Recombination and deletion of the Ctgf conditional allele in the uterus and ovary. A and B, PCR analyses for Ctgf cKO genotyping. Representative PCR images are shown. Flox/− and Flox/+ are abbreviated as F/− and F/+, respectively. C and D, Recombination of Ctgf-flox alleles in the genomic DNA from granulosa cells. E and F, Ctgf transcript levels measured in granulosa cells by real-time qPCR in WT (n = 3), control (n = 3), and both Ctgf cKO female mice (n = 3 for each genotype). Levels of Ctgf relative quantity (RQ) are shown relative to the WT. Ctgf expression is efficiently ablated in the granulosa cells of Ctgf cKO female mice. The data are shown as means ± sem. WT values were set to equal 1. *, P < 0.05; ***, P < 0.001 (vs. WT female mice). G, Representative Western blots of total CTGF protein levels in uterine tissues and granulosa cells from control and both Ctgf cKO female mice. Compared with WT and control female mice, CTGF was markedly lower or undetectable in both Ctgf cKO female mice.

Morphological comparison of ovaries from control and both Ctgf cKO female mice. A–C, Comparison of ovarian histology from sexually mature (age 4–5 months) control and both Ctgf cKO female mice shows that Ctgf-mutant ovaries contain follicles at all stages of folliculogenesis as well as corpora lutea. Despite these observations, it is noted that there are reduced numbers of preantral and antral follicles, and the trend toward increased numbers of atretic preantral and atretic antral follicles in Ctgf cKO (Amhr2^cre/+) ovaries. In addition, high numbers of functional corpora lutea are shown in both Ctgf cKO female mice ovaries. D–F, Immunohistochemistry for 3ß-HSD showing functional corpora lutea in both Ctgf cKO female mice ovaries. An asterisk marks the corpus luteum. G, Statistical analysis of the numbers of the follicle compartments in control and Ctgf cKO (Amhr2^cre/+) female mice (n = 6 for each genotype). H, Statistical analysis of the numbers of the follicle populations in control and Ctgf cKO (Pgr^cre/+) female mice (n = 6 for each genotype). Statistical significance was determined by using Student's unpaired and two-tailed t test. Representative sections are shown. P, Primordial and primary follicle; PF, preantral follicle; APF, atretic primordial, primary, and preantral follicle; A, antral follicle; AF, atretic antral follicle; CL, corpus luteum. The data are shown as means ± sem. *, P < 0.05 (vs. control female mice). All bars correspond to 500 μm.

Impaired ovulation, but not cumulus expansion, in Ctgf cKO female mice. A and B, Ovaries from both Ctgf cKO female mice have large lutenized follicles with trapped oocytes. The bars in panels A and B correspond to 150 μm. C, Immature (3 wk) and mature (age 4 months) female mice of control (immature, n = 6; mature, n = 9), Ctgf cKO (Amhr2^cre/+) (immature; n = 5; mature, n = 5), and Ctgf cKO (Pgr^cre/+) (immature, n = 9; mature, n = 7) were subjected to a superovulation regimen. Oocyte/cumulus masses were then surgically extracted from their oviducts, and oocytes were counted after hyaluronidase digestion for 10 min. The numbers of ovulated oocytes was markedly lower in the independent samples from both Ctgf cKO female mice vs. control female mice. The data are shown as means ± sem. *, P < 0.05; **, P < 0.01 (vs. control female mice). D–F, Control (n = 3) and both Ctgf cKO female mice (n = 3 for each genotype) were treated with PMSG/hCG for in vivo cumulus expansion analysis. D, Note the outward radiation pattern of the expanded cumulus cells from the oocytes illustrated. E and F, The cumulus cells of both Ctgf cKO female mice undergo normal expansion in response to PMSG/hCG treatment. G–L, Control and both Ctgf cKO female mice were treated with PMSG alone, or followed by adding EGF, for in vitro cumulus expansion analyses. G–I, COC from control and both Ctgf cKO female mice cultured in the absence of EGF are illustrated. J, In vitro expansion of cumulus cells from control female mice stimulated by EGF (10 ng/ml). Note the expansion pattern of cumulus cells outward from the oocytes in the COC culture. K and L, Cultured COC from both Ctgf cKO female mice show normal expansion in response to EGF (10 ng/ml). The bars in panels D–F correspond to 150 μm. The bars in panels G–L correspond to 100 μm. 3W, 3-wk; 4M, 4-month.

Ctgf Expression and regulation of Adamts1 during ovulatory period. Real-time qPCR analysis of granulosa cell mRNA from 6- to 8-month-old adult control and both Ctgf cKO female mice stimulated with hCG for 11 h after PMSG priming for 46 h. Granulosa cells were collected from five independent control and cKO ovaries. Total RNA was isolated and converted to cDNA. Adamts1 (A and E), Edn2 (B and F), Il6 (C and G), and Prkg2 (D and H) mRNA levels were analyzed. Adamts1 is lower in Ctgf cKO (Amhr2^cre/+) and Ctgf cKO (Pgr^cre/+) granulosa cells than control. There are no differences in other genes regulated by progesterone receptor. The control values were set to equal 1. **, P < 0.01; ***, P < 0.001 (vs. control female mice). RQ, Relative quantity.

A model of PGR-regulated CTGF signaling during ovulation. Ovulation is initiated by the surge of LH, which acts via its receptor on granulosa cells of preovulatory follicles to stimulate the expression of PGR in these cells. PGR induces expression of CTGF, PPARγ, and other PGR-regulated genes. Activated PPARγ induces the expression of a subset of PGR-target genes including Edn2, Il6, and Prkg2. CTGF mediates its effects, at least partially, by inducing the expression of ADAMTS1, which catalyzes the cleavage of versican to an active form. Activated versican participates in the remodeling of the preovulatory follicle wall, ultimately leading to ovulation. LHR, LH receptor.

Fertility reduction in Ctgf cKO female mice. Known fertile males were bred with 6-wk-old female littermates for 6 months. A, Litter sizes of Ctgf cKO (Amhr2^cre/+) female mice (n = 10) and Ctgf cKO (Pgr^cre/+) female mice (n = 10) vs. control female mice (n = 10). The litter sizes of both Ctgf cKO female mice were reduced compared with control female mice. B, Pups/month in Ctgf cKO (Amhr2^cre/+) female mice (n = 10) and Ctgf cKO (Pgr^cre/+) female mice (n = 10) vs. control female mice (n = 10). The pups/month of both Ctgf cKO female mice were also reduced compared with control female mice. C, A time course of the litter sizes in Ctgf cKO (Amhr2^cre/+) female mice (n = 10) and Ctgf cKO (Pgr^cre/+) female mice (n = 10) vs. control female mice (n = 10) during a 6-month testing period. Both Ctgf cKO female mice became subfertile after 1 month of breeding. Ctgf cKO (Pgr^cre/+) female mice were more severe subfertile than Ctgf cKO (Amhr2^cre/+) female mice. D, Total accumulated pups at each month of breeding vs. control female mice during a 6-month testing period. The control female mice (n = 10) have a uniform accumulation of pups at each month, whereas Ctgf cKO (Amhr2^cre/+) female mice (n = 10) and Ctgf cKO (Pgr^cre/+) female mice (n = 10) have fewer pups. The data are shown as means ± sem. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. control female mice).

Increase of apoptotic granulosa cells in Ctgf cKO (Amhr2^cre/+) ovaries. A TUNEL staining of 4- to 5-month-old ovaries from control (D) and Ctgf cKO (Amhr2^cre/+) (A) female mice reveal a significant increase with apoptotic granulosa cells (green/yellow). Nuclei are counterstained with propidium iodide (red). B and C, High magnification of TUNEL-positive follicles in the Ctgf cKO (Amhr2^cre/+) ovary. E, High magnification of TUNEL-negative follicles in the control ovary. F, TUNEL-negative control. Note that granulosa cells are TUNEL positive, whereas oocytes are negative. G, The proportion of TUNEL-positive follicles was quantified and found to be significantly higher in Ctgf cKO (Amhr2^cre/+) ovaries than control ovaries. H, Caspase-3 transcript levels measured in granulosa cells by real-time qPCR in control (n = 5) and Ctgf cKO female mice (n = 5). Caspase-3 mRNA expression is markedly higher in granulosa cells from Ctgf cKO (Amhr2^cre/+) female mice relative to control female mice. The data are shown as means ± sem. The control values were set to equal 1. ***, P < 0.05 (vs. control female mice). In panels A and D, bars correspond to 250 μm. The bars in panels B, C, E, and F correspond to 150 μm.

Gene-expression changes in granulosa cells from adult control and Ctgf cKO (Amhr2^cre/+) ovaries after PMSG stimulation. Adult control and Ctgf cKO (Amhr2^cre/+) female mice (age 6–8 months) were stimulated with PMSG for 46 h. Granulosa cells were then collected from five independent control and cKO ovaries. Total RNA was isolated and converted to cDNA. Real-time qPCR analysis of granulosa cell mRNA was performed using gene-specific primers. The data are shown as means ± sem. Significant changes in gene expression are seen for Ccnd1 (A), Lhcgr (B), Cyp17a1 (C), Hsd3b (D), Hsd17b7 (E), Ptgfr (F), Sfrp4 (G), Kitl (H), and Myc (I). Other genes examined are shown in Table 4. The control values were set to equal 1. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. control female mice). RQ, Relative quantity.

Time-dependent changes of Ctgf and Adamts1 mRNA expression in granulosa cells of Ctgf cKO and Pgr mutant mice during the preovulatory period. Real-time qPCR analysis of granulosa cell mRNA from 6- to 8-month-old adult control and Ctgf cKO (Pgr^cre/+) female mice and from 3-wk-old Pgr^+/− and Pgr^−/− female mice treated with PMSG for 46 h before the stimulation of ovulation by hCG. Granulosa cells were collected at 0 h (only 46 h PMSG), 6 h, and 11 h after administration of hCG from ovaries of five independent mice. Total RNA was isolated and converted to cDNA. Ctgf, Adamts1, and other progesterone-regulated genes-mRNA levels were analyzed over time. A, Ctgf is induced at 6 h after hCG stimulation, followed by a continuing declines at 6 h and 11 h after hCG stimulation in preovulatory granulosa cells from control mice. In contrast, the expression of Ctgf is significantly down-regulated in Ctgf cKO (Pgr^cre/+) female mice compared with the control expression. B, Adamts1 is induced in a time-dependent manner after hCG stimulation in preovulatory granulosa cells from control mice. However, the expression of Adamts1 is significantly down-regulated in Ctgf cKO (Pgr^cre/+) female mice, according with the reduction of Ctgf expression. C–E, The expression of Edn2, Il6, and Prkg2 was induced after hCG stimulation and unchanged between control and Ctgf cKO (Pgr^cre/+) female mice. The control values were set to equal 1. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. control female mice). F, PGR regulates the induction of Ctgf mRNA during ovulation. Pgr^+/− and Pgr^{−/ −} female mice (age 3 wk) were treated with PMSG for 46 h before the stimulation of ovulation by hCG. Granulosa cells were collected at 0 h (only 46 h PMSG) and 6 h after administration of hCG from Pgr^+/− and Pgr^−/− ovaries (n = 3–5). Total RNA was isolated and real-time PCR was used to measure Ctgf mRNA levels. The expression of Ctgf mRNA in Pgr^+/− granulosa cells at 0 h was set to equal 1. **, P < 0.01 (vs. Pgr^+/− female mice). RQ, relative quantity.