Roscovitine in combination with calcium ionophore induces oocyte activation through reduction of M-phase promoting factor activity in mice.

The aim of the present study was to determine oocyte activation and change in M-phase promoting factor (MPF) activity induced by treatment with calcium ionophore and roscovitine in comparison with those induced by treatment with roscovitine alone and treatment with calcium ionophore and puromycin in mice. Freshly ovulated oocytes obtained from 6-8-week-old mice were divided into five groups (no activation treatment; 5 µM calcium ionophore A23187; 50 µM roscovitine; 5 µM calcium ionophore and 10 µg/ml puromycin; and 5 µM calcium ionophore and 50 µM roscovitine) and were incubated for 6 h. Oocyte activation, assessed by morphological changes, and changes in MPF activity in the five groups at 0, 2, 4 and 6 h of incubation were examined. Activated oocytes were defined as oocytes with at least one pronucleus. Oocytes treated with roscovitine alone were not activated during the 6-h incubation period. All of the oocytes in the calcium ionophore with puromycin group and in the calcium ionophore with roscovitine group were activated. The percentage activity of MPF in oocytes treated with roscovitine alone was decreased after 2 h and increased after 4 h of incubation. The percentage activity of MPF in oocytes treated with calcium ionophore and roscovitine was significantly decreased with suppression of MPF activity being maintained for 6 h, and this change was similar to that in oocytes treated with calcium ionophore and puromycin. Roscovitine with calcium ionophore is effective for induction of oocyte activation through suppression of MPF activity in mice.

The signaling pathways linking to lysophosphatidic acid-promoted meiotic maturation in mice.

The signaling pathways linking to lysophosphatidic acid-promoted meiotic maturation in mice were studied. When mouse oocyte-cumulus cells complexes were cultured with 10(-5) M lysophosphatidic acid (the LPA group), the rate of oocyte nuclear maturation was significantly increased. Additions of pertussis toxin, genistein, U73122, Ro320432, PD98059 or SB203580 significantly suppressed the increase in lysophosphatidic acid-stimulated nuclear maturation rate. These results suggested that Gi/o-coupled lysophosphatidic acid receptors activate phosphatidylinositol-specific phospholipase C, and result in ERK and MAP kinase activation, which is triggered by diacylglycerol-dependent protein kinase C. When intracellular cAMP concentrations of oocytes in the LPA and control groups were measured using the acetylation assay, the intracellular cAMP concentration of an oocyte in the LPA group was significantly lower than the control oocyte (0.117+/-0.04 fmol/oocyte vs. 0.176+/-0.036 fmol/oocyte, p<0.05). In conclusion, our results suggested that lysophosphatidic acid stimulates phospholipase C through a Gi-protein linked receptor on the surface of mouse cumulus cells and stimulates both extracellular signal-regulated kinase and p38 mitogen-activated kinase, resulting in the closure or loose of gap junctions between cumulus cells and the oocyte. The resultant early decrease of oocyte cAMP levels may promote nuclear maturation of mouse oocytes in vitro.

Distribution of type VI collagen in the cartilaginous tissue of the proximal tibia in the domestic cat.

To investigate the distribution of the early stage chondrocytes during the formation and closure of epiphyseal growth plate (EGP) of the domestic cat, we examined the EGP of proximal tibiae by immunohistochemistry for type VI collagen. In the epiphyseal cartilage without the secondary ossification center (SOC) and EGP in newborn cats aged 1 and 10 days, type VI collagen-positive chondrocytes were located around the cartilage canals and articular surface. In the epiphyseal cartilage with the SOC and EGP in young cats aged 1 to 3 months, type VI collagen-positive chondrocytes were located in the upper resting zone of the EGP, and then increased throughout the resting zone along with maturation. In the adult cats with the partially closed EGP, type VI collagen-positive chondrocytes were distributed throughout the remaining EGP. These findings indicate that the early stage chondrocytes characterized with type VI collagen are continuously located in the EGP during maturation. In addition, the increase of the early stage chondrocytes and the decrease of the reserve chondrocytes in the EGP along with maturation may cause the cessation of the longitudinal growth of the EGP, and finally bring about the EGP closure.

Differential expression of neurofilament 200-like immunoreactivity in the main olfactory and vomeronasal systems of the Japanese newt, Cynops pyrrhogaster.

Expression of neurofilament 200 (NF200)-like immunoreactivity was examined in the main olfactory system and the vomeronasal system of the Japanese newt, Cynops pyrrhogaster, using anti-porcine NF200 monoclonal antibody (clone N52) to investigate the differences in phenotypical characteristics between these systems. The entire nasal cavity was a flattened single chamber consisting of the main nasal chamber (MNC) and the lateral nasal sinus (LNS) communicating with each other. The olfactory epithelium (OE) was present in the MNC, and the vomeronasal epithelium (VNE) was in the LNS. The OE possessed only a small number of NF200-like immunoreactive receptor neurons. The olfactory nerve and the olfactory nerve layer of the main olfactory bulb also contained a small number of NF200-like immunoreactive axons. In contrast, the VNE possessed many NF200-like immunoreactive receptor neurons. The vomeronasal nerve and the vomeronasal nerve layer of the accessory olfactory bulb contained many NF200-like immunoreactive axons. These findings in the Japanese newt indicate that NF200-like immunoreactive receptor neurons constitute a major subpopulation in the VNE and a minor subpopulation in the OE. In addition, NF200-like immunoreactivity seems to be a useful marker to distinguish the vomeronasal system from the other nervous systems including the main olfactory system in the Japanese newt. The localization of a few NF200-like immunoreactive receptor neurons in the OE might indicate that pheromone-sensitive receptor neurons are intermingled in the OE of the Japanese newt.

Sperm-immobilizing antibodies suppress an increase in the plasma membrane fluidity of human spermatozoa.

OBJECTIVE: To clarify the mechanism by which capacitation is blocked by sperm-immobilizing antibodies, changes in the plasma membrane fluidity of human spermatozoa exposed to sperm-immobilizing antibodies were evaluated. DESIGN: In vitro cell culture study using human spermatozoa. SETTING: Department of Obstetrics and Gynecology, School of Medicine, The University of Tokushima. PATIENT(S): Semen samples were obtained from four healthy, fertile volunteers. INTERVENTION(S): The internalization of [3H]lyso-platelet activating factor (lyso-PAF) across the plasma membranes of human spermatozoa, which were exposed to sperm-immobilizing antibodies (antisperm group) or not exposed (control group), was measured at 20 and 60 minutes after the addition of a phospholipid probe using the modified albumin-back extraction method. MAIN OUTCOME MEASURE(S): The percentage of internalization of [3H]lyso-PAF across the plasma membrane of human spermatozoa. RESULT(S): Although the percentages of internalization of [3H]lyso-PAF (mean +/- SE) in the antisperm and control groups 20 minutes after addition of [3H]lyso-PAF were not significantly different (6.6% +/- 1.5% and 9.2% +/- 2.1%, respectively), at 60 minutes after the addition, the percentage in the antisperm group (9.0% +/- 1.3%) was significantly lower than that in the control group (13.4% +/- 1.3%). This inhibitory effect was diminished when spermatozoa exposed to sperm-immobilizing antibodies were incubated in an antibody-free medium. CONCLUSION(S): Sperm-immobilizing antibodies suppress the increase in internalization of an alkyl ester lysophospholipid probe in plasma membranes of human spermatozoa, and this inhibitory effect is reversible. Therefore, sperm-immobilizing antibodies suppress the fluidity of the plasma membranes of human spermatozoa, thus blocking capacitation.

Human spermatozoa capacitated with progesterone or a long incubation show accelerated internalization by an alkyl ether lysophospholipid.

OBJECTIVE: To evaluate changes that occur in sperm plasma membranes during capacitation, the internalization of [(3)H]lyso-platelet activating factor ([(3)H]lyso-PAF) across the plasma membrane of human spermatozoa was measured as a function of incubation time or exposure to progesterone (P). DESIGN: In vitro cell culture study using human spermatozoa. SETTING: Department of Obstetrics and Gynecology, School of Medicine, the University of Tokushima, Japan. PATIENT(S): Semen were obtained from three fertile healthy volunteers. INTERVENTION(S): The internalization of [(3)H]lyso-PAF across the plasma membranes of human spermatozoa that were incubated for an extended period or exposed to P was measured at 5, 20, 60, and 120 minutes after the addition of the phospholipid probe using the modified albumin back-exchange method. MAIN OUTCOME MEASURE(S): The percentage of capacitated and acrosome-reacted sperm and the proportion of internalization of lyso-PAF across the plasma membrane. RESULT(S): A 6-hour incubation period significantly increased the percentage of capacitated spermatozoa and the proportion of internalization of [(3)H]lyso-PAF across the plasma membrane of human spermatozoa compared with controls (capacitated spermatozoa, 20.3 +/- 10.6% vs. 8.5 +/- 1.8%; internalization 120 minutes after the addition of the phospholipid probe, 25.6 +/- 2.5% vs. 11.6 +/- 3.0%) (mean +/- SEM). Exposure to P significantly increased the percentage of capacitated spermatozoa compared with controls (19.6 +/- 6.8% vs. 11.0 +/- 2.4%) and also significantly accelerated the internalization of [(3)H]lyso-PAF compared with controls (internalization 120 minutes after the addition of the phospholipid probe, 26.2 +/- 1.8% vs. 21.4 +/- 1.1%). CONCLUSION(S): The administration of P or a long incubation increased the proportion of internalization and consequently induced capacitation in human spermatozoa.

Increased production of bioactive lysophosphatidic acid by serum lysophospholipase D in human pregnancy.

Lysophosphatidic acid (LPA) is a prototype of the lysophospholipid mediator family and has multiple effects in the female reproductive system. Although several metabolic routes have been reported for intracellular formation of LPA, a unique route involving lysophospholipase D, an extracellular enzyme that produces LPA in blood and body fluids, is particularly intriguing for its agonistic role. In this study, using an assay with radioactive palmitoyl-lysophosphatidylcholine, we found that lysophospholipase D activity producing palmitoyl-LPA in human serum gradually increased during pregnancy. Elevated activity of lysophospholipase D was not caused by changes in levels of their precursors, lysophosphatidylcholines, in nonpregnant women or in pregnant women at different gestational periods. With increasing length of gestation, the elevated activity in pregnant women was found to produce increasing proportions of LPA with a palmitoyl group versus other LPAs. These results suggest that LPA formed by increased activity of lysophospholipase D in blood might participate in maintenance of pregnancy.

Lysophosphatidic acid stimulates nuclear and cytoplasmic maturation of golden hamster immature oocytes in vitro via cumulus cells.

Lysophosphatidic acid (LPA), a member of the phospholipid autacoid family, is induced in incubated human follicular fluid by lysophospholipase D. It is well known that LPA functions as a growth factor and the hypothesis that LPA in human follicular fluid takes a part in meiosis of oocytes is quite plausible. We studied the effects of LPA on the maturation of golden hamster immature oocytes in vitro. Hamster oocytes with a germinal vesicle were cultured in Tyrode's albumin lactate pyruvate (TALP) medium with 10(-5) M LPA, 10 ng/ml epidermal growth factor (EGF), 30 ng/ml insulin-like growth factor-1, 1 ng/ml tumor growth factor-alpha or 1 ng/ml basic fibroblast growth factor. The nuclear maturation rates in the LPA and EGF groups were significantly higher than in the control group and the other growth factors did not show any stimulatory effect (LPA group; 74.3% [75/101], EGF group; 82.4% [89/108] vs. control group; 60.2% [59/98], p < 0.05, p < 0.01, respectively). When the cells of cumulus were removed, EGF and LPA did not increase the nuclear maturation rates. Cotreatment EGF and LPA did not significantly enhance the stimulatory effect observed with LPA alone on maturation in vitro. The penetration rate determined by the zona-free hamster oocyte test was significantly higher in the LPA group than in the control group (26.7% vs. 13.2%, p < 0.05) and was comparable with that of oocytes matured in vivo. In conclusion, LPA stimulates the nuclear and cytoplasmic maturation of hamster immature oocytes via cumulus cells.

Production of human mast cell chymase in human myometrium and placenta in cases of normal pregnancy and preeclampsia.

OBJECTIVE: To investigate whether the human mast cell chymase-endothelin-1(1-31) system was present in human myometrium, chorion and umbilical cord in normal pregnancy. METHODS: Myometrium, placenta and umbilical cord were obtained from five normal pregnant women and 10 with preeclampsia. Each tissue was stained with antibodies against hMC and ET-1(1-31). RESULTS: Routine cells were located mainly around vessels. The number of hMC-positive cells and production of ET-1(1-31) were significantly higher in myometrium from patients with severe preeclampsia compared to those from normal pregnant women. In contrast, their numbers were significantly lower in placenta and umbilical cord in patients with severe preeclampsia. CONCLUSIONS: These results suggest that the hMC-ET-1(1-31) system is active in normal pregnancy. Overproduction of hMC and ET-1(1-31) in the myometrium may be involved in the pathogenesis of severe preeclampsia, and in such cases some defense mechanism may operate in the fetus to cope with the pathological effect of the hMC-ET-1(1-31) system.

The obesity in bilateral ovariectomized rats is related to a decrease in the expression of leptin receptors in the brain.

We investigated the expression levels of leptin receptors in the brain of ovariectomized (OVX) rats. The mean expression level of ob mRNA in adipose tissues of OVX rats was significantly (P < 0.01) lower than that in the SHAM operation group rats, and the mean body weight of OVX rats was significantly (P < 0.01) greater than that in the SHAM group rats. However, there were no differences between serum leptin concentrations in these two groups. The mean level of leptin receptor (OB-R) mRNA expression in the brain tissue and the mean level of long form type OB-R (OB-RL) mRNA expression in the hypothalamus of the OVX rats were significantly (P < 0.05) lower than those in the SHAM group rats. These changes were cancelled by supplementation with 17 beta-estradiol in OVX rats. These results suggested that not only changes in the expression level of ob mRNA in adipose tissue and the serum leptin concentration but also changes in the OB-R mRNA in the brain are involved in the body weight increase in OVX rats and that a decrease in OB-R makes transmission of signals to suppress the amount of food intake difficult, thus leading to an increase in body weight.

Involvement of carbohydrate molecules on zona pellucida in human fertilization.

Although the involvement of several receptors and ligand molecules in sperm-zona interaction in many species have been proposed, there has been a little analysis of the kinetics between these molecules during the interaction. In the present study, we applied the detection method using surface plasmon resonance (SPR) by a BIAcore apparatus for the analysis of the putative receptor-ligand interaction of sperm-egg binding. Mannose-BSA or [man](5)-[GlcNAc](2)-Asp was immobilized on the surface of a sensor chip. When concanavalin A (Con A) was delivered to each of two different sensor chips to evaluate their usefulness, the resonance signal after sample injection onto a [man](5)-[GlcNAc](2)-Asp-fixed chip decreased rapidly than the mannose-BSA-fixed chip. However, the amount of binding for Con A during the injection onto the [man](5)-[GlcNAc](2)-Asp-fixed chip was high. When acid sperm extracts (acid extracts) and fractions through a CM column, containing protease activity (protease fractions), were delivered to the mannose-BSA-fixed chip, the SPR signal during the injection was not obviously changed compared with that of the control. However, when sperm samples were delivered to the [man](5)-[GlcNAc](2)-Asp-fixed chip, the SPR response during the injection was enormous. These results suggest that the [man](5)-[GlcNAc](2)-Asp-fixed chip is more useful than the mannose-BSA-fixed chip for investigating the interactions with sperm extracts and that the sensitive method using SPR by a BIAcore apparatus is applicable for the analysis of the putative receptor-ligand interaction of sperm-egg binding.