The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor and was shown to be allelic with the white-spotting locus (W) of the mouse. Mutations at the W locus have pleiotropic effects on the development of hematopoietic stem cells, melanoblasts, and primordial germ cells. In order to elucidate the role of c-kit protein in gametogenesis and oocyte maturation, we have examined immunohistochemically the expression of c-kit in the ovaries of mice at late fetal and postnatal stages, and in early embryos. By the avidin-biotin-peroxidase (ABC) method using rat anti-mouse c-kit monoclonal antibody, the c-kit protein was detected in ovaries after the time of birth, but not before. The expression of c-kit was observed mainly on the surface of oocytes, but not in granulosa cells nor in interstitial regions. Oocytes of primordial to fully grown Graafian follicles showed the c-kit protein. When ovulation was induced by hCG, the expression of c-kit in ovulated unfertilized oocytes was weaker than in oocytes of Graafian follicles. In 1-cell embryos the c-kit protein was still observed, but with cell division its expression further decreased, and it was not detected in embryos of 4-cell, 8-cell, and morula stages. In summary, the highest expression of c-kit was observed on the surface of oocytes arrested in the diplotene stage of meiotic prophase. With ovulation and the resumption of meiotic maturation, its expression declined. These results suggest that the c-kit protein may play some role in meiotic arrest, oocyte growth, and oocyte maturation.

Leukemia inhibitory factor (LIF), a cytokine that induces macrophage differentiation in the murine M1 myeloid leukemia cell line, is essential for blastocyst implantation in mice. However, its expression and the role it plays in the human uterus are unknown. To clarify these issues, we examined LIF gene expression in the human uterus by Northern blot hybridization and by a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. Analysis of LIF mRNA showed two hybridization bands, with estimated mRNA sizes of about 4.0-kb pairs and 1.8-kb pairs. LIF mRNA was detected at high levels in endometrial tissue and decidua, but at low levels in the chorionic villus in first trimester and term placenta. In the secretory phase, the endometrial tissue showed higher LIF expression than in the proliferative phase (9.5-fold; p < 0.01). The endometrial tissues were separated into a stroma-enriched fraction (SF) and an epithelium-enriched fraction (EF), and the LIF mRNA levels in each fraction were examined by quantitative RT-PCR. These levels were higher in the EF than in the SF (3.3-fold; p < 0.05). These findings suggest that, in humans, LIF plays a role in uterine function during the menstrual cycle, as well as during pregnancy.

The immunohistochemical localization of the androgen receptor in the human endometrium at various stages of the menstrual cycle and post-menopausal period, in decidua and placenta of early pregnancy, and in several pathological conditions of the endometrium has been investigated. At any phase of the menstrual cycle, both endometrial glandular cells and endometrial stromal cells showed positive nuclear staining. Endometrial stromal cells of the functional layer showed stronger staining than those of the basal layer, but endometrial glandular cells of both layers showed the same staining intensity. There was little staining in myometrium. Even after menopause, endometrial glandular and stromal cells showed the same staining pattern as the basal layer of pre-menopausal endometrium and the staining intensity of endometrial stromal cells was weak. In decidua and placenta of early pregnancy, decidual and trophoblastic cells showed positive staining and there was no staining in the stromal cells of placenta. The expression of the androgen receptor was also detected in adenomyosis, endometriosis and endometrial carcinoma. Although the proliferation and differentiation of endometrium are mediated mainly by oestrogen and progesterone receptors, the androgen receptor may play some role in modulating these changes. These results suggest that it may be involved in both physiological and pathological changes of the endometrium.

In order to elucidate the role of androgen receptors (AR) in human ovaries, we examined their immunohistochemical localization, in comparison with oestrogen receptors (ER) and progesterone receptors (PR), at various stages of the menstrual cycle and follicular development. Primordial and primary follicles did not express AR. In granulosa and thecal cells of secondary follicles there was weak nuclear staining for AR. Granulosa cells of dominant follicles showed moderate nuclear staining for AR, which was stronger than that in thecal cells. In the luteal phase, the staining intensity for AR was strongest in the early luteal phase just after ovulation and declined gradually thereafter. Thecal cells of atretic follicles showed moderate nuclear staining for AR, which was a little stronger than that in dominant follicles. There was weak nuclear staining for AR in stromal cells surrounding follicles. Though there was variation in the staining intensity, AR were present at almost all stages of the menstrual cycle. There is a possibility that androgens, mediated by AR, may play an essential role in follicular growth and maturation, atresia and luteinization as autocrine or paracrine agents.

Although regioselective removal of 6-O-sulfate groups of heparin has been undertaken by several researchers, complete 6-O-desulfation with little side reaction has not been attained successfully. In this work, a modified method with a certain silylating reagent, N-methyl-N-(trimethylsilyl)trifluoroacetamide, has been established to produce completely 6-O-desulfated heparin with few other chemical changes. The degrees of 6-O-desulfation were estimated by means of chemical disaccharide analyses and/or 13 C NMR spectra. Although the completely 6-O-desulfated heparin lost about 20% of 2-O-sulfate groups, any other chemical changes and depolymerization were not detected. The completely 6-O-desulfated heparin displayed strong inhibition of COS-1 cell adhesion to basic fibroblast growth factor (bFGF)-coated well in a dose-dependent manner, as was clarified by the competitive cell-adhesion assay. Furthermore, the completely 6-O-desulfated heparin was shown to promote in vitro A31 fibroblast proliferation in a dose-dependent manner in the presence of bFGF. These results suggest that signal transduction through bFGF/bFGF receptor in A31 cells occurs in the absence of 6-O-sulfate groups in heparin. The involvement of 6-O-sulfate group(s) of heparin/heparan sulfate in the promotion of bFGF mitogenic activity was reported by several groups. This discrepancy between our results and those of other groups would be due to the differences in molecular size of heparin/heparan sulfate derivatives and/or cell species used for the assay. Heparin and heparan sulfate (HS)1 are known as glycosaminoglycan (GAG) components of extracellular matrix-forming connective tissues of animals. The former GAG, heparin, is exclusively distributed in mast cells, and the latter GAG, HS, is widely distributed in animal tissues. With the accumulation of the information concerning biological roles of heparin and HS, it has been revealed that their biological functions mostly depend upon interaction between polysaccharides and physiologically active molecules, although the biological roles of heparin and HS are highly diverged. For instance, they interact with lipoprotein lipase (1, 2), anti-thrombin III (3, 4), basic fibroblast growth factor (bFGF) (5-8), etc. Furthermore, minimum structures of heparin and/or HS necessary for binding with antithrombin III and/or bFGF have been determined (9, 12). Chemical modification of heparin has been undertaken by several researchers, focusing on the elucidation of the mechanism underlying interaction between heparin and the physiologically active molecules as described above. Specific removal of major sulfate groups of heparin such as 2-O-sulfate, 6-Osulfate, and N-sulfate groups would be useful in order to clarify the backbone structures of oligosaccharides bearing specific array of sulfate groups responsible for the interactions with physiologically active molecules. For instance, selective removal of 6-O-sulfate groups from glucosamine residues of heparin is of great importance in order to evaluate the involvemen...