An essential role for C/EBPbeta in female reproduction

Abstract

A large number of intercellular signaling molecules have been identified that orchestrate female reproductive physiology. However, with the exception of steroid hormone receptors, little information exists about the transcriptional regulators that mediate cellular responses to these signals. The transcription factor C/EBP beta (CCAAT/enhancer-binding protein beta) is expressed in ovaries and testes, as well as many other tissues of adult mice. Here we show that mice carrying a targeted deletion of the C/EBP beta gene exhibit reproductive defects. Although these animals develop normally and males are fertile, adult females are sterile. Transplantation of normal ovaries into mutant females restored fertility, thus localizing the primary reproductive defect to the ovary proper. In normal ovaries, C/EBP beta mRNA is specifically induced by luteinizing hormone (LH/hCG) in the granulosa layer of preovulatory antral follicles. C/EBP beta-deficient ovaries lack corpora lutea and fail to down-regulate expression of the prostaglandin endoperoxidase synthase 2 and P450 aromatase genes in response to gonadotropins. These findings demonstrate that C/EBP beta is essential for periovulatory granulosa cell differentiation in response to LH. C/EBP beta is thus established as a critical downstream target of G-protein-coupled LH receptor signaling and one of the first transcription factors, other than steroid hormone receptors, known to be required for ovarian follicle development in vivo.

Figure 1

Targeted mutation of the C/EBPβ gene in mice. (A) Diagram of the targeting vector, wild-type allele, and mutated allele. The entire coding region and parts of the promoter were replaced by a neomycin resistance gene. Homologous recombination at the 5′ side of the gene was screened by a probe (P5′) that detects the conversion of a 4.5-kb EcoRI fragment into 5.0 kb and, at the 3′ side, a probe (P3′) that detects the alteration of a 7.0-kb BamHI fragment into a 5.5-kb fragment. (B) BamHI; (X) XbaI; (N) NotI; (Nh) NheI; (R) EcoRI. (B) Southern blot analysis of BamHI digested genomic mouse DNA probed with P3′ (see A). The 7.0-kb fragment diagnostic of the wild-type allele (+/+) and the 5.5-kb fragment diagnostic of the mutated allele (−/−) are indicated. (C) Western blot analysis of liver nuclear extracts from heterozygous or homozygous mutant animals probed with a C/EBPβ-specific antiserum (arrow). The asterisk identifies a nonspecific cross-reactivity of the antiserum.

Figure 2

Seminal vaginal plug frequency in 5 C/EBPβ-deficient females. Mutant females were mated to males of proven fertility and analyzed for the presence of seminal vaginal plugs every morning for 31 days. (−) No plug; (+) plugged; (·) no record.

Figure 3

Morphology of normal and C/EBPβ-deficient ovaries from untreated animals or after treatment with gonadotropins in vivo. Photomicrographs (40×) of sections of hematoxylin/eosin-stained ovaries from heterozygous (A) and homozygous (B) mutant ovaries of 2- to 3-month old mice, and of heterozygous (C) and mutant (D) ovaries from mice treated for 2 days with PMSG and subsequently for 14 hr with hCG (see Table 2). The arrow indicates an oocyte that has failed to be expelled. (CL) Corpus luteum.

Figure 4

Morphology of transplanted ovaries. Photomicrographs (50×) of sections of hematoxylin/eosin-stained ovaries from a heterozygous ovary (A) 3 months after transplantation into a homozygous mutant host (see Table 3A) and a mutant ovary (B) 2 months after transplantation into a wild-type host animal (see Table 3B). (CL) Corpus luteum.

Figure 5

C/EBPβ expression is induced in granulosa cells 4–7 hr after hCG treatment. Bright-field (A,C), dark-field (B,D), and bright- plus dark-field (E,F) photomicrographs of sections from normal adult ovaries after in situ hybridization to a C/EBPβ-specific antisense cRNA probe. The animals were treated for 2 days with PMSG and then with hCG for 4 hr (A,B) and 7 hr (C,D), respectively. E and F show high magnification of the two follicles shown at the bottom of C and D; silver grains (predominantly over granulosa cells) are in pink. The scale bar in E represents 10 μm. (A,B) C/EBPβ^+/− ovaries; (C–F) C/EBPβ+/+ ovaries. (CL) Corpus luteum; (t) theca layer. Magnifications, 62.5× (A–D); 100× (E); 320× (F).

Figure 6

Gene expression analysis of normal and mutant ovaries after gonadotropin treatment. The animals were treated for 2 days with PMSG and with hCG for 4 hr (normal, C/EBPβ^+/−) and 7 hr (normal, C/EBPβ^+/+), as indicated. One ovary from each animal was fixed for histological analysis (Fig. 5); total RNA was isolated from the second ovary and 10 μg was analyzed by Northern blotting with sequential hybridization to radioactively labeled cDNA probes for the indicated genes.