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.

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.

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.

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.

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.

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.

Vaginal mucus in mice: developmental and gene expression features of epithelial mucous cells during pregnancy†.

The vagina is the site of copulation and serves as the birth canal. It also provides protection against external pathogens. In mice, due to the absence of cervical glands, the vaginal epithelium is the main producer of vaginal mucus. The development and differentiation of vaginal epithelium-constituting cells and the molecular characteristics of vaginal mucus have not been thoroughly examined. Here, we characterized vaginal mucous cell development and the expression of mucus-related factors in pregnant mice. The vaginal mucous epithelium layer thickened and became multilayered after Day 12 of pregnancy and secreted increasing amounts of mucus until early postpartum. Using histochemistry and transmission electron microscopy, we found supra-basal mucous cells as probable candidates for precursor cells. In vaginal mucous cells, the expression of TFF1, a stabilizer of mucus, was high, and some members of mucins and antimicrobial peptides (MUC5B and DEFB1) were expressed in a stage-dependent manner. In summary, this study presents the partial characterization of vaginal epithelial mucous cell lineage and expression of genes encoding several peptide substances that may affect vaginal tissue homeostasis and mucosal immunity during pregnancy and parturition.

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.

Improvement in identification of pro-estrous mice by using a novel method of detecting vaginal mucous cells.

Accurate identification of the murine estrous cycle using vaginal exfoliative cytology is the initial and crucial step for controlled reproduction of this species. However, it is generally difficult to discriminate each stage of the cycle, and thus to select pro-estrous mice for mating. To increase the accuracy of identification of the pro-estrous stage, we re-evaluated the vaginal fold histology and modified the method of exfoliative cytology. Tissue fixation using methanol in Carnoy's solution but not paraformaldehyde, combined with Alcian blue staining but not the conventional Giemsa staining, resulted in better manifestation of mucosal cell layers in the vaginal epithelium just above the keratinized layer. This mucous layer in the fold histology was found to form specifically in the pro-estrous and late di-estrous stages, and the mucous cells exfoliated in smear samples only in the pro-estrous stage. This novel method was found, by a blinded test, to increase the rate of accurate identification of the pro-estrous stage compared to the conventional method (80% vs 50%). Consistent with this finding, the mating experiment with "pro-estrous" females selected by the novel method revealed a significantly higher success rate than that with the conventional method (78.0% vs 47.5%). Thus, our study demonstrates vaginal exfoliative mucous cells as a better potential marker to detect the "receptive" state of female mice that leads to an improved success rate of mating.

Augmentation of Nr4a3 and Suppression of Fshb Expression in the Pituitary Gland of Female Annexin A5 Null Mouse.

GnRH enhances the expression of annexin A5 (ANXA5) in pituitary gonadotropes, and ANXA5 enhances gonadotropin secretion. However, the impact of ANXA5 regulation on the expression of pituitary hormone genes remains unclear. Here, using quantitative PCR, we demonstrated that ANXA5 deficiency in female mice reduced the expression of Fshb and Gh in their pituitary glands. Transcriptome analysis confirmed a specific increase in Nr4a3 mRNA expression in addition to lower levels of Fshb expression in ANXA5-deficient female pituitary glands. This gene was then found to be a GnRH-inducible immediate early gene, and its increased expression caused protein to accumulate in the nucleus after administration of a GnRH agonist in LßT2 cells, which are an in vitro pituitary gonadotrope model. The increase in ANXA5 protein levels in LßT2 cells clearly suppressed Nr4a3 expression. siRNA-mediated inhibition of Nr4a3 expression increased Fshb expression. The results revealed that GnRH stimulates Nr4a3 and Anxa5 sequentially. NR4A3 suppression of Fshb may be necessary for later massive secretion of FSH by GnRH in gonadotropes, and Nr4a3 would be negatively regulated by ANXA5 to increase FSH secretion.

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.

In vivo evidence for possible up-regulating roles of lysophosphatidic acid around fertilization in rats.

Lysophosphatidic acid (LPA) produced by autotaxin (ATX) is recognized as a multi-functional mediator in mammalian reproduction. This study focused on possible effect(s) of LPA on ovulated cumulus-oocyte complexes (COCs) around fertilization in rats in vivo. Immunohistochemistry revealed the cell-type-dependent localization of candidates of synthetic enzymes, ATX and two phospholipases A2 isofroms, and LPA receptors LPA1-4 in ovulated COCs and in oviductal epithelium. The eggs ovulated with a form of COCs became denuded of cumulus cells and underwent fragmentation in the absence of fertilization. In vivo experiments of local administration in non-copulated rats demonstrated that eggs denudation was increased by LPA and decreased by anti-ATX antibody and that fragmentation was inhibited by LPA and stimulated by an ATX chemical inhibitor. Furthermore, LPA administration in adult copulated rats increased the rate of cleaved embryos significantly. Obtained results suggest the presence of LPA synthesis and action system in ovulated COCs within the oviductal ampulla and positive actions of LPA possibly at multiple sites around fertilization in rats.

Annexin A1 is a novel target gene of gonadotropin-releasing hormone in LßT2 gonadotrope cells.

Gonadotropin-releasing hormone (GnRH) regulates gonadotropin secretion. We previously demonstrated that the expression of annexin A5 (ANXA5) is stimulated by GnRH in gonadotropes and has a significant role in gonadotropin secretion. It is therefore of interest to know whether other members of the ANXA family, which consists of twelve structurally related members, are also regulated by GnRH. Therefore, the expression of all annexins was examined in LßT2 gonadotrope cells. ANXA4, A5, A6, A7 and A11 were detected in LßT2 cells. The expression of ANXA5 and A1 mRNA was stimulated by a GnRH agonist. An increase in ANXA1 protein by this agonist was demonstrated by western blotting. Immunohistochemistry showed that ANXA1 was present in the nucleus and to a lesser extent in the cytoplasm of some rat pituitary cells. The GnRH agonist induced translocation of ANXA1 to the periphery of LßT2 cells. The presence of ANXA1 in gonadotropes and its increase upon GnRH agonist treatment were confirmed in a primary pituitary cell culture. ANXA1 expression was also demonstrated in the ovary, the testis, the thyroid gland and the pancreas in a different manner to that of ANXA5. These data suggest that ANXA1 is a novel GnRH target gene in gonadotropes. ANXA1 also may be a target of local GnRH in peripheral tissues and may have a different role than that of ANXA5.

Possible role of PPARγ in the negative regulation of ovulatory cascade and luteal development in rats.

Peroxisome proliferator-activated receptor γ (PPARγ), a member of a nuclear receptor family, has been shown to be implicated in various reproductive processes. Here, we evaluated possible roles of PPARγ in ovulation and luteal development in a gonadotropins-primed immature rat model. Immunoreactive PPARγ was expressed in granulosa cells of eCG-stimulated mature follicles, and its expression level decreased following ovulatory hCG stimulus. Intra-bursal treatment with rosiglitazone (a PPARγ agonist) simultaneously with subcutaneously administered hCG blocked the induction of cyclooxygenase-2 and steroidogenic acute regulatory protein (StAR) in preovulatory follicles. Consistently, tissue levels of their respective products, prostaglandin (PG) E2 and progesterone (P4), were reduced, leading to significantly decreased ovulation rate. GW9662, a PPARγ antagonist, was almost ineffective to alter those values. Local treatment with rosiglitazone 24 hr after hCG administration caused reductions in the size, StAR expression and P4 secretion of corpus luteum 48 hr later. Obtained data are possible functional evidence with rats for granulosa cell PPARγ as a negative regulator of PG and P4 synthesis during follicle rupture and transformation to luteal tissue. LH/hCG-induced decreases in PPARγ expression and its activity would be an early component in the proper induction of following ovulatory cascade and luteal development.

Type-dependent differences in Fas expression and phagocytes distribution in rat corpora lutea during natural regression: an immunohistochemical evidence.

Though Fas/Fas ligand (FasL) system-dependent apoptosis is considered to be the primary form of cell death in regressing corpus luteum (CL), the cellular identity and regulation of expression of the ligand and receptor molecules are not fully understood. Here, we focused on immunohistochemical determination of Fas expression during natural regression with comparison of three different types of rat CLs. Detected Fas was in good spatial association with cleaved caspase-3 and FasL proteins and with macrophages and neutrophils. In CLs of the cycle and pseudopregnancy, Fas-positive cell types included large and small luteal (steroidogenic) cells and capillary endothelial cells mainly, and blood-derived immune cells occasionally. Fas signals were abundant at multiple focal inflammatory-like sites. In contrast, Fas signals in CL of pregnancy did not localize in steroidogenic cells, but almost exclusively in endothelial cells and granulocytes. The signals scattered evenly throughout the CL tissue as phagocytes also did. In all CLs types, the numbers of Fas-expressing cells increased transiently after functional inactivation and at the early phase of structural regression. This observation revealed spatio-temporally regulated expression of Fas that was highly associated with apoptotic and phagocytotic systems and type-dependent differences in Fas expression and phagocytes dynamics in naturally regressing CL of rats.

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.

Prolactin Suppression of Gonadotropin-Releasing Hormone Initiation of Mammary Gland Involution in Female Rats.

It has been demonstrated that mammary gland involution after lactation is initiated by accumulation of milk in alveoli after weaning. Here, we report that involution is also dependent on mammary GnRH expression that is suppressed by PRL during lactation. Reduction of plasma prolactin (PRL) by the withdrawal of suckling stimuli increased GnRH and annexin A5 (ANXA5) expression in the mammary tissues after lactation with augmentation of epithelial apoptosis. Intramammary injection of a GnRH antagonist suppressed ANXA5 expression and apoptosis of epithelial cells after forcible weaning at midlactation, whereas local administration of GnRH agonist (GnRHa) caused apoptosis of epithelial cells with ANXA5 augmentation in lactating rats. The latter treatment also decreased mammary weight, milk production, and casein accumulation. Mammary mast cells were strongly immunopositive for GnRH and the number increased in the mammary tissues after weaning. GnRHa was shown to be a chemoattractant for mast cells by mammary local administration of GnRHa and Boyden chamber assay. PRL suppressed the mammary expression of both ANXA5 and GnRH mRNA. It also decreased mast cell numbers in the gland after lactation. These results are the first to demonstrate that GnRH, synthesized locally in the mammary tissues, is required for mammary involution after lactation. GnRH is also suggested to introduce mast cells into the regressing mammary gland and would be in favor of tissue remodeling. The suppression of these processes by PRL is a novel physiological function of PRL.

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.