hybridization with specific cDNA antisense probes (left column, brightfield; right column, darkfield). A and B, Ptx3 is detected at low levels in mural granulosa cells (MC) and cumulus granulosa cells (CC) by 1 h after hCG injection. CF, By 5 h after hCG injection, expression has increased in the cumulus cells and in mural granulosa cells in contact with the follicular antrum. G and H, By 12 h after hCG injection, expression is localized to expanded cumulus cells. Oocytes (O) do not express Ptx3.
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Figure 3. Localization of Tnfip6 mRNA in the Ovary after Superovulation
In situ hybridization analysis was performed using a specific Tnfip6 antisense probe (left column, brightfield; right column, darkfield). A and B, One hour after hCG injection Tnfip6 mRNA localizes to both mural and cumulus granulosa cells. CF, By 5 h after hCG injection, expression has increased in both the mural granulosa cells and the cumulus granulosa cells. G and H, By 12 h after hCG injection, Tnfip6 expression is localized to the expanded cumulus cells, and lower levels are detected in the mural granulosa cells. Oocytes (O) do not express Tnfip6.
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Previous studies demonstrated that Ptx3 was induced in the ovary (as well as in heart, skeletal muscle, lung, and thymus) by an acute phase response triggered by ip injection of bacterial lipopolysaccharide (LPS) (19). To determine whether the inflammatory stimulus and the LH surge induced Ptx3 expression in a similar fashion, we performed in situ hybridization on ovaries from mice treated with PMSG and hCG and/or LPS. Five hours after hCG injection, Ptx3 was present in the cumulus cells and mural granulosa cells that line the antral cavity of preovulatory follicles (Fig. 4
, A and B). Surprisingly, after hormonal treatment (PMSG and hCG) and LPS injection, Ptx3 was also detected in the bursa, the epithelial layer that surrounds the ovary, and in adipose tissue connected to the ovary (Fig. 4
, C and D). Ovaries from females injected with PMSG and saline show low levels of Ptx3 signal in granulosa cells of antral follicles (Fig. 4
, E and F), whereas PMSG injection followed 48 h later by LPS injection (Fig. 4
, G and H) induce a low level of expression of Ptx3 mRNA in the stroma of the ovary as well as high levels in the bursa and adipose tissue. The mice injected with LPS showed clear signs of an ongoing acute phase response (i.e. ruffled fur and trembling). Northern blot analysis of RNA from the hearts of injected mice confirmed that the LPS had induced Ptx3 mRNA in extrahepatic sites (data not shown). Thus, these findings suggest that LPS has no direct effects on the ovarian follicle and mainly regulated Ptx3 in the surrounding bursa and adipose tissue.

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Figure 4. Localization of Ptx3 mRNA in the Ovary after Superovulation and during an Acute Phase Response
Three-week-old female CD1 mice were injected with PMSG and 48 h later with hCG (AD). A and B, By 5 h after hCG, Ptx3 signal is detected in cumulus granulosa cells (CC) and some mural granulosa cells (MC). C and D, At the time of hCG injection, mice were also given 0.8 mg of LPS (Escherichia coli strain 0111: B4, Sigma, St. Louis, MO) ip, and ovaries collected 5 h later. The presence of LPS induces Ptx3 mRNA in ovarian bursa (B), and adipose (A) tissue, and does not change the pattern of expression in cumulus granulosa cells. EH, In the control experiment, 3-wk-old female CD1 mice were injected with PMSG and 48 h later with saline (E, F) or 0.8 mg of LPS (G, H) and ovaries were collected 5 h later. E and F, Low levels of Ptx3 mRNA are detected in granulosa cells of antral follicles in the saline treated mice. G and H, After LPS injection, the signal localizes to stromal cells, adipose tissue, and bursa. Left column, Brightfield; right column, darkfield.
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Targeted Mutation of the Ptx3 Gene in Embryonic Stem (ES) Cells and Generation of Ptx3 Null Mutant Mice
To determine the role of Ptx3 in vivo, we used gene targeting technology in ES cells to generate mice carrying a null mutation in the Ptx3 gene. The Ptx3 gene consists of 3 exons, spanning 4 kb on mouse chromosome 3 (19). We used a genomic clone isolated from a 129/SvEv genomic library to engineer a targeting vector to delete exons 1 and 2 (Fig. 5A
). Homologous recombination of the targeting vector at the Ptx3 locus was predicted to yield a null mutation because exon 1 encodes the initiation methionine codon as well as the signal peptide that is required for protein secretion. The Ptx3 targeting vector was electroporated into AB2.2 ES cells, and 29 of 95 (30%) of the ES cell clones analyzed were targeted at the Ptx3 locus. Three of these cell lines were used to produce chimeric male mice that were fertile and transmitted the Ptx3 mutant allele (Ptx3tm1Zuk; herein called Ptx3-) to the F1 progeny. Intercrossing of the F1 heterozygotes yielded F2 progeny including 91 wild-type mice (24.9%), 172 heterozygous (Ptx3+/-) mice (47.2%), and 102 homozygous (Ptx3-/-) mice (27.9%) out of 48 litters analyzed. Thus, the mutated allele is transmitted with the expected Mendelian frequency of 1:2:1, and the male:female ratio is approximately 50:50 (Fig. 5B
, top). Southern blot analysis using a probe spanning exons 1 and 2 demonstrated a lack of hybridization in the DNA derived from Ptx3-/- mice, thereby confirming that the mutated allele that we generated is null (Fig. 5B
, bottom).

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Figure 5. Targeted Disruption of the Ptx3 Gene
A, The Ptx3 replacement-targeting vector was electroporated into AB2.2 ES cells. After selection with hypoxantine, aminopteridine, thimidine, and 1-(2'-deoxy-2'fluoro-ß-D-arabinofuranosyl)-5-iodouracil), positive 5' and 3' recombination events were confirmed by Southern blot analysis with 3' external and 5' internal probes. B, Southern blot analysis of tail DNA derived from 11 offspring of a litter from a heterozygous mating. Top panel, The probe spanning exon 3 detects an 8.5-kb wild-type band and a 6-kb recombinant band. Bottom panel, Analysis of the same tail DNA with a probe spanning exons 1 and 2 detects only the 8.5-kb band in DNA derived from heterozygous (+/-) and wild-type (+/+) mice, but not in the homozygous (-/-) null offspring. RV, EcoRV; BII, BglII; S, SpeI.
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Fertility of Ptx3 Mutant Mice
To assess the role of PTX3 in fertility, heterozygous and homozygous mutant males and females were bred for 6 months. Homozygous Ptx3-/- male displayed normal fertility. Likewise, mating of ten Ptx3+/- females with Ptx3+/- males over a period of 6 months resulted in 61 litters with an average litter size of 8.2 ± 0.1 pups per litter (n = 61), not statistically different from the litter sizes of wild-type mice in previous experiments [8.4 ± 0.2 pups per litter, n = 107 (Ref. 20)]. These findings demonstrate that Ptx3+/- females show normal fertility. Twenty-two Ptx3-/- females bred to wild-type males over the same 6-month period displayed significantly reduced fertility, with fewer pups born in each litter and approximately one-half the litters per month when compared with heterozygous females (Table 2
). Breeding of 129 inbred Ptx3-/- females (n = 3) over a 6-month period failed to produce any offspring in contrast to 129 inbred Ptx3-/- females (n = 7), which yielded 32 litters (0.76 ± 0.1 litters/month) with an average litter size of 5.69 ± 0.33. Thus, Ptx3 plays an important role in female fertility.
Ovarian Physiology and Analysis of Cumulus-Oocyte Integrity in Ptx3 Knockout Mice
To understand the cause of the reduced fertility in Ptx3-/- female mice, we first analyzed the mutant ovaries grossly and histologically. Ptx3-/- ovaries were indistinguishable from wild-type or Ptx3+/- ovaries. Histological analysis demonstrated that in mature females at 12 wk of age, the stages of folliculogenesis up to the large antral follicle stage are normal as expected based on the spatiotemporal expression of Ptx3 in the ovary. In addition, corpora lutea were present, confirming that ovulation occurs in the absence of PTX3 (Fig. 6
, A and B). The ovulatory function of Ptx3-/- mice was then assayed by determining the ovarian response to superovulation with exogenous gonadotropins. Nineteen to 20 h hours after hormonal (hCG) stimulation, oocytes were recovered from the ampulla of the oviduct. Fewer oocytes were released by Ptx3-/- females (9.9 ± 0.5) compared with Ptx3+/- (31.2 ± 0.6) or wild-type females (22.2 ± 0.9) [P < 0.001]. Histological analysis of the mutant ovaries in these experiments indicated that the presence of oocytes in luteinized follicles is a rare event (Fig. 6C
) and does not account for the reduction in the number of eggs released into the oviduct. Surprisingly, oocytes isolated from Ptx3-/- females were denuded of cumulus granulosa cells (Fig. 7B
), whereas oocytes released from Ptx3+/- (Fig. 7A
) and wild-type females (data not shown) retained the cumulus cells, which form a compact 3-dimensional mucoelastic structure surrounding the oocyte.

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Figure 6. Histological Analysis of Ptx3+/- Ovaries
A and B, Ovaries from Ptx3+/- (A) and Ptx3-/- females at 12 wk of age showing normal follicular development and corpora lutea (PF, primary follicle; AF, antral follicle; CL, corpus luteum). C, Luteinized follicle with a trapped oocyte (O) from a superovulated Ptx3-/- mouse, 20 h post-hCG. DF, Ovaries isolated from Ptx3-/- mice 8 h post-hCG are enriched in preovulatory follicles. D, Follicle where a normal cumulus expansion has occurred. E, Absence of cumulus cells adherent to the oocyte. F, Follicle with randomly scattered cumulus cells in the antrum surrounding the oocyte.
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Figure 7. Defects in Cumulus Cell Adherence in Ptx3 Null Mice
A, Oocytes (OO) isolated from the oviducts of Ptx3+/- females after hormonal stimulation are embedded in a three-dimensional extracellular matrix. B, In contrast, the cumulus cells (CC) of Ptx3-/- mice are loosely attached to the oocyte and readily fell off the oocyte. Panel B was photographed at twice the relative magnification of panel A.
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To assess whether the loss of cumulus cells occurs before or after ovulation, we superovulated Ptx3+/- and Ptx3-/- females and collected the ovaries 8 h after the hCG injection. Histologically, these ovaries show preovulatory follicles with oocytes surrounded by cumulus cells, some of which have apparently undergone expansion (Fig. 6D
) similar to Ptx3+/- controls. However, in several preovulatory follicles, the oocyte is not surrounded by cumulus cells (Fig. 6E
) or the expanded cumulus mass appears to be randomly scattered around the oocyte (Fig. 6F
).
Several reports indicate that the cumulus mass that accompanies the ovulating oocyte into the oviduct plays a critical role in successful fertilization of the released ovum (3, 15, 21, 22). To determine the fertilization rate in Ptx3-/- mice, we superovulated 3-wk-old females and mated them to stud males. Approximately 20 h after the hCG injection, we isolated oocytes and one-cell embryos from the ampulla of the oviduct. Fifty-five percent of the oocytes from Ptx3+/- females successfully developed into two-cell embryos after 24 h of culture (Table 3
). In contrast, only 1.3% of the oocytes from Ptx3-/- female mice developed to the two-cell stage, demonstrating a dramatic reduction in fertilization rate in the absence of PTX3. Thus, defects in cumulus cell adherence to the oocyte in the periovulatory period reduce the ovulation and fertilization rates, resulting in subfertility of the Ptx3-/- female mice.
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DISCUSSION
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We used gene chip technology to identify and confirm genes that are regulated by GDF-9 in the periovulatory stage of folliculogenesis. Our previous and current studies have demonstrated that GDF-9 induces the expression of several key genes known to be involved in ovulation. Our major new findings were that TNFIP6, a hyaluronic acid binding protein, and PTX3, an acute-phase response protein involved in inflammatory processes, are induced by GDF-9 in our granulosa cell culture system. In the ovary, Ptx3 mRNA is detected in cumulus granulosa cells after the LH surge, with a peak of expression at 8 h after the exogenous hormonal stimulation. Using a gene knockout approach, we show that Ptx3 null female mice develop normally but are subfertile due to defects in ovulation and cumulus cell-oocyte integrity post ovulation.
In the periovulatory period, the oocyte secretes one or more factors that affect the surrounding cumulus cells (4). Our laboratory has presented data that the oocyte-secreted protein GDF-9 stimulates hyaluronan synthase 2, cyclooxygenase 2, and the EP2 PGE2 receptor mRNA and inhibits urokinase plasminogen activator and LH receptor mRNA in preovulatory granulosa cells (10, 13). Thus, GDF-9 appears to be a key oocyte-secreted regulator of cumulus cell gene expression and cumulus cell function in the periovulatory period. In this study, we also show that Tnfip6 and Ptx3 are two additional genes, which are regulated by GDF-9 in our granulosa cell expression system. Both of these proteins appear to be integral factors in the cumulus cell-oocyte complex after the LH surge (see below).
After the LH surge, the cumulus cells of the preovulatory follicle lose contact with each other and the oocyte. However, the cumulus cells and oocyte are embedded in a mucoelastic extracellular matrix (23) rich in hyaluronic acid, the product of HAS2 from the cumulus cells (11). The matrix contains a number of proteins which stabilize the hyaluronic acid matrix. The hyaluronic acid surface receptor, CD44, appears to anchor the matrix to the cumulus cells (24). Multiple proteoglycans, which bind hyaluronic acid, are also present in the cumulus cell secreted matrix (25). The hyaluronic acid chains are held together by various cumulus cell-derived and serum-derived link proteins. In vitro, newly synthesized hyaluronic acid chains are retained in the cumulus cell-oocyte complex only if serum is present. In particular, serum contains inter-
-trypsin inhibitor (ITI), a complex of bikunin (called urinary trypsin inhibitor) and one or two heavy chains (HC1, HC2, or HC3) linked through a chondroitin-4-sulfate chain of bikunin (23). After the LH surge, the blood-follicle barrier is open to ITI, and the heavy chains of ITI are transferred to the cumulus cellproduced hyaluronic acid to form a covalent complex. The complex appears to be strengthened further by TNFIP6, a hyaluronic acid binding protein that is covalently attached to an ITI heavy chain and strongly interacts with hyaluronic acid (26).
Knockout models lacking several of the above factors have demonstrated that cumulus expansion and the integrity of the hyaluronic acid-rich matrix are critical for female fertility. Cumulus cell-oocyte complexes from mice lacking cyclooxygenase 2 (27) and the EP2 PGE2 receptor (15) fail to demonstrate cumulus expansion. Preovulatory follicles from bikunin knockout mice demonstrate disorganized cumulus cell-oocyte complexes resulting in dramatically reduced fertility (28, 29). Although a viable knockout of Tnfip6 has not yet been reported, our studies here suggest that the oocyte, and specifically GDF-9 from the oocyte, plays a critical role in the induction of Tnfip6 from the cumulus cells of periovulatory follicles and that this oocyte regulation of Tnfip6 is not activated until after the LH surge in mice (our studies here and Ref. 26) and rats (30). In addition, nonoocyte factors must play a role in Tnfip6 induction because the gene is also expressed in the mural granulosa cells.
What function does PTX3 play in this process? Ovaries and cumulus cell-oocyte complexes within the Ptx3 knockout ovaries appear relatively intact before the breakdown of the follicle wall. Because periovulatory action of GDF-9 induces the synthesis of other components of the cumulus cell-oocyte extracellular matrix, our findings that GDF-9 induces the secreted protein PTX3 in the same time period suggests that PTX3 also becomes part of this extracellular matrix. In the mouse, Ptx3 is induced in cells and at other sites by proinflammatory signals such as IL-1 and TNF as well as LPS. PTX3 has been characterized as a marker of human inflammatory conditions such as septic shock (31), rheumatoid arthritis (32), Castlemans disease (33), and acute myocardial infarction (34). A protective role of PTX3 at these sites could be hypothesized based on the findings that PTX3 binds apoptotic cells in the endothelium, thereby reducing their clearance by antigen-presenting dendritic cells and preventing the eventual onset of an autoimmune reaction (35). Mice overexpressing the Ptx3 gene under its own promoter have improved survival in response to endotoxic shock and sepsis (36), and Ptx3-/- mice have been reported to undergo more severe seizure-related neuronal damage (37). Our findings of reduced ovulation and defects in cumulus cell-oocyte integrity suggest that PTX3 functions to bind to the cumulus cell-oocyte extracellular matrix to protect the oocyte and extracellular matrix from proteolytic enzymes present at the apex of the follicle during the extrusion from the ovary and in the oviductal environment. Proteolytic degradation of the extracellular matrix is a physiological process that starts after ovulation, leading to progressive oocyte denudation that correlates with a decline in the ability of the oocyte to be fertilized (38, 39). Proteases produced by the preovulatory follicle and present in the oviductal environment have been reported to destabilize the cumulus matrix by degrading proteins required for hyaluronan stabilization (40). An untimely release of proteases, as well as a lack of antiprotease activity in the extracellular matrix (e.g. absence of PTX3), might account for early oocyte denudation. Thus, PTX3 appears to protect the cumulus mass that is vital for the capture of the oocyte by the oviductal fimbria and its efficient entry into the oviduct (41, 42). An alternative explanation for the decrease in ovulation in the Ptx3-/- knockout mice is that PTX3 on the surface of the cumulus cell-oocyte complex acts to directly bind the complex to the fimbria of the oviduct to shuttle the complex into the oviduct.
The cumulus mass has previously been shown to play a role in fertilization, facilitating egg-sperm interactions and the subsequent acrosomal reaction (3, 15). Because the cumulus mass surrounding the oocytes of Ptx3-/- mice is disrupted, this is the likely (indirect) cause for their reduced fertilization. Thus, the oocytes in Ptx3-/- mice appear to lose their optimal extracellular environment and show a lower efficiency of ovulation and fertilization.
Our in vitro studies demonstrate that GDF-9 stimulates cumulus expansion of oocytectomized complexes and can regulate 7 genes that are differentially expressed in cumulus granulosa cells (Has2, Cox2, EP2 receptor, Ptx3, and Tnfip6) vs. mural granulosa cells (urokinase plasminogen activator and LH receptor) in the periovulatory follicle (Refs. 6, 13 , and the present studies). Interestingly, the LH surge is the trigger for this differential expression pattern but must do so indirectly because LH receptors are not present on cumulus granulosa cells. This suggests that a factor from the mural granulosa cells or the serum, upon stimulation from LH, positively induces a GDF-9 signal transduction cascade. This could be accomplished by 1) processing of an inactive GDF-9 precursor that is bound to the cumulus cell extracellular matrix or present in the oocytes; 2) stimulating the degradation of an inhibitory protein or complex; 3) up-regulation of a cumulus cell receptor or cytoplasmic cascade; or 4) inducing a transcriptional cofactor, which acts along with GDF-9 regulated transcription factors. Interestingly, mice lacking the type I BMP receptor also have defects in the integrity of cumulus cell-oocyte complexes and have fertility defects but display up-regulated Cox2 mRNA levels (43). Our recent studies have shown that the knockout of the Bmp15 gene, which encodes a member of the TGFß superfamily closely related to Gdf9, results in reduced female fertility. The Bmp15-/- phenotype is exacerbated when the mice also carry a mutation in one of the Gdf9 alleles (i.e. Bmp15-/-Gdf9+/-). Oocytes recovered from these double or single mutant mice are also denuded of cumulus cells, thereby phenocopying the postovulatory defects in the Ptx3-/- mice. Although we do not know whether BMP-15 plays a role in the regulation of Ptx3 gene expression, clearly both GDF-9 and BMP-15 appear to play important roles in the periovulatory ovary. The identification of the receptors involved in GDF-9 and BMP-15 or GDF-9:BMP-15 heterodimer signaling and the possible roles of type I BMP receptor in these pathways (positive or negative) and the regulation of PTX3 and COX2, will require further studies.
In conclusion, we have identified PTX3 as a novel protein downstream of the GDF-9 signal transduction cascade in the preovulatory follicle. Using knockout technology, we demonstrate that PTX3 play key roles in the efficient delivery of the cumulus-oocyte complex to the oviduct and successful fertilization of the egg. These studies further confirm important roles of the cumulus cells and oocyte-somatic cell communication in reproductive physiology.
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MATERIALS AND METHODS
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DNA Microarray Hybridization and Analysis
Ten micrograms of total RNA were quantitatively amplified and biotin-labeled (44). Briefly, RNA was converted to double-stranded cDNA using an oligo-deoxythymidine primer that has a T7 RNA polymerase site on the 5' end (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T24)-3'). The cDNA was then used directly in an in vitro transcription reaction in the presence of biotinylated nucleotides Bio-11-UTP and Bio-11-CTP (Enzo, Farmingdale, NY). To improve hybridization kinetics, the labeled antisense RNA was fragmented by incubating at 94 C for 35 min in 30 mM MgOAc, 100 mM KOAc. Hybridization to Mu11K and Mu19K GeneChips (Affymetrix) displaying probes for 30,000 mouse genes/expressed sequence tags was performed at 40 C overnight in a mix that included 10 µg fragmented RNA, 6x SSPE [sodium chloride, sodium phosphate, EDTA (1x SSPE, 0.18 M NaCl, 10 mM NaH2PO4, 1 mM EDTA, pH 7.4)], 0.005% Triton X-100, and 100 µg/ml herring sperm DNA in a total volume of 200 µl. Chips were washed, stained with SA-PE and read using an Affymetrix GeneChip scanner and accompanying gene expression software. Labeled bacterial RNAs of known concentration were spiked into each chip hybridization mix to generate an internal 11-member standard curve, allowing normalization between chips and conversion of raw hybridization intensity values to mRNA frequency (mRNA molecules per million). The data were filtered for genes modulated at least 3-fold (using mRNA frequency values) when comparing GDF-9-treated cells to those treated with media alone. For genes selected for further study, tiles on the chip were visually inspected for manufacturing defects, and expression was comfirmed by Northern blot analysis.
RNA Isolation, Granulosa Cell Culture, and Northern Blot Analysis
Mural granulosa cells were isolated from large antral follicles of CD1 mice treated with PMSG for 48 h and placed into culture. The experimental groups were freshly isolated granulosa cells (time 0), cells cultured for 5 h in presence of CHO cell conditioned media containing 50 ng/ml recombinant GDF-9, and cell cultured for the same amount of time with CHO cell conditioned media lacking GDF-9. Total RNA was isolated and used for oligonucleotide array hybridization. RNA isolation and Northern blot analysis were performed as previously described (6). The recombinant GDF-9 medium and the CHO control medium lacking GDF-9 were produced under the same conditions as described previously in the presence of heparin (6). Similar induction of Ptx3 and Cox2 gene expression is observed in the presence of GDF-9containing conditioned medium containing 1% FCS and the absence of heparin (Jorgez, C., and M. M. Matzuk, unpublished data).
ES Cell Manipulation and Southern Blot Analysis
About 15 kb of genomic sequence encompassing the mouse Ptx3 gene was isolated from a 129/SvEv genomic library. This genomic sequence was used to generate a targeting vector to mutate the Ptx3 gene in ES cells. The targeting vector contained 5.8 kb of genomic DNA upstream of Ptx3 exon 1, a selectable marker [the phosphoglycerate kinase promoter, hypoxanthine-guanine phosphoribosyl transferase minigene (PGKhprt) expression cassette], 2 kb of Ptx3 intron 2, and a negative selectable marker (the MC1tk expression cassette) (Fig. 3A
). The linearized vector was electroporated into the hprt-negative AB2.2 ES cell line, clones were selected in hypoxantine, aminopteridine, thimidine, and 1-(2'-deoxy-2'fluoro-ß-D-arabinofuranosyl)-5-iodouracil], and DNA from the clones analyzed by Southern blot as described (45, 46, 47). Targeted ES cell clones were injected into blastocysts, as described (46). Thirty percent of the ES cells were targeted at the Ptx3 locus, and three of these ES cell clones (Ptx3-134-A5, Ptx3-134-B2, Ptx3-134-D4) were injected into mouse blastocysts. Male chimeras derived from all three targeted ES cell clones transmitted the mutant Ptx3 allele to F1 offspring. F1 heterozygous mice were intercrossed to produce Ptx3-/- mice. Chimeras were mated to either C57BL6/J females to produce 129/SvEv/C57BL6/J hybrid mice or to 129/SvEv females to produce 129/SvEv inbred mice. Southern blot analysis was used for genotype analysis of all Ptx3 mutant offspring as shown (Fig. 3B
).
Superovulation and Isolation of Oocytes/Embryos
Twenty-two to 25-d-old Ptx3+/- and Ptx3-/- female mice were injected with PMSG (ip, 7.5 IU/mouse), and given hCG (ip, 5 IU/mouse) 48 h later. Mice were then either caged overnight or bred to C57/129 hybrid stud males. The following morning eggs and/or embryos were recovered in M2 medium, counted, and cultured in vitro for 24 h in M16 medium.
Statistical Analysis
When appropriate, statistical significance was calculated by one-way ANOVA. Data are expressed as average ± SEM.
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ACKNOWLEDGMENTS
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We thank Dr. Lei Chen and Mr. Julio Agno for technical help, Dr. T. Rajendra Kumar and Ms. Kathleen Burns for critical reading of the manuscript, and Ms. Shirley Baker for manuscript formatting.
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FOOTNOTES
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We would especially like to thank Dr. Lou DePaolo and the other members of NICHD who helped to support these experiments through NIH Grant HD-33438 after Tropical Storm Allison. During the initial stages of these studies, J.A.E. was a student in the Medical Scientist Training Program supported in part by NIH Grants GM-07330 and GM-08307.
Abbreviations: BMF-15, Bone morphogenetic protein-15; COX2, cyclooxygenase 2; ES, embryonic stem; GDF-9 or Gdf9, growth differentiation factor-9; Has2, hyaluronan synthase 2; HC, heavy chain; hCG, human CG; hprt, hypoxanthine-guanine phosphoribosyl transferase; ITI, inter-
-trypsin inhibitor; LPS, lipopolysaccharide; PGK, phosphoglycerate kinase; Ptx3 or PTX3, pentraxin 3; Tnfip6, TNF-induced protein 6.
Received for publication December 17, 2001.
Accepted for publication March 14, 2002.
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