Luteal phospholipase A2 activity increases during functional and structural luteolysis in pregnant rats.

We determined cytosolic phospholipase A2 activity of the corpus luteum during luteolysis in pregnant and post-partum rats. Phospholipase A2 activity and its metabolite prostaglandin F2alpha in the corpus luteum remarkably increased just before parturition and further rose transiently during post-partum structural luteolysis. The absence of a pups' suckling stimulus delayed corpus luteum involution, being associated with an altered fluctuation in phospholipase A2 activity and depressed prostaglandin F2alpha levels. Exogenous prolactin had a reversal effect. Pharmacological and immunochemical characterization suggests multiple isoforms of phospholipase A2 in a pregnant corpus luteum. These results show the increased phospholipase A2 activity and its possible implication in luteolysis in pregnant rats.

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 microg) caused a dose-dependent inhibition of decidualization. These results show the presence of cPLA2 and its probable implication in decidualization in rat uterus.

Ovariectomy enhances the expression and nuclear translocation of annexin 5 in rat anterior pituitary gonadotrophs.

An observation of abundant annexin 5, a novel calcium and phospholipid binding protein, in gonadotrophs of the anterior pituitary gland of ovariectomized rats (Kawaminami et al., 1997 (in press)) led us to investigate the effect of ovariectomy on the subcellular distribution and synthesis of annexin 5. Gonadotrophs, which were identified by immunocytochemistry with anti LHbeta antiserum, dramatically increased their size three weeks after ovariectomy. These 'castration cells' were shown to contain abundant annexin 5 associated with the plasma membrane, nuclear envelope and nucleoplasm. True localization within the nucleus was shown by optical sectioning with a confocal microscope. Northern blot analysis showed that annexin 5 mRNA in the anterior pituitary gland was increased 24 h after ovariectomy. It further increased in parallel with LHbeta mRNA at three weeks and it decreased in parallel with LHbeta mRNA when estradiol (250 microg/animal per day) was given for 3 days. These results show that the expression of pituitary annexin 5 is controlled by ovarian estradiol and imply that annexin 5 plays a physiological role in the nucleus of activated gonadotrophs.

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.

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.

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.

Biphasic change in correlation between ovarian lipid peroxides and progestational activity during pseudopregnancy induced in immature rats.

We measured ovarian lipid peroxide (LP) levels and plasma progestins, progesterone (P4) and 20alpha-dihydroprogesterone, throughout pseudopregnancy in gonadotropin-primed immature rats. Plasma P4 fluctuated, with two peaks on days 5 (PSP5) and 8 of pseudopregnancy, and then declined to the basal level by PSP12. Ovarian LP increased from PSP1 to PSP4, decreased temporarily until PSP8, and then rose gradually until PSP14. From PSP1 through PSP7, ovarian LP was positively correlated with total progestins according to the Spearman ranked correlation coefficient (r=+0.829, p<0.05). In contrast, a negative correlation between ovarian LP and plasma P4 was apparent (r=-0.816, p<0.05) from PSP8 to PSP14. These results show the biphasic correlation of LP with luteal progestational activity depending on the luteal stage.

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 F2alpha 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.

Differences in pathological characteristics and prognosis of clinical A2 prostatic cancer from A1 and B disease.

A retrospective study was done of 53 cases of clinical stages A1 to B2 prostatic carcinomas staged by pelvic lymphadenectomy. The study compared the histologic differentiation, degree of lymphocytic infiltration, incidence of lymph node metastases and type of cellular response of clinical stage A2 to stages A1 and B disease. The available data pertaining to the incidence and survival of patients with stage A2 prostatic carcinoma were analyzed. Our study indicates that 1 of every 3 unsuspected carcinomas is of clinical stage A2. The stage A2 tumors are diffused, with a higher degree of undifferentiation and a higher incidence of lymph node metastases than tumors classified clinically as stages A1 and B1. Also the survival of patients with clinical stage A2 tumors is lower than the survival of patients with clinical stage B1 disease. Clinical stage A2 tumors are more advanced biologically than clinical stage B1 tumors.

Association of annexin V with prolactin in the rat anterior pituitary gland.

When pituitary extracts were subjected to non denaturing polyacrylamide gel electrophoresis, an unknown protein was found to associate with a proportion of the prolactin. This protein was dissociated from prolactin by sodium dodecyl sulfate. The protein was purified and sequenced. As the amino terminus was blocked, the amino acid sequences of three peptide fragments were determined. The obtained sequences of 41 amino acids were identical to partial sequences of a known protein, rat Annexin V. The molecular mass, 36 kDa, was also the same as the molecular weight of Annexin V. The existence of Annexin V mRNA in rat pituitary glands was also confirmed by polymerase chain reaction. These results show that Annexin V, a member of the calcium-dependent phospholipid binding proteins, is synthesized in the rat pituitary gland, and suggest its association with prolatin in the gland.

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

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.

Intrapituitary distribution and effects of annexin 5 on prolactin release.

Annexin 5 is expressed by rat anterior pituitary cells and a depolarizing stimulus results in increased extracellular display and, depending on local calcium concentrations, potential release into the extracellular environment. In order to further investigate the role of annexin 5 in anterior pituitary function, we have examined the intracellular distribution by immunocytochemistry and the effects of annexin 5 on the release of a major secretory product, prolactin. Prolactin was chosen because we could easily monitor effects on basal release and effects on the immediate and sustained phases of thyroid stimulating hormone releasing hormone (TRH)-stimulated release. Immunocytochemical localization of annexin 5 showed staining of the majority of anterior pituitary cells. Labeling was predominantly on the nuclear envelope and plasma membrane. For the chosen secretory product, prolactin, annexin 5 was found in most, but not all prolactin positive cells. When recombinant annexin 5 (50 ng/mL) was added to a 3 h static culture incubation of rat anterior pituitary cells, prolactin release was inhibited by about 30% (p<0.05). A lower dose had a reduced effect and higher doses had no further inhibitory effect, indicating that the effect was specific to annexin 5 and not a nonspecific toxic effect of some contaminant in the preparation. This interpretation was further strengthened in a time-course experiment demonstrating that when TRH and annexin 5 were added together, there was no effect of annexin 5 on the amount of prolactin released. After a 3 h preincubation in annexin 5, however, prolactin release, in response to TRH, was suppressed by about 30% in both the acute and sustained phases. These data suggest that annexin 5 may be a local regulator of release in the anterior pituitary, but a slow onset effect on both phases of TRH-stimulated release suggests that this is not an effect at the plasma membrane such as local extracellular calcium depletion by plasma membrane-bound annexin 5.

Secretion of recombinant rat annexin 5 by insect cells in a baculovirus expression system.

Rat annexin 5 was expressed in insect cells using a baculovirus vector, Autographa californica nuclear polyhedrosis virus. The rat annexin 5 cDNA was prepared by a polymerase chain reaction using mRNA from rat pituitary glands and placed under the control of the polyhedrin promoter. The gene product was 36 k dalton and was recognized by anti-rat annexin 5 serum. The calcium dependent binding of the recombinant annexin 5 to membranes was confirmed. The recombinant protein appeared in the medium by 21 hours post-inoculation in high amount and this was specific to this recombinant virus. High potassium milieu (20 mM KCl) for two hours increased the release of the recombinant protein but not for the recombinant beta-galactosidase prepared for a control. These results reveal that the product of the annexin 5 gene, which lacks a signal sequence, follows a secretory pathway in insect cells.