Sachiko Saito

Production of activin-binding protein by rat granulosa cells in vitro.

We have developed an assay method for activin-binding protein, which exploits its high affinity for sulfated polysaccharides. We used this method to investigate the production of activin-binding protein by rat ovarian granulosa cells, in vitro. The production of activin-binding protein by granulosa cells was dependent on the cell density; the maximum was observed at 6 x 10(5) cells/ml. Follicle-stimulating hormone (FSH), but not luteinizing hormone (LH), enhanced production significantly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ligand blotting analyses of the activin-binding protein secreted by rat granulosa cells demonstrated it was the same protein molecule as that purified from rat ovaries. It is inferred from these results that the granulosa cell is a source of ovarian activin-binding protein and that its secretion is regulated by FSH.

Anti-activin A Antibody (IgY) Specifically Neutralizes Various Activin A Activities

Abstract Activin A (βAβA), originally isolated from ovarian follicular fluids as a follicule-stimulating hormone (FSH) secretion stimulator, has also been identified as an erythroid differentiation factor (EDF), a neuron survival factor and a mesoderm-inducing factor. Thus, activin A is a multifunctional factor, and further studies on its physiological function are important. However, it is very difficult to produce a specific antibody to neutralize the activity of activin A because of its highly conserved amino acid sequence across mammalian species. In this study, we succeeded in generating an antibody against activin A, which can neutralize several activities of activin A, such as the stimulation of FSH secretion from pituitary cells and the induction of the differentiation of erythrocytes in vitro. This antibody did not affect the activity of activin B (βBβB), which induces the differentiation of erythrocytes in vitro, and the activity of inhibin A (αβA), which inhibits FSH secretion from pituitary in vitro, but slightly neutralized that of activin AB (βAβB). Western blotting analysis showed that this antibody recognized both dimeric and monomeric forms of the βA subunit of activin and inhibin. These results suggest that this antibody recognizes the βA subunit of activin and specifically neutralizes the activity of a dimer of the βA subunit, activin A. Furthermore, by the addition of this antibody to the culture medium, the development of murine embryos was suppressed, suggesting that endogenous activin A plays an important role in murine development. These results indicate the usefulness of this antibody for studies of endogenous activin actions. [P.S.E.B.M. 1996, Vol 211]

Syntheses and biological evaluation of analogs of luteinizing hormone-releasing hormone (LH-RH) modified in position 2, 3, 4 or 5

Abstract Syntheses by the conventional methods as well as the chemical, physical and biological properties are described of the following analogs of the LH-releasing hormone (LH-RH): [Leu3]-LH-RH, [Phe3]-LH-RH, [Trp2] [His3]-LH-RH, Des-Trp3-LH-RH, Des-His2-[Phe5]-LH-RH, [Ala4]-LH-RH, [Phe5]-LH-RH and [Ala4] [Phe5]-LH-RH. In vivo assays showed that [Leu3]-LH-RH did not release LH in doses as high as 5 – 25 μg, having less than 0.0008% of LH-RH activity, while [Phe3]-LH-RH had 0.43% of the LH-RH activity of natural LH-RH. The LH-RH activities of [Trp2] [His3]-LH-RH, Des-Trp3-LH-RH and Des-His2-[Phe5]-LH-RH were extremely low. On the other hand, [Ala4]-LH-RH, [Phe5]-LH-RH and [Ala4] [Phe5]-LH-RH had significant LH-RH activity. The structure-activity relationship of LH-RH is discussed on the basis of these findings.

Expression of the signal transducing regions of CD4-like and lck genes in murine egg.

We previously demonstrated that the MHC class II molecule on murine sperm and CD4-like molecule on the egg vitelline membrane are involved in fertilization. In this study, the RNA transcripts in murine eggs corresponding to parts of the CD4 gene and lck gene in thymocytes were demonstrated by means of the reverse transcriptase (RT)/polymerase chain reaction (PCR) followed by the sequencing of the PCR products. The deduced sequences potentially encode the amino acid sequence of the transmembrane to the cytoplasmic domain of CD4 and that of the N-terminal domain of p56lck respectively. These findings indicate that a signal transducing complex similar to that in immune T cells is expressed at the transcriptional level in murine eggs.

Syntheses and LH- and FSH-RH activities of LH-RH analogs substituted at position 8

Abstract Syntheses by the conventional method are described of [Gln8]-LH-RH, [Leu8]-LH-RH and [Pro8] [Arg9]-LH-RH. These peptides were purified by column chromatography on CM-Sephadex and gelfiltration on Sephadex G-25 and proved to be homogeneous. The LH-RH and FSH-RH activities of these peptides were compared with those of natural LH-RH in vivo and in vitro . [Gln8]-LH-RH had significant LH- and FSH-RH activities, while [Leu8]-LH-RH and [Pro8] [Arg9]-LH-RH had lower activities.

Lacational Anovulation in Rats and Its Dependency on Progesterone

Abstract The relationship between prolactin (PRL) secretion and anovulation in lactating rats was studied. Normal lactating rats and lactating rats treated with antiserum against luteinizing hormone-releasing hormone at the time of postpartal ovulation were used. Normal lactating rats were treated with either a dopamine agonist (CB-154, 150 μg/rat) on Day 10 or 13, or pups removal on Day 7 or 10, and thereafter luteolysis and inhibition on PRL secretion were assessed. With the CB-154 treatment, the incidence of luteolysis increased as the lactational period advanced (42% vs 72%), whereas it decreased (73% vs 14%) with the pups removal. Thus, dopamine effectively inhibited PRL secretion during the later lactational stage, but could not do so during the earlier stage when there were mechanisms other than dopamine stimulating PRL secretion. Following luteal regression induced by CB-154, ovulation did not occur if the rats were treated with CB-154 on Day 10, whereas 50% of the rats ovulated within 4 days if treated on Day 13. Furthermore, in the lactating rats treated with anti-luteinizing hormone-releasing hormone serum during late pregnancy, ovulation was not observed until Day 10 of lactation. Since the serum progesterone levels were low in these rats due to the absence of ovulation and lactational corpora lutea, the blockade of ovulation was not due to elevated circulating progesterone during the early lactational period. The mechanism of ovulation blockade during lactation thus seems to shift from being progesterone independent to progesterone dependent at a similar period when the neuroendocrine control of PRL secretion shifts from dopamine independent to dependent.