Fibroblast growth factor-9, a local regulator of ovarian function.

Fibroblast growth factor 9 (FGF9) is widely expressed in embryos and fetuses and has been shown to be involved in male sex determination, testicular cord formation, and Sertoli cell differentiation. Given its male gender bias, the ovary has not been reported to express FGF9, nor has a role in ovarian function been explored. We report here that FGF9 mRNA and protein are present in the rat ovary and provide evidence that supports a role for FGF9 in ovarian progesterone production. FGF9 mRNA levels as determined by real-time PCR were high in 4-d-old rat ovaries, thereafter declining and stabilizing at levels approximately 30% of d 4 levels at d 12-25. Levels of FGF9 mRNA in the ovary were significantly higher than that present in adult testis, at all ages studied. The FGF9 receptors FGFR2 and FGFR3 mRNAs were present in postnatal and immature rat ovary and appeared to be constitutively expressed. FGF9 protein was localized to theca, stromal cells, and corpora lutea and FGFR2 and FGFR3 proteins to granulosa cells, theca cells, oocytes, and corpora lutea, by immunohistochemistry. Follicular differentiation induced by gonadotropin treatment reduced the expression of FGF9 mRNA by immature rat ovaries, whereas the estrogen-stimulated development of large preantral follicles had no significant effect. In vitro, FGF9 stimulated progesterone production by granulosa cells beyond that elicited by a maximally stimulating dose of FSH. When the granulosa cells were pretreated with FSH to induce LH receptors, FGF9 was found not to be as potent as LH in stimulating progesterone production, nor did it enhance LH-stimulated production. The combined treatments of FSH/FGF9 and FSH/LH, however, were most effective at stimulating progesterone production by these differentiated granulosa cells. Analyses of steroidogenic regulatory proteins indicate that steroidogenic acute regulatory protein and P450 side chain cleavage mRNA levels were enhanced by FGF9, providing a mechanism of action for the increased progesterone synthesis. In summary, the data are consistent with a paracrine role for FGF9 in the ovary.

Expression patterns of hyaluronan, hyaluronan synthases and hyaluronidases indicate a role for hyaluronan in the progression of endometrial cancer.

OBJECTIVE: The extracellular glycosaminoglycan hyaluronan (HA) and its degradative enzymes, hyaluronidases (Hyal), play important roles in tumor metastasis and angiogenesis. HA promotes tumor cell adhesion and migration, while its cleaved fragments stimulate angiogenesis. The aims of this study were to assess the levels of HA and how it might be regulated in endometrial cancer. METHODS: Endometrial carcinomas were grouped according to histologic grade (Grade 1-3). HA histochemistry utilized a biotinylated HA binding peptide (n = 15), while HA synthase (HAS) immunohistochemistry utilized an antibody recognizing HAS1, HAS2 HAS3 (n = 24). Real-time RT-PCR was used to determine the mRNA expression of Hyal 1, Hyal 2 (n = 13) and Hyal 3 (n = 11) in endometrial carcinomas. RESULTS: HA, its synthases and degradative enzymes were identified in endometrial carcinomas of all histologic grades. HA was predominantly localized to tumor-associated stroma. Semiquantitative analysis revealed increased HA levels with tumor grade, however, this increase only attained significance in Grade 2 carcinomas (P < 0.05). HA staining intensity scores were significantly associated with the presence of myometrial invasion (P < 0.05). Alternatively, HAS was predominantly localized in tumor epithelial cells, and its levels did not vary with tumor grade. Expression of Hyal 3 and Hyal 2 mRNA were >1000-fold and >30-fold greater respectively than that of Hyal 1 mRNA, the major Hyal expressed in other cancers. No Hyal type varied with tumor grade. CONCLUSION: This is the first study to demonstrate the cellular localization of HA and its synthases and that Hyal 3 mRNA is predominant in endometrial cancer. The results suggest a role for elevated HA in endometrial cancer progression.

The influence of ovarian steroids on ovine endometrial glycosaminoglycans.

The ovine endometrium is subjected to cyclic oscillations of estrogen and progesterone in preparation for implantation. One response to fluctuating hormonal levels is the degree of hydration of the tissue, suggesting cyclical alterations in glycosaminoglycan/proteoglycan content. The aim of the present study was to quantitate and characterize glycosaminoglycans in the ovine endometrium during estrogen and progesterone dominant stages. Endogenous endometrial glycosaminoglycan content was determined by chemical analysis and characterized by enzyme specific or chemical degradation. [(35)S]-sulphate and [(3)H]-glucosamine labeled proteoglycans/glycosaminoglycans were extracted by cell lysis or with 4M guanidine-HCl. Extracts were purified by anion exchange and gel chromatography and characterized as above. Estrogen and progesterone dominant endometrium contained 3.2 +/- 0.1 and 2.1 +/- 0.1 mg endogenous glycosaminoglycan/g dehydrated tissue, respectively. Characterization of endogenous glycosaminoglycan showed chondroitin sulphate and hyaluronan contributing over 80%. The major difference between hormonal dominant tissue was a higher estrogenic hyaluronan percentage and a higher progestational keratan sulphate percentage (p < 0.001). Estrogen dominant tissue incorporated 1.6-1.9 fold more radiolabeled proteoglycans/glycosaminoglycans (p < 0.001). Analysis of newly synthesized proteoglycans/glycosaminoglycans revealed a heparan/chondroitin sulphate ratio of 1:2.2-2.5. Keratan sulphate was not detected. Estrogenic hyaluronan was 1.6 fold greater in [(3)H]-labeled tissue. Analysis of labeled proteoglycans/glycosaminoglycans revealed two size classes with apparent molecular weights >2.0 x 10(6) and 0.8-1.1 x 10(5) and a charge class eluting between 0.1-0.5 M NaCl. The greater glycosaminoglycan content (particularly hyaluronan) and synthesis in estrogen dominant tissue supports a role for steroid hormones in endometrial glycosaminoglycan/proteoglycan regulation and consequent tissue hydration. It also suggests a role for these macromolecules in endometrial function and possibly the implantation process.