Previous studies from our laboratory established that large M(r) mucin glycoproteins are major apically disposed components of mouse uterine epithelial cells in vitro. The present studies demonstrate that Muc-1 represents one of the apically disposed mucin glycoproteins of mouse uterine epithelia, and that Muc-1 protein and messenger RNA (mRNA) expression are regulated in the periimplantation mouse uterus by ovarian steroids. Muc-1 expression is exclusive to the epithelial cells of the uterus under all conditions examined. Muc-1 expression is high in the proestrous and estrous stages and decreases during diestrous. Both Muc-1 protein and mRNA decline to barely detectable levels by day 4 of pregnancy, i.e. before the time of blastocyst attachment. In contrast, Muc-1 expression in the cervix and vagina is maintained during this same period. Delayed implantation was established in pregnant mice by ovariectomy and maintained by the administration of exogenous progesterone (P). Initiation of implantation was triggered by coinjection of P-maintained mice with a nidatory dose of 17 beta-estradiol (E2). Muc-1 levels in the uterine epithelia of P-maintained mice declined to low levels similar to those observed on day 4 of normal pregnancy. Coinjection of E2 did not alter Muc-1 expression, suggesting that down-regulation of Muc-1 is a P-dominated event. This was confirmed in ovariectomized nonpregnant mice, which displayed stimulation of Muc-1 expression after 6 h of E2 injection. E2-Stimulated Muc-1 expression was inhibited by the pure antiestrogen, ICI 164,384. Although P alone had no effect on Muc-1 expression, it antagonized the action of E2. Injection of pregnant mice with the antiprogestin, RU486, a known implantation inhibitor, on day 3 of pregnancy restored high level expression of Muc-1 mRNA on day 4, indicating that down-regulation of Muc-1 is P receptor mediated. Collectively, these data indicate that Muc-1 expression in mouse uterine epithelium is strongly influenced by ovarian steroids. It is suggested that the loss of Muc-1 contributes to generation of a receptive uterine state.

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Connective tissue growth factor (CTGF) is a growth and chemotactic factor for fibroblasts encoded by an immediate early gene that is transcriptionally activated by transforming growth factor ss. Although the primary translational product of the pig CTGF gene is predicted to be of approximate Mr 38 000, pig uterine luminal flushings (ULF) contained 10- to 20-kDa CTGF proteins that were heparin-binding and mitogenic, whereas 38-kDa CTGF was not apparent. The N-termini of two microheterogeneous forms of 16-kDa CTGF, as well as 18-kDa and 20-kDa forms of CTGF, commenced at, respectively, Cys199, Ala197, Asp186, and Asp186 and did not correspond to intron-exon boundaries in the CTGF gene. Northern blotting revealed a single porcine (p) CTGF transcript of 2.4 kilobases in endometrium from Day 10 to 16 cycling or pregnant pigs. Ten- to twenty-kilodalton pCTGF proteins in ULF were stable for 48 h at 37 degreesC whereas native 38-kDa pCTGF was degraded within 10 min under the same conditions. CTGF-degrading activity in pig ULF was heat-sensitive and concentration- and time-dependent. Ten- to twenty-kilodalton CTGF levels in ULF peaked on Day 16 of the cycle and on Day 12 of pregnancy and were highly correlated with the levels of proteolytic activity for 38-kDa CTGF. Collectively these data suggest that bioactive 10- to 20-kDa CTGF proteins are generated in utero through limited proteolysis of the 38-kDa CTGF primary translational product.

Previous studies from our laboratory established that large M(r) mucin glycoproteins are major apically disposed components of mouse uterine epithelial cells in vitro. The present studies demonstrate that Muc-1 represents one of the apically disposed mucin glycoproteins of mouse uterine epithelia, and that Muc-1 protein and messenger RNA (mRNA) expression are regulated in the periimplantation mouse uterus by ovarian steroids. Muc-1 expression is exclusive to the epithelial cells of the uterus under all conditions examined. Muc-1 expression is high in the proestrous and estrous stages and decreases during diestrous. Both Muc-1 protein and mRNA decline to barely detectable levels by day 4 of pregnancy, i.e. before the time of blastocyst attachment. In contrast, Muc-1 expression in the cervix and vagina is maintained during this same period. Delayed implantation was established in pregnant mice by ovariectomy and maintained by the administration of exogenous progesterone (P). Initiation of implantation was triggered by coinjection of P-maintained mice with a nidatory dose of 17 beta-estradiol (E2). Muc-1 levels in the uterine epithelia of P-maintained mice declined to low levels similar to those observed on day 4 of normal pregnancy. Coinjection of E2 did not alter Muc-1 expression, suggesting that down-regulation of Muc-1 is a P-dominated event. This was confirmed in ovariectomized nonpregnant mice, which displayed stimulation of Muc-1 expression after 6 h of E2 injection. E2-Stimulated Muc-1 expression was inhibited by the pure antiestrogen, ICI 164,384. Although P alone had no effect on Muc-1 expression, it antagonized the action of E2. Injection of pregnant mice with the antiprogestin, RU486, a known implantation inhibitor, on day 3 of pregnancy restored high level expression of Muc-1 mRNA on day 4, indicating that down-regulation of Muc-1 is P receptor mediated. Collectively, these data indicate that Muc-1 expression in mouse uterine epithelium is strongly influenced by ovarian steroids. It is suggested that the loss of Muc-1 contributes to generation of a receptive uterine state.

A role for connective tissue growth factor (CTGF) in reproductive function has been suggested from recent studies in the pig. To extend these findings, we have analyzed the immunohistochemical localization of CTGF during the estrous cycle and early pregnancy in mice. During the diestrous and early proestrous stages, CTGF was localized at high levels to both luminal and glandular uterine epithelial cells and at much lower levels in the stroma or myometrium. Epithelial expression of CTGF was considerably reduced at estrus. On Days 1.5-3.5 of pregnancy, CTGF was localized mainly to the uterine epithelial cells, which showed a substantially reduced level of CTGF on Day 4.5. On Days 5.5 and 6.5, CTGF was present at high levels in uterine decidual cells. CTGF was detected in the trophectoderm and inner cell mass of the preimplantation embryo on Day 4.5 and became preferentially localized to embryonic endoderm and mesoderm on Days 5.5-6.5. Multiple mass forms of CTGF (Mr 14 000-38 000) were present in endometrial extracts and uterine luminal flushings. Collectively, these data support a role for CTGF in uterine cell growth, migration, adhesion, and extracellular matrix production during the estrous cycle and early pregnancy, as well as in early development of the embryo.

Connective tissue growth factor (CTGF) is a 38 kDa modular protein that is involved in processes such as cell proliferation, survival, migration, adhesion and extracellular matrix production. Target cells for CTGF include fibroblasts, smooth muscle cells and endothelial cells. Using a specific peptide antibody, CTGF was localized in tissue sections obtained from mouse embryos between days 7.5 and 14.5 of gestation. On day 7.5, CTGF was present at high levels in the embryonic ectoderm, mesoderm, and chorion, with weaker levels in the squamous endoderm. No CTGF was detected in Reichert's membrane, parietal endoderm, and visceral endoderm. Decidual cells of the maternal uterus stained strongly for CTGF. There was no specific staining for CTGF on day 11.5 but by day 13.5, the protein was detectable in a limited number of structures, most notably the liver, thymus, lung, and intestine. By day 14.5, staining for CTGF had become extensive and was present at high or moderate levels in the thymus, aorta, trachea, liver, lung, adrenal gland, kidney, iliac sinus, olfactory region, tongue, pharynx, esophagus, thyroglossal duct, choroid plexi, Rathke's pouch, stomach, pancreas, and midgut. Relatively weak staining was present in the heart and dorsal root ganglia. There was no staining in the vitelline duct or in the outer cortical or medullary regions of the brain. Among specific cell types, CTGF was localized at high levels in secretory and absorptive epithelial cells and liver parenchyma cells, at moderate levels in vascular endothelial cells and myoblasts and at low levels in mesenchymal and connective tissue cells. These data show that various tissues and organ systems produce CTGF in a specific temporo-spatial pattern during embryogenesis and support a role for CTGF in cellular differentiation and development during prenatal life.

Connective tissue growth factor (CTGF) is a 38kDa mitogen and chemotactic factor for fibroblasts that is transcriptionally activated by serum or transforming growth factor-beta and may play a role in wound healing and various skin diseases. In these studies, pig endometrium was shown to contain a single CTGF transcript of 2.4kb and to produce a 38kDa CTGF-immunoreactive protein. Cloning and sequencing of a 1.5kb pig uterine CTGF cDNA revealed that the predicted pCTGF primary translation product displayed 92% identity to human CTGF and 93% identity to mouse CTGF. The pCTGF cDNA encoded a 26 amino acid signal peptide followed by a 323-residue sequence containing 38 highly conserved cysteine residues. In common with mouse and human CTGF proteins, pCTGF is predicted to resemble a multi-functional mosaic protein that contains four distinct modules.

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