Masahiro Tahara

RhoA/Rho-Kinase Cascade Is Involved in Oxytocin-Induced Rat Uterine Contraction

The RhoA/Rho-kinase cascade is involved in various cellular functions, including migration, proliferation, and smooth muscle contraction. We examined the potential role of this pathway in oxytocin-induced uterine contraction. The specific Rho-kinase inhibitor Y-27632 inhibited oxytocin-induced rat uterine contraction on d 21 of pregnancy in a concentration-dependent manner, whereas the extent of this inhibition was reduced in the nonpregnant uterus. Y-27632 had no effect on oxytocin-induced intracellular Ca 2 mobilization in myometrial cells. Immunoblot analysis showed that oxytocin increased the level of myosin light chain phosphorylation, and this increase was attenuated by Y-27632. Oxytocin increased the phosphorylation of myosin-binding subunit of myosin phosphatase, one of the major substrates of Rho-kinase, and this increase was reduced by Y-27632. The expression of Rhokinase protein was shown to increase in the uterus during pregnancy compared with the nonpregnant uterus, whereas the expression of RhoA protein remained at the same level during pregnancy. RT-PCR and Northern blot analysis showed that the expression of Rho-kinase was up-regulated at the transcriptional level during pregnancy. These results suggest that the RhoA/Rho-kinase pathway may have an important role in oxytocin-induced uterine contraction, and that up-regulation of Rho-kinase is involved in the mechanism underlying the increased contractility of the pregnant myometrium. (Endocrinology 143: 920 –929, 2002)

SNAP-25 is essential for cortical granule exocytosis in mouse eggs

Synaptosome-associated protein of 25 kDa (SNAP-25) has been shown to play an important role in Ca2+-dependent exocytosis in neurons and endocrine cells. During fertilization, sperm-egg fusion induces cytosolic Ca2+ mobilization and subsequently Ca2+-dependent cortical granule (CG) exocytosis in eggs. However, it is not yet clear whether SNAP-25 is involved in this process. In this study, we determined the expression and function of SNAP-25 in mouse eggs. mRNA and SNAP-25 were detected in metaphase II (MII) mouse eggs by RT-PCR and immunoblot analysis, respectively. Next, to determine the function of SNAP-25, we evaluated the change in CG exocytosis with a membrane dye, tetramethylammonium-1,6-diphenyl-1,3,5-hexatriene, after microinjection of a botulinum neurotoxin A (BoNT/A), which selectively cleaves SNAP-25 in MII eggs. Sperm-induced CG exocytosis was significantly inhibited in the BoNT/A-treated eggs. The inhibition was attenuated by coinjection of SNAP-25. These results suggest that SNAP-25 may be involved in Ca2+-dependent CG exocytosis during fertilization in mouse eggs.

Decrease in neonatal suckled milk volume in diabetic women

To clarify the influence of diseases with hormonal disturbance on puerperal lactation, we studied the amounts of milk obtained by suckling and manual extraction during the first 6 days after delivery and the rate of breast-feeding in 40 patients with diabetes mellitus, 40 with hyperthyroidism, 28 with hypothyroidism and 57 with ovulatory disturbance, and in 40 healthy women. The mean body weight of the patients with diabetes mellitus was 7 kg more than those in other groups, and delivery occurred one gestational week earlier than in other groups. The amounts of suckled milk produced by patients with diabetes mellitus and hypothyroidism were significantly (p less than 0.05) less than those in other groups; there was no significant difference in milk productions in the other groups. One month after delivery, 35.5% of the babies in the diabetic group were receiving complete breast-feeding, which was significantly (p less than 0.05) lower than the percentages (52-55%) in the other groups. These data suggest that neonatal suckled milk volume in diabetic women is decreased, and that hypothyroidism may influence the initiation of lactation even when the patients are euthyroid due to treatment.

Geranylgeranylacetone inhibits lysophosphatidic acid‐induced invasion of human ovarian carcinoma cells in vitro

Lysophosphatidic acid (LPA) induced a dose‐dependent increase of cancer cell invasion by promoting Rho/Rho‐associated kinase signaling. Prenylation of Rho is essential for regulating cell growth, motility, and invasion. Geranylgeranylacetone (GGA), an isoprenoid compound, is used clinically as an antiulcer drug. Recent findings suggested that GGA might inhibit the small GTPase activation by suppressing prenylation. The authors hypothesized that the anticancer effects of GGA result from the inhibition of Rho activation.

EXPRESSION OF RAB3A IN THE CORTICAL REGION IN MOUSE METAPHASE II EGGS

Rab3A, a member of the small GTP-binding protein superfamily, has been implicated in regulated exocytosis at presynapses. At fertilization, sperm-egg fusion induces cytosolic calcium mobilization and cortical granule (CG) exocytosis in the egg. However, it is not yet clear whether Rab3A is involved in this process. We previously reported that Rabphilin-3A functions in calcium-dependent CG exocytosis. Rabphilin-3A is known to bind with Rab3A, Rab3B, and Rab3C. In this study, we clarified which member of the Rab3 was expressed in mouse metaphase II eggs. Messenger RNA encoding Rab3A but not Rab3B or Rab3C, was detected in unfertilized metaphase II eggs by RT-PCR. Rab3A protein was also detected in unfertilized metaphase II eggs by immunoblot analysis. Next the expression and the localization of Rab3A in eggs at various stages of development were determined by immunofluorescence analysis using confocal laser scanning microscopy. Rab3A protein was specifically distributed in the cortical region in eggs from before fertilization to the two-cell stage. However, it was not detected at the three- or four-cell stage 40 hr after fertilization. These results suggest that Rab3A may function with Rabphilin-3A in CG exocytosis.

Roles of prostaglandins and intracellular free calcium mobilisation in epidermal growth factor-induced proliferation of human amnion cells

Objective To investigate the mechanisms which regulate the growth of human amnion cells.

Mechanism and Control of Mammalian Cortical Granule Exocytosis

ABSTRACT Polyspermy is penetration of the egg cytoplasm by more than a single spermatozoon, and in humans, polyspermy usually results in spontaneous abortion. Following sperm penetration in mammals, cortical granules (CGs), special organelles in eggs, release their contents into the perivitelline space. The CG exudates act on the zona pellucida, causing biochemical and structural changes that result in zona sperm receptor modification and zona hardening, and thus block polyspermic penetration. Significant advances have been made in elucidating signal molecules and signal transduction cascades that play important roles in the CG exocytosis and subsequent polyspermy block. Ca2+ oscillation is necessary and sufficient for CG exocytosis as well as for other events of egg activation. The Ca2+-dependent pathways and the proteins involved in membrane fusion may play pivotal roles in the regulation of CG exocytosis. Mammalian oocytes develop their ability to undergo CG exocytosis during maturation. This article reviews the signal molecules and signal transduction cascades involved in CG exocytosis.