Tomohiro Yonezawa

Requirement of the Fas ligand-expressing luteal immune cells for regression of corpus luteum

Apoptosis in corpus luteum (CL) is induced by prolactin (PRL) in female rats. PRL‐induced apoptosis in CL is mediated by the Fas/Fas ligand (FasL) system. The CL consists of steroidogenic and non‐steroidogenic cells, including immunocytes. Fas mRNA was detected only in the luteal steroidogenic cells, and FasL mRNA was expressed only by the non‐steroidogenic CD3‐positive luteal immunocytes. Removing the luteal immune cells from the luteal cells inhibited PRL‐induced luteal cell apoptosis effectively. Thus, FasL‐expressing non‐steroidogenic luteal immunocytes are required for PRL‐induced luteal cell apoptosis and heterogeneous induction of apoptosis by Fas/FasL in CL.

Induction of granulin precursor gene expression by estrogen treatment in neonatal rat hypothalamus.

Our previous research has demonstrated that androgen treatment during the perinatal period increases granulin (grn) precursor mRNA levels in the neonatal rat hypothalamus. To elucidate whether exogenous estrogen increases grn mRNA in the neonatal hypothalami, expression of grn gene in the neonatal hypothalamus was studied by the competitive reverse transcription-polymerase chain reaction method. At 6 and 10 days of age, grn gene expression was significantly increased in the hypothalamus of pups whose dam has been dietarily administrated ethinyl estradiol from day 15 of gestation to the day of sampling. The subcutaneous injection of estradiol benzoate to neonatal rats at 2 days of age significantly increased grn gene expression on day 10. It was shown that estrogen, as well as androgen, was able to induce grn gene expression in the neonatal hypothalamus.

Loss of Maternal Annexin A5 Increases the Likelihood of Placental Platelet Thrombosis and Foetal Loss

Antiphospholipid syndrome is associated with an increased risk of thrombosis and pregnancy loss. Annexin A5 (Anxa5) is a candidate autoantigen. It is not known, however, whether endogenous Anxa5 prevents foetal loss during normal pregnancy. We found significant reductions in litter size and foetal weight in Anxa5-null mice (Anxa5-KO). These changes occurred even when only the mother was Anxa5-KO. A small amount of placental fibrin deposition was observed in the decidual tissues, but did not noticeably differ between wild-type and Anxa5-KO mice. However, immunoreactivity for integrin beta 3/CD61, a platelet marker, was demonstrated within thrombi in the arterial canals only in Anxa5-KO mothers. Subcutaneous administration of the anticoagulant heparin to pregnant Anxa5-KO mice significantly reduced pregnancy loss, suggesting that maternal Anxa5 is crucial for maintaining intact placental circulation. Hence, the presence of maternal Anxa5 minimises the risk of thrombosis in the placental circulation and reduces the risk of foetal loss.

Evaluation of NT-Pro BNP and CT-ANP as Markers of Concentric Hypertrophy in Dogs with a Model of Compensated Aortic Stenosis

BACKGROUND Serum C-terminal atrial natriuretic peptide (CT-ANP) and N-terminal pro B-type natriuretic peptide (NT-pro BNP) concentrations have not been measured serially in dogs with chronic pressure overload of the heart. HYPOTHESIS We investigated whether serial evaluation of CT-ANP and NT-pro BNP concentrations is a useful guide to the risk of cardiac remodeling in dogs with a model of aortic stenosis. ANIMALS Six male Beagles. METHODS After anesthesia, the aorta was constricted with a polyester band and mean left ventricular systolic pressure (LVPs) was 50 mmHg above baseline. Echocardiographic and intracardiac catheter examinations and blood sampling were performed before surgery and 3 and 6 months after surgery. RESULTS LVP and left ventricular end-diastolic pressure (LVEDP) were significantly higher at 6 months. Compared with baseline, end-diastolic intraventricular septum thickness (IVSd), left ventricular posterior wall thickness (LVPWd), and relative wall thickness (RWT) were significantly increased 3 and 6 months after aortic constriction. Serum CT-ANP concentrations were increased significantly at 3 months and serum NT-pro BNP concentrations were significantly higher 3 and 6 months after aortic constriction. Serum NT-pro BNP concentration was significantly correlated with LVEDP and IVSd whereas serum CT-ANP concentration was not correlated with any measurement. Stepwise regression analysis showed that LVEDP, IVSd, and RWT could predict serum NT-pro BNP. CONCLUSIONS AND CLINICAL IMPORTANCE This study indicated the differential regulation of NT-pro BNP and CT-ANP concentrations during pressure overload. NT-pro BNP assay may be used as an additional screening method to stratify early-stage ventricular remodeling because of aortic constriction.

Inhibition of ovulation by a lipoxygenase inhibitor involves reduced cyclooxygenase-2 expression and prostaglandin E2 production in gonadotropin-primed immature rats

Potential roles of cyclooxygenase (COX) pathway of arachidonic acid (AA) metabolism are established in a murine model of induced ovulation. Pharmacological inhibition of an alternative lipoxygenase (LOX) pathway has been shown to cause defective ovulation, but the mechanism is still undefined. This study investigated the effects of two LOX inhibitors and their time dependency on ovulation and COX activity in gonadotropins (eCG and human chorionic gonadotropin (hCG))-primed immature rats. Intra-ovarian bursal treatment with a general LOX inhibitor nordihydroguaiaretic acid (NDGA) at 0 h post-hCG (hCG0h) dose dependently inhibited ovulation rate. The drug was still but less effective when treated at hCG6h. A more specific inhibitor, 3,4-dihydroxyphenyl ethanol (DPE) was also inhibitory when treated at hCG0h but not at hCG6h. Interestingly, treatment with DPE at hCG0h resulted in attenuated expression of immunoreactive PTGS2 in granulosa layers and concomitant decrease in ovarian prostaglandin E(2) (PGE(2)) content at hCG8h. NDGA treatment reduced immunoreactive PTGS2. Ovulatory impairment by both inhibitors was prevented by systemic administration of PGE(2) at hCG6h. Immunohistochemistry revealed the expression of ALOX5 and ALOX12 in both thecal and granulosa layers of preovulatory follicles and, notably, the augmented immunoreactivities during 8 h after hCG treatment. Our results indicate the probable presence of multiple LOX isoforms and that specific inhibition of LOX at an early stage of hCG-signaling led to reduced PTGS2 activity and thus defective ovulation. They reveal a probable relationship between two pathways of AA metabolism and account at least partly for the mechanism by which the LOX inhibitor causes impaired ovulation.

Matrix metalloproteinase-2 stimulates collagen-I expression through phosphorylation of focal adhesion kinase in rat cardiac fibroblasts

Collagen-I is thought to be the main component of the extracellular matrix in cardiac fibrosis, the accumulation of which occurs with excessive activation of matrix metalloproteinase-2 (MMP-2). MMP-2 degrades the extracellular matrix; however, the relative importance of MMP-2 to collagen-I synthesis in cardiac fibroblasts remains unclear. We investigated whether extracellular activation of MMP-2 regulates collagen-I synthesis and phosphorylation of focal adhesion kinase (FAK) in rat cardiac fibroblasts. Primary cultures of rat cardiac fibroblasts were incubated with purified active MMP-2 to determine whether extracellular MMP-2 affects collagen-I synthesis and FAK phosphorylation in cardiac fibroblasts. Exogenous MMP-2 significantly stimulated FAK (Tyr397) phosphorylation and induced collagen-I expression in a time-dependent manner. Simultaneous treatment with the FAK inhibitor PF573228 abolished exogenous MMP-2-enhanced FAK (Tyr397) phosphorylation and collagen-I expression. Cells were then stimulated with norepinephrine (NE) to investigate whether endogenous MMP-2 could also induce collagen-I expression through FAK (Tyr397) phosphorylation. NE-stimulated endogenous MMP-2 activation in conditioned medium was significantly attenuated by simultaneous treatment with the MMP inhibitor PD166793. Similarly, NE-induced FAK (Tyr397) phosphorylation and collagen-I expression were significantly inhibited by simultaneous treatment with PD166793 or PF573228. Furthermore, MMP-2 knockdown induced by small interfering RNA (siRNA) significantly abolished endogenous MMP-2 expression and activation. MMP-2 siRNA significantly abolished NE-induced FAK (Tyr397) phosphorylation and collagen-I expression. These findings suggest that the extracellular activation of MMP-2 accelerated collagen-I synthesis in rat cardiac fibroblasts and that FAK phosphorylation (Tyr397) plays a pivotal role in MMP-2-stimulated collagen-I synthesis.

Augmentation of Metastin/Kisspeptin mRNA Expression by the Proestrous Luteinizing Hormone Surge in Granulosa Cells of Rats: Implications for Luteinization

ABSTRACT Variations in mRNA levels and sources of metastin/kisspeptin, neurokinin B (NKB), dynorphin, and kisspeptin receptor GPR54 were examined in the ovaries of cycling rats. Kisspeptin and dynorphin mRNAs dramatically increased at 2000 h of the proestrous day. NKB mRNA also increased, but the peak was delayed by 6 h. GPR54 mRNA declined inversely with kisspeptin. Whole-ovary expressions of kisspeptin and dynorphin mRNAs, but not of NKB mRNA, were augmented by the administration of human chorionic gonadotropin (hCG). By means of laser-capture microdissection, kisspeptin mRNA was shown mostly in follicles at 2000 h of proestrus, whereas NKB and dynorphin were expressed mainly in interstitial tissues. GPR54 mRNA was detected equally in follicles, corpora lutea, and interstitial tissues. The hCG stimulated the follicular expression of kisspeptin and interstitial tissue expression of dynorphin mRNA. In primary cultures of granulosa cells prepared from equine chorionic gonadotropin-pretreated immature rats, hCG stimulated the expression of kisspeptin, dynorphin, and NKB mRNAs. Distortion of the corpus luteum and surrounding tissue borders was sometimes seen after intra-ovarian bursa administration of kisspeptin antagonist p234 for 3 days from proestrus. Progesterone production stimulated by hCG in granulosa cell culture was suppressed by p234. These data demonstrate that significant amounts of kisspeptin are synthesized in granulosa cells and dynorphin in interstitial tissues, in response to the proestrous luteinizing hormone surge, whereas granulosa cells also contain dynorphin and NKB, suggesting at least a role for kisspeptin in the luteinization of granulosa cells.

Distribution of Aromatase and Sex Steroid Receptors in the Baculum During the Rat Life Cycle: Effects of Estrogen During the Early Development of the Baculum

The baculum, also called os penis, plays an important role during copulation. However, the hormonal regulation of its development remains to be elucidated. To determine the direct involvement of sex steroids in the development of the baculum of rats, the distributions of androgen receptors (ARs), aromatase, and estrogen receptor alpha (ESR1) were observed immunohistochemically. On Postnatal Day 1, the rudiment of the baculum expressed ARs, aromatase, and ESR1. In the proximal segment of the baculum of neonatal rats, ARs were expressed in the parosteal layer but not in the periosteum or osteoblasts. Aromatase was expressed from the parosteal layer to the endosteum, particularly in the inner osteogenic layer. ESR1 was also abundantly expressed in almost all cells from the parosteal layer to the endosteum. ARs, aromatase, and ESR1 were all abundantly expressed during the neonatal period in the hyaline cartilage of the proximal segment and in fibrocartilage of the distal segment of the baculum. Expression in all the tissues was attenuated in an age-dependent manner and became quite weak at puberty. To determine the effect of estrogen on the growth of the baculum, the aromatase inhibitor 1,4,6-androstatrien-3,17-dione (ATD) was subcutaneously injected daily into pregnant rats from Days 19 to 23 of gestation and into pups on postnatal Days 1, 3, 5, 7, and 9. On Day 10, the length of the baculum in the ATD-treated rats was significantly shorter than that in the controls, although the body weight did not change. These findings suggest that not only androgen but also locally aromatized estrogen is involved in the early growth and development of the baculum.

Inhibitory Effects of Oral Disulfiram on Endotoxin-Induced Uveitis in Rats

Purpose: Disulfiram (DSF) exhibits a wide variety of biological activities, including an anti-inflammatory action, on which we focused our attention. The aim of the present study was to investigate the effect of oral DSF on endotoxin-induced uveitis (EIU) in rats. Methods: We investigated its effect upon cellular infiltration and protein leakage, as well as on the concentration of tumor necrosis factor-α (TNF-α), nitric oxide (NO), and prostaglandin E2 (PGE2) in the anterior chamber. Some eyes were enucleated for histologic examination and immunohistochemical analysis. EIU was induced in male Lewis rats by a footpad injection of lipopolysaccharide (LPS). One hour before the LPS injection, either 250, 500, or 750 mg/kg DSF was administered orally. Twenty-four hours later, the aqueous humor was collected from both eyes, and the number of infiltrating cells and protein concentration in the aqueous humor were determined. Levels of TNF-α, NO, and PGE2 were determined by enzyme-linked immunosorbent assay. Immunohistochemical analysis in the iris ciliary body (ICB) cells was perfomed to determine the expression of activated nuclear factor kappa B (NF-κB), inducible-nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Results: The oral administration with DSF suppressed, in a dose-dependent manner, the number of inflammatory cells, the protein concentration, and the levels of TNF-α, NO, and PGE2 in the aqueous humor and improved the histiologic status of the ocular tissue. The expression of activated NF-κB-positive cells in the ICB was significantly inhibited by oral administrated with DSF 3 hr after the LPS injection. The LPS-induced increased expressions of iNOS and COX-2 proteins in the ICB were also inhibited by oral DSF 24 hr after LPS injection. Conclusions: The present results indicate that oral DSF suppresses the inflammation in EIU by inhibiting the NF-κB-dependent pathway and the subsequent production of pro-inflammatory mediators.

Appeasing Pheromone Inhibits Cortisol Augmentation and Agonistic Behaviors During Social Stress in Adult Miniature Pigs

Pairing and physical confrontation in adult sows causes social stress reactions and aggressive behaviors. Recently, maternal pig skin secretions were isolated and a mixture containing several fatty acids, now called pig appeasing pheromone (PAP), was synthesized. In this study, we investigated the effects of PAP on social and immune stress response in adult female miniature pigs. PAP or vehicle solvents were sprayed into the pens of individually housed adult sows. A two-week exposure to the pheromone did not alter basal salivary cortisol levels or circadian rhythms. Following this treatment, the animals were paired and placed in a new pen that was divided with a wire-mesh fence. Although salivary cortisol increased markedly in the vehicle-treated group, the PAP-treated group exhibited a drastic inhibition of cortisol secretion. This effect was sustained even after they were allowed to physically interact following fence removal. Moreover, the latency time of agonistic behaviors, such as escaping or biting, was significantly extended after PAP exposure. When lipopolysaccharide was injected intramuscularly, cortisol levels, rectal temperatures, and lying time lengths increased substantially. No differences were observed between the pheromone-treated and untreated groups. These results suggest that this synthetic pheromone alleviates social stress in adult pigs, although it does not affect immune stress responses. Our findings demonstrate the potential benefit of this pheromone in field applications and clinical disciplines relating to adult female pigs.