Parallel expression patterns of NR4A nuclear receptor family genes in the pituitary gland of proestrus rats.

The NR4A nuclear receptor family (NR4As), encompassing NR4A1, NR4A2, and NR4A3, exerts pivotal roles in cellular processes through intricate expression patterns and interactions. Despite the influence of some NR4As on anterior pituitary functions regulated by the hypothalamus, their physiological expression patterns remain unclear. In our prior work, we demonstrated the specific upregulation of NR4A3 in the rat anterior pituitary gland during the proestrus afternoon, coinciding with a gonadotropin surge. In this study, we investigated changes in pituitary Nr4a gene expression throughout the estrous cycle in rats and a gonadotropin surge-induced model. Nr4a1 and Nr4a2 gene expression significantly increased during proestrus, aligning with previous observations for Nr4a3. Furthermore, prolactin gene expression increased sequentially with rising Nr4a gene expression, while thyroid-stimulating hormone beta gene expression remained stable. Immunohistochemistry revealed a widespread and differential distribution of NR4A proteins in the anterior pituitary, with NR4A1 and NR4A3 being particularly abundant in thyrotrophs, and NR4A2 in gonadotrophs. In estrogen-treated ovariectomized rats, elevated luteinizing hormone secretion corresponded to markedly upregulated expression of Nr4a1, Nr4a2, and Nr4a3. In gonadotroph and somatomammotroph cell lines, gonadotropin- and thyrotropin-releasing hormones transiently and dose-dependently increased the expression of Nr4a genes. These findings suggest that hypothalamic hormone secretion during proestrus may induce the parallel expression of pituitary Nr4a genes, potentially influencing the pituitary gene expression program related to endocrine functions before and after ovulation.

Thyrotropin-releasing hormone stimulates NR4A3 expression in the pituitary thyrotrophs of proestrus rats.

The secretion of several hypothalamic peptide hormones is activated during the preovulatory period. Hypothalamic thyrotropin-releasing hormone (TRH) is one such hormone with reproductive and/or metabolic significance. However, it remains unclear whether thyroid-stimulating hormone (TSH)-producing thyrotrophs are produced during the preovulatory period. We previously found a transient increase in the expression of the nuclear receptor NR4A3, a well-known immediate early gene, in the proestrus afternoon in the anterior pituitary glands of rats. To investigate the relationship between TRH secretion and pituitary NR4A3 expression during proestrus, we used proestrus and thyroidectomized rats to identify NR4A3-expressing cells and examined the regulation of Nr4a3 gene expression via the hypothalamus-pituitary-thyroid (HPT) axis. The percentage of NR4A3-expressing cells increased in thyrotrophs at 14:00 h of proestrus. Incubation of rat primary pituitary cells with TRH transiently stimulated Nr4a3 expression. Thyroidectomy to attenuate the negative feedback effects led to increased serum TSH levels and Nr4a3 gene expression in the anterior pituitary, whereas thyroxine (T4) administration conversely suppressed Nr4a3 expression. Additionally, the administration of T4 or TRH antibodies significantly suppressed the increase in Nr4a3 expression at 14:00 h of proestrus. These results demonstrate that pituitary NR4A3 expression is regulated by the HPT axis, and that TRH stimulates thyrotrophs and induces NR4A3 expression during the proestrus afternoon. This suggests the potential involvement of NR4A3 in the regulation of the HPT axis during pre- and post-ovulatory periods.

Lysophosphatidic acid stimulates rat uterine contraction in vitro.

Lysophosphatidic acid (LPA) has been implicated in the uterine endometrial functions of implantation and decidualization; however, not much is known about its myometrial contractile function. Herein we characterized the uterotonic effects of LPA in non-pregnant (estrus) and peri-parturient rats in vitro. LPA dose-dependently (0.01-10 µM) stimulated the amplitude and integral, but not the frequency, of the uterine strip contraction of estrous rats. The stimulatory effect of LPA was enhanced 1 day before parturition but was lost 1 day postpartum. LPA did not cause the de novo synthesis of prostaglandin (PG) F2α but stimulated contractions cooperatively with the PG. LPA-induced contractions were significantly inhibited by an LPA1/2/3 antagonist in the uteri of estrous rats but not in term rats. This study characterized the uterotonic effect of a natural LPA that occurs at physiological concentrations, changes with reproductive states, and is independent of mediation by the newly synthesized PG.

Activin A specifically suppresses the expression of annexin A5 mRNA and augments gonadotropin-releasing hormone stimulation of A1 expression in LßT2 gonadotrope cells.

Recently, we reported that gonadotropin-releasing hormone (GnRH) stimulates annexin A1 (Anxa1) and A5 (Anxa5) mRNA expression through the GnRH-receptor-mitogen-activated protein kinase cascade in LßT2 cells. As LßT2 cells respond to activin, we examined the effect of activin A on Anxa1 and a5 expression in LßT2 cells. Activin A (0.4 and 4 ng/mL) treatment decreased Anxa5 mRNA levels in a dose-dependent manner, but did not affect Anxa1 mRNA levels at concentrations up to 40 ng/mL. After activin A treatment (4 ng/mL), Anxa5 mRNA levels significantly decreased at 6 h, gradually declined until 24 h, and remained low until 48 h, whereas Anxa1 mRNA levels did not significantly change following treatment. Pretreatment with activin A for 24 h increased GnRH agonist (GnRHa)-induced Anxa1 increase by approximately 7-fold compared with GnRHa stimulation alone, but Anxa5 was not affected. As previously reported, these activin A treatments increased gonadotropin ß subunit and GnRH receptor mRNA levels and slightly decreased common α-glycoprotein subunit mRNA levels. Furthermore, we examined the effect of activin A on Nr4a3, which is repressed by ANXA5 and which reduces Fshb expression, and found that activin A augmented Nr4a3 expression and slightly decreased the GnRHa-induced increase in Nr4a3. These results suggest that in gonadotrope cells, the mechanism regulating Anxa1 and a5 expression is differentially coupled with activin A signal transduction. Activin A suppresses Anxa5 expression under increased Nr4a3 expression, whereas activin A and GnRH synergistically stimulate Anxa1 expression. These GnRH-inducible annexins may have different specific functions in gonadotropes.

The expression of Annexin A1 and A5 mRNA by gonadotropin-releasing hormone in LßT2 gonadotrope cells.

Gonadotropin-releasing hormone (GnRH) stimulation of annexin A1 (ANXA1) and A5 (ANXA5) mRNA expression was analyzed in LßT2 gonadotrope cells. Quantitative polymerase chain reaction results showed that a GnRH analog (GnRHa) stimulated the expression of both ANXA1 and A5 mRNA with a peak at 12 h of incubation; however, ANXA1 mRNA was extremely stimulated (60 folds). Immunocytochemical analysis confirmed these findings. A GnRH antagonist inhibited the effect of GnRHa. ANXA1 and A5 mRNA levels were significantly increased by protein kinase C (PKC) activator (12-O-Tetradecanoylphorbol-13-acetate; TPA), but not by dibutyryl cAMP. GnRHa-stimulated induction of ANXA1 and A5 mRNA was inhibited by PKC (GF109203) and MEK inhibitors (PD98059). TPA increased ANXA1 and A5 mRNA expression in a dose-dependent manner (1 nM to 10 µM), while the extent of the increase was much greater in ANXA1. After stimulation with 10 nM or 1 µM TPA, ANXA1 and A5 mRNA levels were increased at 6 h. ANXA1 mRNA levels were higher in the 1 µM TPA than in the 10 nM TPA treatment, whereas 1 µM TPA did not show further stimulation of ANXA5 mRNA compared to 10 nM TPA. These results clearly show that ANXA1 mRNA expression is stimulated by GnRH through PKC like ANXA5, and the response of ANXA1 is much larger than that of ANXA5. A close relationship between these annexins and a significant role for ANXA1 in GnRH action at gonadotropes is suggested.

Sequential preovulatory expression of a gonadotropin-releasing hormone-inducible gene, Nr4a3, and its suppressor Anxa5 in the pituitary gland of female rats.

Functional relationship between nuclear receptor subfamily 4 group A member 3 (Nr4a3) and annexin A5 (Anxa5), which are two gonadotropin-releasing hormone (GnRH)-inducible genes, has been established while evaluating pituitary gonadotropes in relation to follicle-stimulating hormone beta (Fshb) expression. However, the physiological variations that arise due to the differential expression of these genes in the pituitary gland during rat estrous cycle remain unknown. This study aimed to evaluate the Nr4a3 and Anxa5 mRNA expression during the estrous cycle in rats in comparison with the expression of the gonadotropin subunit genes, luteinizing hormone beta (Lhb) and Fshb. Nr4a3 mRNA expression showed a single peak at 1400 h of proestrus during the 4-d estrous cycle. Anxa5 mRNA level was elevated along with increased Fshb mRNA expression after the decline of Nr4a3 mRNA until 2300 h. Lhb mRNA expression levels were not significantly changed during the estrous cycle. Notably, addition of a GnRH antagonist at 1100 h completely eradicated luteinizing hormone secretion at 1400 h and 1700 h of proestrus, and significantly reduced the Nr4a3 mRNA expression level at both the time points. These results suggest that GnRH is, at least partly, responsible for the increase in pituitary Nr4a3, and that the interaction between NR4A3 and ANXA5 is required to regulate Fshb expression during the preovulatory gonadotropin surge.

Effects of gonadotropin-releasing hormone agonist on human chorionic gonadotropin activity in granulosa cells of immature female rats.

Although the expression of gonadotropin-releasing hormone (GnRH) in the ovaries is well established, its physiological role remains unknown. The aim of this study was to determine whether ovarian GnRH mediates the actions of human chorionic gonadotropin (hCG) in the granulosa cells of immature female rats. Follicular growth was induced by administration of pregnant mare serum gonadotropin (PMSG, 15 IU/0.15 ml) on day 25 after birth, and hCG (20 IU/0.2 ml) was administered on day 27 revealing the increase of plasma progesterone level. Primary cultures of granulosa cells were established from large follicles 2 days after PMSG treatment. Progesterone synthesis was augmented by hCG in a dose-dependent manner. Annexin A5 (ANXA5), a biomarker of GnRH, was expressed in the granulosa-luteal cells after hCG treatment, as shown by immunohistochemistry, suggesting that hCG treatment induced GnRH action. The GnRH mRNA level was increased by hCG, and treatment with GnRH agonist (GnRHa) increased ANXA5 mRNA levels in the primary cultures of granulosa cells. Concomitant incubation of GnRH (10-7 M) or GnRHa (fertirelin acetate, 10-8 M) with hCG suppressed progesterone synthesis during a 3 h incubation period. The mRNA expression of luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) was synergistically stimulated and suppressed by hCG and GnRHa, respectively. GnRHa stimulated p21 expression, and GnRHa and hCG synergistically reduced the mRNA expression levels of p27 and FOXO1. These data suggest that GnRH induced by LH may have a role for the LH-mediated luteinization of granulosa cells. In addition, ANXA5 may be involved in GnRH action. GnRH-ANXA5 would be an important mechanism in cell differentiation.

Augmentation of gonadotropin-releasing hormone receptor expression in the post-lactational mammary tissues of rats.

Gonadotropin-releasing hormone (GnRH) is a neurohormone of the hypothalamus controlling pituitary gonadotropin secretion and hence gametogenesis. While it has also been believed that GnRH is synthesized and functions in various peripheral tissues, the expression of GnRH receptor (GnRH-R) in peripheral tissues is not well-described. We previously found that annexin A5, which is increased in the pituitary gonadotropes by GnRH, is dramatically increased in rat mammary epithelial cells after weaning, suggesting that local GnRH is responsible for this increase. Annexin A5 is a member of the annexin family of proteins and is thought to be involved in various regulatory mechanisms, including apoptosis. In the present study, we examined GnRH-R expression in the mammary tissues after weaning. Although GnRH-R mRNA was not detected in the mammary tissues during lactation, it was dramatically increased after weaning. Forced weaning at mid-lactation (day 10) also promoted the expression of GnRH-R transcripts in mammary tissues within 2 days. Furthermore, western blotting analysis with anti-GnRH-R showed that the expression of an immuno-positive 60-kDa protein, whose size was equivalent to that of rat GnRH-R, was confirmed to increase after weaning. These findings clarified the induction of GnRH-R in the mammary tissues after weaning and suggest that GnRH is involved in the involution and tissue remodeling of post-lactating rat mammary tissues.

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

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.

Gonadotropin-releasing hormone is prerequisite for the constitutive expression of pituitary annexin A5.

Annexin A5 (ANXA5), a member of the structurally related family of annexin proteins, is expressed in pituitary gonadotropes. We previously reported that ANXA5 expression is stimulated by gonadotropin-releasing hormone (GnRH). In the present study, we investigated ANXA5 expression in the anterior pituitary gland of GnRH-deficient mutant hypogonadal (hpg) mice. RT-PCR demonstrated that luteinizing hormone ß subunit (LHß) and ANXA5 mRNA levels were both lower in the pituitary gland of hpg mice than in wild-type mice. Immunohistochemistry showed that ANXA5 expression throughout the pituitary gland was very low in hpg mice, suggesting that ANXA5 is diminished in gonadotropes and also in other cell types. Subcutaneous administration of a GnRH analogue, des-gly10 (Pro9)-GnRH ethylamide (1 µg/day for 7 days), augmented the expression of LHß and ANXA5 in the pituitary gland in hpg mice. However, LHß- and ANXA5-positive cells did not show exactly matched spatial distributions. These findings suggest that GnRH is necessary for constitutive ANXA5 expression in the pituitary gland, not only in gonadotropes but also in other pituitary gland cell types. A close relationship between ANXA5 and LHß expression was confirmed. It is suggested that a significant role of ANXA5 in the physiologic secretion of LH.

A comprehensive immunohistochemistry of prostaglandins F2α and E2 synthetic enzymes in rat ovary and uterus around parturition.

A comprehensive immunohistochemistry with the isoform-distinguishable antibodies against prostaglandin (PG) F2α and PGE2 biosynthetic enzymes was undertaken to identify the cellular types and enzyme isoforms in rat ovary and uterus around parturition. In general ovarian and uterine cells showed positive immunoreactions for phospholipase A2 groups 4A and 6A, but not group 2A, and cyclooxygenase (COX)-1 rather than COX-2. Their immunoreactions for PGF2α synthase and PGE2 synthase were cell type-dependently variable. The putative PGF2α and PGE2 producing cell types included, as expected, ovarian luteal cells, uterine endometrial epithelium and myometrium, and cervical connective tissue and, unexpectedly, ovarian stromal cells and basal lamina of cervical endometrium. Obtained data indicate the generation of PGF2α and PGE2 by multiple sites, which are entirely the same as established sites of actions, in parturition processes and tissue-dependent differential usage of PG biosynthetic pathway.

Enhanced social reward response and anxiety-like behavior with downregulation of nucleus accumbens glucocorticoid receptor in BALB/c mice.

Social anhedonia is a psychological state with difficulty in experiencing pleasure from social interactions and is observed in various diseases, such as depressive disorders. Although the relationships between social reward responses and anxiety- and depression-like behaviors have remained unclear, a social reward conditioned place preference (SCPP) test can be used to analyze the rewarding nature of social interactions. To elucidate these relationships, we used 5-week-old male mice of AKR, BALB/c, and C57BL/6J strains and conducted behavioral tests in the following order: elevated plus-maze test (EPM), open field test (OFT), SCPP, saccharin preference test (SPT), and passive avoidance test. The nucleus accumbens of these mice were collected 24 hr after these behavioral tests and were used for western blotting to determine the levels of receptors for brain-derived neurotrophic factors and glucocorticoids. BALB/c mice displayed the highest levels of anxiety-like behavior in EPM and OFT as well as physical anhedonia-like behaviors in SPT. They also showed increased responses to social rewards and huddling behaviors in SCPP, with downregulated glucocorticoid receptor (GR). Regression analysis results revealed positive influences of anxiety- and physical anhedonia-like behaviors and expressions of GR on social reward responses. Collectively, temperament associated with anxiety and physical anhedonia may affect social reward responses, which possibly is influenced by the expression of GR that can modify these psychological traits.

Expression of lysophosphatidic acid receptors in the rat uterus: cellular distribution of protein and gestation-associated changes in gene expression.

Though lysophosphatidic acid (LPA) shows a variety of regulatory roles in reproduction, its action mechanisms in the gestational organs are still largely unknown. We here characterized cellular distribution of its six kinds of specific receptors (LPA1-6) in rat uteri by immunohistochemistry and quantitatively analyzed changes in Lpar1-6 mRNAs expression throughout pregnancy. Among LPA1-6, evident expression of LPA3, LPA4, and LPA6 was immunologically detected and less expression of immunoreactive LPA1 and LPA2 was also found. Luminal and glandular epithelial cells, stromal cells, and myometrial cells are sites of positive immunoreactions, and they are all likely to express three or more subtypes. All of Lpar1-6 mRNAs were expressed, and their alterations were variable depending on subtypes and gestational age. The present information suggests that diverse actions of LPA in the uterus involve varied expression of LPA receptors dependent on tissue/cell types, receptor subtype(s), and organ reproductive states and helps to understand uterine biology of LPA.

Mast Cell Dynamics in the Ovary Are Governed by GnRH and Prolactin.

Gonadotrophin releasing hormone (GnRH) facilitates the migration of mast cells (MCs) into the involuting mammary gland. As GnRH is also expressed in the ovary, we examined changes in ovarian MCs. MCs in the ovary were mainly in interstitial tissue and their number increased during the estrous cycle to produce 2 peaks, one at diestrus 2 (20:00 hours) and another at proestrus (17:00 hours). Laser microdissection demonstrated that GnRH mRNA is expressed throughout ovarian tissues (corpora lutea, follicles, and interstitial tissues). GnRH immunoreactivity was also ubiquitous, but MCs were the most strongly immunostained. Analysis of GnRH mRNA in the ovary showed it to fluctuate similarly to the variation in MC number during the estrous cycle, and MCs also expressed GnRH. Local administration of a GnRH agonist (GnRHa) into the hemilateral ovarian bursa increased MCs in the administered ovary. MC number and GnRH mRNA were significantly lowered in the pregnant ovary. Prolactin administration suppressed the normal peaks in MC number in the ovary at both diestrus and proestrus. By contrast, a dopamine agonist, administered when prolactin was elevated during pseudopregnancy, increased ovarian MC number. Furthermore, prolactin inhibited GnRHa-induced peritoneal MC migration in a Transwell assay. These data clearly demonstrate that ovarian MC number is regulated positively by local GnRH expression and negatively by prolactin. The suppressive effect of prolactin on GnRH and MCs would be part of its luteotrophic action.

Group IVA phospholipase A2 regulates testosterone biosynthesis by murine Leydig cells and is required for timely sexual maturation.

In the present paper, we report that PLA2G4A (Group IVA phospholipase A2) is important in the development and function of rodent testes. Interstitial cells of rat testes had high PLA2 (phospholipase A2) activity that was very sensitive to the PLA2G4A-preferential inhibitor AACOCF3 (arachidonyl trifluoromethyl ketone). PLA2G4A protein was expressed primarily in the interstitial cells of wild-type mouse testes throughout maturation. Although Pla2g4a knockout (Pla2g4a-/-) male mice are fertile, their sexual maturation was delayed, as indicated by cauda epididymal sperm count and seminal vesicle development. Delayed function of Pla2g4a-/- mice testes was associated with histological abnormalities including disorganized architecture, swollen appearance and fewer interstitial cells. Basal secretion of testosterone was attenuated significantly and steroidogenic response to hCG (human chorionic gonadotropin) treatment was reduced in Pla2g4a-/- mice compared with their Pla2g4a+/+ littermates during the sexual maturation period. Chemical inhibition of PLA2G4A activity by AACOCF3 or pyrrophenone significantly reduced hCG-stimulated testosterone production in cultured rat interstitial cells. AACOCF3 inhibited forskolin- and cAMP analogue-stimulated testosterone production. These results provide the first evidence that PLA2G4A plays a role in male testes physiology and development. These results may have implications for the potential clinical use of PLA2G4A inhibitors.

Changes in the expressions of annexin A1, annexin A5, inhibin/activin subunits, and vitamin D receptor mRNAs in pituitary glands of female rats during the estrous cycle: correlation analyses among these factors.

Pituitary gonadotropin secretion is regulated by several pituitary factors as well as GnRH and ovarian hormones. To elucidate the regulatory mechanisms of pituitary gonadotropin secretions, we observed changes in the mRNA levels of pituitary factors, namely annexin A1 (Anxa1) and Anxa5, inhibin/activin subunits, follistatin (Fst), and vitamin D receptor (Vdr), in female rat pituitary glands during the estrous cycle. Additionally, levels of LHß subunit (Lhb), FSHß subunit (Fshb), and GnRH receptor (Gnrh-r) mRNA were examined. During proestrus, Anxa1, Anxa5, Vdr, and inhibin α-subunit (Inha) exhibited the lowest levels, while during estrus, activin ßB-subunit (Actbb), Lhb, and Gnrh-r were the lowest. Moreover, Fshb exhibited the highest value during metestrus, whereas Fst did not differ significantly. Correlation analyses revealed 16 statistically significant gene combinations. In particular, four combinations, namely Anxa5 and Inha, Anxa5 and Actbb, Inha and Vdr, and Inha and Actbb, were highly significant (P<0.0001), while four combinations, Anxa1 and Anxa5, Anxa1 and Vdr, Anxa5 and Vdr, and Lhb and Gnrh-r, were moderately significant (P<0.001). The remaining eight combinations that exhibited statistical significance were Anxa1 and Inha, Anxa1 and Actbb, Vdr and Actbb, Anxa1 and Fshb, Inha and Lhb, Actbb and Fshb, Actbb and Lhb, and Fst and Fshb (P<0.05). These results highlight strong correlations among Anxa1, Anxa5, Vdr, Inha, and Actbb, thereby suggesting that an interaction among ANXA1, ANXA5, and VDR may lead to further communications with inhibin and/or activin in the pituitary gland.

Gonadotropin-releasing hormone stimulation of annexin A5 expression in the thymus of male rats.

As gonadotropin-releasing hormone (GnRH) is expressed in the thymus, its direct action on thymic cells, including thymic involution, has been suggested. Annexin A5 (ANXA5), a biomarker of GnRH, was used to determine whether GnRH affects the thymus of male rats. Immunohistochemistry showed positive reactions for ANXA5 in large medullary epithelial cells at 30 days of age, and the expression continued until 180 days of age. Organ culture of thymus pieces was performed to examine the direct action of a GnRH agonist (GnRHa) on the expression of Anxa5 and Gnrh mRNA. Thymus tissues obtained from male rats (40-60 days old) were cut into small pieces (2-3 mm3) and incubated for 3 hr with the GnRHa. The expression levels of Anxa5 and Gnrh mRNA were augmented by the GnRHa. Immunohistochemistry of these tissue fragments showed that ANXA5 expression was enhanced, especially in medullary epithelial cells. These results revealed that GnRH synthesis in the thymus could affect thymic epithelial cells after puberty.

Changes in the expression of annexin A1 in the anterior pituitary gland after ovariectomy in rats.

The expression of annexin A1 (ANXA1) is augmented by gonadotrophin releasing hormone (GnRH) in LßT2 gonadotroph. We examined the distribution of ANXA1 in the pituitary tissues and the effect of ovariectomy. ANXA1 was mainly stained on folliculostellate cell-like irregular shaped cells with extended process of adult female rats. Large gonadotroph, so called castration cells, appeared two weeks after the ovariectomy. ANXA1 in castration cells exists around cells although another GnRH responsive annexin, ANXA5, was apparent also in the cytoplasm. The pituitary expression of ANXA1 after ovariectomy was significantly higher than intact rats. These difference in tissue distribution of two annexins suggest ANXA1 and ANXA5 bear different physiological function in the gonadotroph under GnRH regulation.

Anti-ovulatory effects of RU486 and trilostane involve impaired cyclooxygenase-2 expression and mitotic activity of follicular granulosa cells in rats.

To explore the mechanism for anti-ovulatory effects of blockade of preovulatory synthesis and action of progesterone, we focused on cyclooxygenase (COX)-2 induction and mitotic activity of granulosa cells in gonadotropins-treated rats. Treatment with RU486 (a progesterone receptor antagonist) or trilostane (a 3ß-hydroxysteroid dehydrogenase inhibitor) just prior to or 4h after human chorinonic gonadotropin (hCG) (hCG4h) decreased ovulation rates and circulating progesterone level. Human CG induction of immunoreactive COX-2 in the granulosa layer of mature Graafian follicles at hCG8h was reduced by RU486 treatment at hCG0h and trilostane treatment at hCG4h. RU486 treatment further attenuated ovarian prostaglandin E(2) (PGE(2)) level significantly. Cell proliferative activity in mural granulosa layer of the inhibitors-treated follicles was significantly lower than in intact group. Obtained results show that inhibition of synthesis and action of progesterone caused attenuated COX-2/PGE(2) system and dysregulated mitotic response of granulosa cells, resulting in decreased ovulation.

Prostaglandins F2α and E2 in rat placenta and fetal membrane: a comprehensive immunohistochemistry of their synthetic enzymes and in vivo tissue levels during normal pregnancy.

We determined a comprehensive immunohistochemistry of putative isoforms of enzymes for prostaglandin (PG) F2α and PGE2 biosynthesis and these PGs levels in placenta and fetal membrane of normal pregnant rats in vivo. Placenta and fetal membrane showed positive immunoreactions for phospholipase A2 group 4A, but not group 2A, and cyclooxygenase (COX)-1 rather than COX-2. They showed positive immunoreactions for at least one isoform of each of PGF synthase and PGE synthase with tissue-dependent variations. PGF2α and PGE2 levels in both tissues were highest on day 12 and declined and remained low thereafter. Obtained data would be the basic information on the primary PGs synthesis in rat placenta and fetal membrane in normal pregnancy.