Mitsumori Kawaminami

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.

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.

Involvement of cytosolic phospholipase A2 in the ovulatory process in gonadotropin-primed immature rats.

The preovulatory LH surge induces a remarkable increase in ovarian prostaglandins (PGs) which help to mediate the ovulatory process. We investigated whether cytosolic phospholipase A2 (cPLA2) has a role in this PG production in PMSG/hCG-primed immature rats. The immunoreactive signal for cPLA2 was localized in both thecal and granulosa layers of mature follicles and became evident in response to gonadotropins. The PLA2 activity in the whole ovarian cytosol rose slightly after PMSG stimulation, persisted relatively constant until 24 h after hCG injection and thereafter increased gradually. Intra-ovarian bursal injection of arachidonyl trifluoromethyl ketone, a specific inhibitor for cPLA2 ( 1.0-3.0 mg/ovary), significantly reduced ovarian PGE2 content and the ovulation rate. These results suggest that cPLA2 exists in periovulatory follicles and functions in PG production related to the ovulation process.

Immunocytochemical localization of annexin 5, a calcium-dependent phospholipid-binding protein, in rat endocrine organs.

Abstract Annexin 5, a unique calcium- and phospholipid-binding protein, has been investigated for its specific distribution in rat endocrine organs by immunocytochemistry with a specific antiserum to recombinant rat annexin 5. Follicular epithelial cells and parafollicular cells of the thyroid gland, adrenocortical cells of the zona fasciculata and zona reticularis, luteal cells, testicular interstitial cells, and Sertoli cells were shown to contain annexin 5. To examine whether the synthesis of annexin 5 would be affected by a change in humoral signal, the distribution of annexin 5 in the anterior pituitary was examined three weeks after ovariectomy. The withdrawal of ovarian hormones induced huge castration cells in the anterior pituitary gland, which contained abundant annexin 5. Annexin 5 was not detected in the pineal gland, the parathyroid gland, the islet of Langerhans, the adrenal medulla, zona glomerulosa cells, and granulosa cells. Since annexin 5 was shown to exist in many of the endocrine tissues examined, to be localized in specific cell types, and to be abundant in castration cells, it is suggested that annexin 5 contributes to secretory cell functions, which may be common to endocrine cells secreting chemically different hormones.

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.

Annexin 5 Messenger Ribonucleic Acid Expression in Pituitary Gonadotropes Is Induced by Gonadotropin-Releasing Hormone (GnRH) and Modulates GnRH Stimulation of Gonadotropin Release

Our previous studies on annexin 5, a member of the annexin family of proteins, have shown its expression in the anterior pituitary gland, its preferential distribution in gonadotropes, and its increase after ovariectomy. In the present study, we examined (1) whether annexin 5 is synthesized in gonadotropes, (2) whether its expression is under the control of gonadotropin-releasing hormone (GnRH), and (3) the effect of annexin 5 on gonadotropin release. Large cells, also called castration cells, appeared in anterior pituitary tissue 3 weeks after ovariectomy. These cells have been confirmed to be hyperfunctioning gonadotropes and are easily discriminated from other pituitary cells without immunostaining. Using in situ hybridization with a digoxigenin-labeled ribonucleic acid probe, enhanced expression of annexin 5 messenger ribonucleic acid (mRNA) in these gonadotropes was clearly demonstrated. Northern blot analysis showed an increase in the level of annexin 5 mRNA expression 3 weeks after ovariectomy. It was lessened 3 h after the injection of Cetrorelix (GnRH antagonist, 10 µg i.v.). Administration of a GnRH analog [GnRHa; Des-Gly 10 (Pro9) GnRH ethylamide, 0.2 ml of 2.5 µg/ml saline ten times intraperitoneally at 30-min intervals] significantly increased pituitary annexin 5 mRNA. In primary cultures of anterior pituitary cells, recombinant rat annexin 5 stimulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release in a dose-dependent manner. Concomitant administration of annexin 5 (1 µg/ml) and GnRHa augmented the LH and FSH release induced by GnRHa. After a 1-hour incubation, cycloheximide (10 µg/ml) apparently inhibited the LH response to GnRHa, while annexin 5 (2 µg/ml) moderated this inhibition. Further, the antisense oligodeoxynucleotide to annexin 5 mRNA blunted the LH response to GnRHa. It is thus concluded that annexin 5 is synthesized in the gonadotropes under the effect of GnRH, and it is suggested that annexin 5 synthesis mediates at least partly GnRH receptor signaling to stimulate gonadotropin secretion.

Sustained activity of luteal cytosolic phospholipase A2 during luteolysis in pseudopregnant rats: its possible implication in tissue involution.

We investigated the expression and activity of cytosolic phospholipase A2 (cPLA2) in the corpus luteum during spontaneous and induced luteolysis in pseudopregnant rats. In both models, luteal PLA2 activity rose in association with functional regression and persisted during the following structural regression. Tissue concentration of prostaglandin F2α with a luteolytic potency showed a similar fluctuation. The enzyme activity was almost completely suppressed by a cPLA2-specific inhibitor. Expression of cPLA2, analyzed by immunohistochemistry, became enhanced during luteolysis with preferential localization to phagocytotic and fibrotic replacement sites. Taken together with our previous finding, the data indicate a persistent elevation in luteal cPLA2 expression and activity that may affect tissue involution in vivo.

Expression and cellular distribution of cytosolic phospholipase A2 in the rat ovary

Cellular expression of cytosolic phospholipase A2 (cPLA2) was investigated in the rat ovary in different endocrine states. Its mRNA expression was detected by RT-PCR. The immunohistochemistry identified an intense signal for cPLA2 in oocytes. Granulosa and thecal cells in growing follicles were negative, but turned positive during the periovulatory period, whereas those in atretic follicles were highly immunoreactive. The immunoreactive signal was modest in newly formed corpora lutea (CL) but intensified in functionally and morphologically regressing CL. These results show a broad but specific distribution of cPLA2 in ovarian cell types, and suggest its role in ovulation, CL regulation and apoptotic processes.

Role of cytosolic phospholipase A2 in eicosanoid generation by corpora lutea of pseudopregnant rats: effects of its specific inhibitor

This study was performed to investigate whether 85 kDa cytosolic phospholipase A2 (cPLA2) functions in eicosanoid generation in rat corpora lutea (CL) using its specific inhibitor, arachidonyl trifluoromethyl ketone (ATK). In both immature and adult pseudopregnant rats, PLA2 activity in the cytosol of CL, measured by the liposome-vesicle assay, increased from day 6 of pseudopregnancy (PSP6) to PSP12. 10 microM ATK potently inhibited all of these activities to 10-20% and the rate of the inhibition by ATK was much higher on PSP12. ATK also reduced arachidonic acid (AA) release from luteal cells of PSP12 prelabelled with 3H-AA. Furthermore, the production of prostaglandin E2 by cultured luteal cells was mostly suppressed by the drug. These results suggest the augmentation of cPLA2 activity with the luteal age of pseudopregnant rats and its principal role in eicosanoid generation in CL.

Cytosolic phospholipase A2 in rat decidual cells: evidence for its role in decidualization.

We investigated the existence and possible role of cytosolic phospholipase A2 (cPLA2) in rat decidualized uteri. PLA2 activity in the cytosol of a decidualized uterine horn, induced by intraluminal oil infusion, was significantly higher than that in contralateral intact horn. The activity was almost completely depressed by cPLA2 inhibitors including arachidonyl trifluoromethyl ketone (ATK). The immunoreactive signals for cPLA2 were intense in decidua and glandular epithelial cells. In vivo administration of ATK (0.1–100 μg) caused a dose‐dependent inhibition of decidualization. These results show the presence of cPLA2 and its probable implication in decidualization in rat uterus.