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Tumor necrosis factor‐α (TNF‐α) is known as a pluripotent cell mediator, and it is implicated in the control of uterine cell growth, differentiation and function during estrous cycle and pregnancy. In this study, we investigated the effect of TNF‐α on endometrial stromal cells derived from rat uterus (rat endometrial stromal cells, RES). RES were isolated from rat endometrium at day 5 of pregnancy. Proliferation activities of RES were measured by using bromodeoxyuridine (BrdU) labeling kit, the productions of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) were measured by enzyme immunoassay kits and the production of matrix metalloproteinases (MMPs) was analyzed by gelatin‐zymography. TNF‐α, as well as epidermal growth factor and fibroblast growth factor‐2, significantly increased the proliferation activity of RES (P<0.05). TNF‐α selectively stimulated the production of PGE2 in RES (P<0.05), but not the production of PGF2α. Additionally, TNF‐α did not stimulate the production of MMPs in RES at the concentration of 5 ng/mL, compared with the control groups (P>0.05). In conclusion, this study demonstrates several regulational functions of TNF‐α on RES using in vitro culture system. The effects of TNF‐α on proliferation and MMP production of RES have been shown for the first time. We believe that these results demonstrate part of the functions of TNF‐α in endometrium and contribute to the better understanding of endometrial functions. J. Exp. Zool. 307A:699–707, 2007. © 2007 Wiley‐Liss, Inc.

Abstract: The present study was performed to establish a real-time monitoring of the cAMP response element binding protein (CREB) signalling using granulosa cells, and to assess the modulation of CREB activity by potential ovarian autocrine/paracrine and oocyte-derived factors. Granulosa cells were isolated from porcine follicles and cultured for 2 days, and then transfected with CRE-containing pGL3. The cells were directly stimulated or cultured with FSH, LH, forskolin, or a permeable cAMP analog, and/or IGF-I, EGF, bFGF, TGF-β2 or TNF-α, or cumulus-oocyte complex (COCs) for the real-time monitoring of CREB signaling. The activation pattern of CREB signaling consisted of three distinct phases, i.e., burst, attenuation and refractory. In contrast to FSH, LH, and forskolin, a cAMP analog induced the prolonged activation, although three distinct phases were observed at its high concentration. Of all the autocrine/paracrine factors, only IGF-I slightly induced CREB activity. On the other hand, TGF-β2 and TNF-α significantly repressed FSH-stimulated transcriptional activation of CREB by 30% (P < 0.05) and 45% (P < 0.05), respectively. Additionally, coculture with COCs caused a significant suppression of transcriptional activation of CREB signaling stimulated by FSH. These results indicate that ovarian autocrine/paracrine factors such as IGF-I, TGF-β2, TNF-α and oocyte-derived factors modulate the CREB signaling. The present study provides a new approach for direct signaling study on transcription factors under the influences of potential factors.

A multifunctional growth and differentiation factor TGF‐β is expressed at various developmental stages, and its principle role may be involvement in organogenesis. The present study was performed to evaluate the temporal and spatial expression of TGF‐β2 mRNA in developing somites of chicken embryos during their early developmental periods. TGF‐βs were expressed in various tissues of the whole embryo obtained at stage 26 (5 days of incubation) as revealed by whole‐mount in situ hybridization. TGF‐β2 mRNA was predominantly expressed in somites as well as the head, branchial arch, wing buds, and leg buds. TGF‐β2 mRNA first appeared in the rostral somites on E4, and its expression sites expanded to the middle range of somites at stage 26. At stages 29–31 (6–7 days), expression in the rostral somites disappeared, and it appeared in the caudal somites. TGF‐β2 expression was also analyzed in sections of the embryo by in situ hybridization. The expression sites of TGF‐β2 were clearly observed in the myotomal somite tips as well as the neural tube. RT‐PCR analysis showed that TGF‐β2 expression was very low in the blastocyte stage embryo and thereafter increased linearly in the whole trunk until stage 26. These data indicate that TGF‐β2 may be a regulatory factor participating in the somitogenesis of chicken embryos. J. Exp. Zool. 303A:323–330, 2005. © 2005 Wiley‐Liss, Inc.

Gene silencing using small interfering RNA (siRNA) is not established in avian species. The present study was performed to evaluate RNA interference (RNAi) in the chicken embryo by using a dual fluorescence reporter assay, a plasmid encoding green fluorescent protein (GFP) and a plasmid encoding red fluorescent protein (RFP). The siRNA targeting the GFP mRNA sequence (GFP‐siRNA) with both plasmids was introduced into cultured cells and whole embryos by lipofection and microelectroporation, respectively. GFP‐ and RFP‐expressed cells and embryos were observed under fluorescent microscopy and analyzed by flow cytometer, and their mRNAs were analyzed by reverse transcription‐PCR (RT‐PCR). The strong fluorescence was observed by introducing both plasmids into cells. The intensity of the green fluorescence generated by GFP was greatly suppressed by introducing GFP‐siRNA. RT‐PCR analysis showed that introducing GFP‐siRNA also decreased GFP mRNA levels. In contrast to GFP, the intensity of the red fluorescence generated by RFP and the RFP mRNA levels remained unchanged. In whole embryos, also, introducing GFP‐siRNA specifically suppressed GFP expression, and the suppression was maintained for at least 72 h. Consequently, it was concluded that the gene silencing using siRNA is applicable to analyzing the function of genes of interest during avian embryogenesis. J. Exp. Zool. 301A:820–827, 2004. © 2004 Wiley‐Liss, Inc.

The endothelial type (NOS-3) of three isoforms of nitric oxide (NO) synthase occurs in porcine oocytes and granulosa cells, but the regulation of NO synthesis in oocytes remains unknown. The present study was designed to evaluate steroid control in the process of oocyte NO synthesis. Cumulus-oocyte complexes (COCs), obtained from small-sized antral follicles of immature porcine ovaries, were cultured in estrogen-deprived medium, and the effect of steroids or steroid-free porcine follicular fluids on the NO release from oocytes was investigated. Oocytes that were isolated from cultured COCs were incubated with 1 μM ionomycin. The NO metabolites were identified using a NO detector-high-pressure liquid chromatography system. Oocytes from COCs cultured with 10 nM 17β-estradiol (E2) released NO in response to ionomycin, whereas progesterone and testosterone had little effect on the synthesis of NO. An inhibitor of NOS suppressed the synthesis of NO. The maximal synthesis was observed after a 15 h-culture with E2. However, oocytes freshly obtained from antral follicles did not response to ionomycin, and the E2action was suppressed by the addition of steroid-free follicular fluids. Analyses of RT-PCR and Western blotting showed that E2did not increase NOS-3 expression. In addition, estrogen receptor β was detected in oocytes and cumulus cells, and estrogen receptor α was detected only in cumulus cells. These findings suggest that oocyte NOS-3 is promoted for the synthesis of NO by E2without increases in NOS-3 expression, but the synthesis of NO is suppressed, at least in the oocytes of early antral follicles.

The development of chicken embryonic gonads is locally regulated by the systematic action of growth factors. Recently, we used suppressive subtraction cloning to identify transforming growth factor β2 (TGF-β2) as a growth factor gene preferentially expressed in chicken embryonic ovaries and testes during the early periods of development (Hattori et al. 2002a. Prominent expression of transforming growth factor β2 gene in the chicken embryonic gonad as revealed by suppressive subtraction cloning. Gen Comp Endocrinol 125:311–316). In the present study, the function of TGF-β2 in chicken embryonic gonads was investigated using a serum-free culture system in the presence of several growth factors, which may behave as mitogenic or survival factors of primordial germ cells (PGCs). Chicken germinal ridges containing PGCs and germinal ridge stroma cells (GRSCs) were collected from six-day embryos. Addition of TGF-β2 caused a dose-dependent inhibition of the number of co-cultured PGCs and GRSCs in the presence of these growth factors. However, there was no obvious difference between embryonic ovaries and testes in the effects of TGF-β2. Immunocytochemical analysis using anti-SSEA–1 antibody revealed that TGF-β2 induced fragmentation of PGCs. Expression of the TGF-β2 gene was estimated in the co-cultured PGCs and GRSCs by semi-quantitative RT-PCR. The mRNA level of TGF-β2 was significantly suppressed in the presence of the growth factors. These results suggest that TGF-β2 is a gonadal regulator preferentially expressed at the early stages of chicken embryonic development and reduces the growth of PGCs and GRSCs by suppressing proliferation. However, expression of TGF-β2 may be controlled by mitogenic or survival factors of PGCs. J. Exp. Zool. 301A:290-296, 2004. © 2004 Wiley-Liss, Inc.

The present study was designed to investigate the localization of endothelial nitric oxide synthase (eNOS) in porcine oocytes and its possible function during in vitrodevelopment. RT-PCR and immunoblotting analyses revealed the presence of eNOS in the oocytes prepared from small follicles, with an amplified product of 456 bp and an apparent mol wt of 130 kDa, respectively. The synthesis of oocyte NO was suppressed during a 72-h culture of cumulus-oocyte complexes in the presence of follicle-stimulating hormone (FSH), but not luteinizing hormone (LH). However, the decrease in NO synthesis did not result from the levels of eNOS mRNA and its protein, as revealed by analyses of RT-PCR and Western blot analysis, suggesting that expression of oocyte eNOS is not dependent upon gonadotropin stimulation. In proliferated cumulus cells, LH receptor mRNA expression was detected after a 48-h culture with FSH, as revealed by RT-PCR analysis. mRNA expression was inhibited by an NO-releasing agent (S-nitroso-N-acetyl-DL-penicillamine) after an additional 24-h culture. These results suggest that oocytes may release eNOS-derived NO as a signal for somatic cells to steadily suppress the development of cumulus cells, if not FSH stimulation. Conversely, the synthesis of NO is suppressed during the action of FSH on the cumulus cells with no changes in eNOS expression.

Abstract. The present study was performed to evaluate the different carbohydrate structure of rat LH isoelectric components related to their intrinsic biological activities. Terminal sialic acid residues were essential to the formation of multiple LH components observed in the isoelectric focussing profile, which was proved by their interaction with Ricinus communis agglutinin-120 following neuraminidase treatment, and the conversion of component F (pI, 10.0) to less alkaline components after incubation with liver Golgi membrane fraction in the presence of CMP-NeuNAc. The affinity studies using lentil lectin indicated that component F was not an asialo form of component A (pI 8.4). The serial removal of sialic acid residues from these components led to increases in the steroidogenic activity, owing to increases in the activation of the receptor-adenylate cyclase system. The enhancement of the steroidogenic activity by desialylation was very great in component A'(pI, 8.0) (751% increase), and decreased with increasing pI. It can be concluded that the different biological potencies of intact LH components are attributable principally to terminal sialic acid residues. However, the peripheral chains of asialo oligosaccharides of less alkaline components (pI, 8.0, 8.4) seem to prevent the maximal cellular responses, since their desialylated forms did not attain the maximum activity.

The present study was designed to prove the carbohydrate-binding proteins interacting with cell surface sialyllactosylceramide (GM3, NeuAcα2→3Galβ1→4Glcβ1→ 1′Cer), which is highly expressed during differentiation of rat ovarian granulosa cells. As a specific ligand for the sialyllactose (SL)-binding proteins on granulosa cells, we used a radioiodinated multivalent SL-linked albumin (Alb-(SL)17). The specific association of the ligand to the putative proteins on the intact cells was competitively inhibited by GM3more effectively than other gangliosides, sialyllactotetraosylceramide, sialylneolactotetraosylceramide, and several glycoproteins with N-linked oligosaccharides. However, the proteins had no specificity for the side chain (N-acetyl or N-glycolyl forms) of sialic acid in GM3. Scatchard analysis of Alb-(SL)17binding showed high (Kd= 6.4 × 10−10M) and low (Kd= 3.1 × 10−8M) affinity population of binding sites. By direct binding of 125I-Alb-(SL)17to SL-binding proteins on Western blots, the putative proteins with molecular masses of 35, 18, and 14 kDa were detected. The interaction of the multivalent derivative with these binding proteins was differently modulated by Ca2+and Mn2+. The SL-binding proteins occurred in immature granulosa cells and progressively decreased during differentiation, whereas their endogenous ligand GM3increased. These results indicate that relatively low molecular weight SL-binding proteins exist on the surface of immature granulosa cells and that they may serve as receptor sites for newly synthesized GM3during differentiation.

Abstract. Biosynthesis and secretion of LH were investigated using primary cultures of rat gonadotropes following exposure to swainsonine, an inhibitor of N-linked oligosaccharide processing. The inhibition of terminal glycosylation by swainsonine was assessed by the increase in incorporation of [3H]mannose and the decrease of incorporation of [3H]galactose into the trichloroacetic acid-insoluble and anti-ovine LHβ immunoprecipitable fractions. This inhibitor led to an increase in cellular LH with high affinity for concanavalin A and a decrease of LH with less affinity for the lectin, although the cellular LH was not decreased. The swainsonine-treated cells accumulated highly basic LH (pI, 9.6, 10.0) in a time-dependent manner. Although the treated cells showed a decrease in the responsiveness to hormonal stimulation, there were no differenes in the rate and extent of LH secretion induced by maximal stimulation with 100 nmol/I GnRH between the control and treated cells over 24 h. The accumulation of highly basic LH molecules did not result from a decrease in their secretion, since they were secreted in the presence and absence of GnRH. It is concluded that significant changes in N-linked oligosaccharide structure of LH have no effect upon its secretion.

Chicken primordial germ cells (PGCs) dynamically migrate towards the prospective gonadal area through the germinal crescent region and bloodstream at early embryonic stages from stage X (Eyal-Giladi and Kochav) to stage 29 (Hamburger and Hamilton) and they differentiate into oocyte or sperm in gonad. To date, chicken PGCs have been mainly identified by histochemical and immunohistochemical methods such as PAS staining and immunohistochemistry using antibodies against stage-specific embryonic antigen-1 (SSEA-1) and embryonic mouse antigen-1 (EMA-1). Chicken PGCs can be also identified by some morphological aspects, such as large size and relatively big nucleus etc. However, these detection methods are not completely specific for chicken PGCs, and therefore they may not reflect precise characterization of PGCs. Therefore, the origin, migration, and differentiation of chicken germ cells are not fully understood. The objective of this study is to isolate chicken dead end homologue (CDH) whose expression is highly specific to germ cells in other species and investigate expression of CDH mRNA and protein in the chicken germ cell. As revealed by rapid amplification of cDNA ends (RACE)-PCR method, we have isolated the 1456 bp-nucleotide sequence of full-length CDH cDNA coding an open reading frame of 329 amino acids. When compared with dead end homologue proteins of other vertebrates over the entire alignment, a significant homology was obtained in human (54%), mouse (45%), Xenopus (47%), and zebrafish (34%). The homology of only RNA binding motif present in CDH at amino acids 54–133 was higher than that of entire alignment. As revealed by RT-PCR, CDH mRNA was detected in PGCs circulating in blood stream at stage 15 and SSEA-1 positive cells at early blastoderm, which were separated by micromanipulator and magnetic cell separation system (MACS), respectively. The temporal and spatial expression of CDH mRNA was also investigated by in situ hybridization (ISH) on PGCs circulating in blood stream, section of gonads and whole-mount embryos. CDH expression was detected in PGCs circulating in blood stream, gonadal anlage at stage 29, and the testis and ovary of E18 chicken embryo, that was similar to the results of chicken vasahomologue expression and PAS staining. Chicken embryos from stage X to stage 20 were also subjected to ISH. The hybridized samples were then sectioned to analyze the translocation of CDH positive cells. CDH positive cells could be counted from stage X to stage 4, with minimally 30 cells at the blastoderm and approximately 260 cells at the germinal crescent. Thus, specific expression of CDH mRNA has been observed in chicken PGCs located at the blastoderm, germinal crescent, and prospective gonadal area by ISH and RT-PCR analyses. We also raised polyclonal antibody against CDH protein. Specific expression of CDH protein in chicken germ cell was confirmed by western blot and immunohistochemical staining. Collectively it is concluded that isolated CDH is specifically expressed in chicken germ cells. This research was supported in part by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Sciences (JSPS; 16380200) (to M-A. H) and Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (No. 09744).

In rodents, implantation involves a direct interaction between embryo and uterus that is initiated by a recognition of the hatched, free embryo, followed by membrane apposition. Then, adhesion between the trophectoderm-trophoblast and endometrial luminal epithelium occur. Finally, the embryo passes through the luminal epithelium and epithelial basement membrane and implants in the endometrial stroma. During implantation, dramatic tissue remodeling occurs in the endometrium, and it is thought that matrix metalloproteinases (MMPs) are the key regulators in this process. MMPs are the enzymes that degrade components of the extracellular matrix and are members of the Zinc Metalloproteinase family, having 28 distinct isoforms. There are several studies showed that MMPs were significantly expressed in the rat uterus during the implantation phase. However, the expressional profile of MMPs and its regulation function is not fully elucidated. Additionally, the expression of MMPs during decidualization which is one of the important physiological process in implantation, are still not fully understood. The purpose of the present study is to investigate the expression of MMPs during the implantation phase in the rat uterus. We firstly analyzed the expressional profile of MMPs during the implantation phase. Then the activity of MMPs during decidualization was analyzed by zymography using in vitro decidualization system of the cultured rat endometrial stromal cells (RES). Uteri were obtained from sexually mature rats during the estrous cycle and various stages of early pregnancy (Day 1.5, 3.5, 5.5 and 7.5 of gestation). Total RNA was collected from these uteri. The expression of ß-actin and MMPs (MMP-1, -2, -3, -7, -9 and -11) mRNA were examined by reverse transcription-polymerase chain reaction (RT-PCR). The expression patterns of MMP-2, -3 and 9 mRNA were evaluated at each stage by quantitative real time RT-PCR. Quantitative real time RT-PCR was carried out using a Roche Light Cycler 1.5 system (Roche). The Taqman Master kit (Roche) in combination with the Universal Probe Library for Rat (Roche) was used to assess gene expression. In vitro decidualization of the RES was introduced by the treatment of medroxyprogesterone acetate (MPA). After the treatment for 24h, conditioned medium was collected and the production of MMPs were measured by gelatin zymography. The quantitative measurement of MMPs was performed by densitometric analysis of zymogram using an ImageJ program (Version 1.36). Six MMPs (MMP-1, -2, -3, -7, -9 and 11) mRNA were expressed at estrus cycle and early pregnancy. Expression of MMP-2, -3 and -9 mRNA was not significantly changed during the estrus cycle and early pregnancy. Additionally, there were no significant differences in the expressions of MMP-2 and -9 mRNA during in vitro decidualization. However, the active form of MMP-2 and -9 was significantly increased after the treatment for in vitro decidualization of the RES. These results indicate that the function of MMPs during the implantation phase is not regulated by mRNA expression, but by the activation of MMPs. It is also suggested that RES produces some factor or factors that can activate MMPs during decidualization. This research was supported by a grant from the Ministry of Education, Culture, Sport, Science, and Technology of Japan (Kiban-kenkyu C 18580282).

Circadian rhythm is generated by genetically determined biological clock with the 24-hour light-dark cycle. In mammals, the central clock is located in the suprachiasmatic nucleus of the hypothalamus. At molecular level, the clock system is composed of interlocked transcriptional and translational feedback loops. The CLOCK and BMAL1, associated as heterodimers, bind to the E-box enhancer and positively drive the expression of Period genes, Cryptochrome genesand Rev-erbAgenes whose proteins, in turn, form multimeric complexes and feed back to repress the transactivation by CLOCK/BMAL1 in the nucleus. Circadian rhythms also exist in a variety of peripheral tissues, and are synchronized by the central pacemaker through complex interaction of neural, humoral, and behavioral cues. Ovarian granulosa cells (GCs) undergo proliferation and differentiation during folliculogenesis under the regulation of FSH and locally produced factors. The granulosa cells are finally differentiated into luteal cells (LCs) after ovulation. However, currently little is known about whether and how circadian clockwork could be affected during folliculogenesis and luteinization. In the present study, the real-time monitoring system was employed to evaluate the clockwork in the GCs and LCs obtained from transgenic rats that have been constructed with Per2promoter (chr1 (-):93289505-93293019)-destabilized luciferase reporter gene. The ovaries were obtained from DES-, and eCG and/or hCG-treated immature rats, and the GCs and LCs were prepared to culture in DMEM/F12 supplemented with 10% FBS. Cultured cells were treated for 2 h with or without dexamethasone in serum-free medium, then the medium was replaced with 2 ml of DMEM/F12 containing 5% FBS, 15 mM HEPES, and 0.1 mM luciferin. The cells were also exposed to gonadotropins (FSH or LH) without dexamethasone. Real-time monitoring of bioluminescence was performed at 37C using Kronos AB-2500 interfaced to computer for continuous data acquisition. The photon counts were integrated for 1 min at intervals of 8 min. In immature GCs from DES-treated rats, no oscillation of Per2expression was observed without dexamethasone treatment, but one oscillation was induced after dexamethasone or FSH treatment. In mature GCs from eCG-treated rats, irregular Per2oscillation was observed without dexamethasone treatment, but persistent oscillation was induced after dexamethasone or LH treatment. In LC from eCG/hCG-treated rats, persistent oscillation was induced with or without dexamethasone or LH treatment. Collectively, it is concluded that circadian clockwork system in ovarian cells is developed during folliculogenesis and luteinization, and synchronized by gonadotropins as well as dexamethasone.

The primordial germ cells (PGC) are progenitors that differentiate into the germ cells in the gonads from which the gametes are ultimately derived. The molecular basis for PGC specification, proliferation and differentiation has attracted interest not only for developmental biology but also for practical utilization of genetic resources such as medicine and animal production by generation of knock-in/knock-out animals. A proliferation system of PGCs should be established to generate the genetic modified chicken. However, the mechanism of PGC proliferation remains to be unknown. So far, germ-cell-specific genes such as vasa, dead end and Deleted in Azoospermia-Like have been isolated in animal species including mouse, human, monkey, Xenopus, chicken and Zebrafish. These genes code RNA binding proteins that are mainly expressed in germ cells. Based on the nucleic acid sequences of these genes, we raised specific polyclonal antibodies against recombinant proteins. In addition, a gene coding chicken neuron-derived neurotrophic factor (NENF), one of the neurotrophin, was isolated from PGCs by subtracted PCR-generated cDNA libraries. In the present study, chicken NENF-GST fusion protein was produced and immunized to generate anti-chicken NENF antibody. The expression and function of chicken NENF were investigated in chicken PGCs. Chicken NENF protein with 164 amino acid sequences of coding region was prepared for the antigen. The sequences were inserted in the pGEX-6P-1 vector. The chicken NENF-GST fusion protein was expressed in Escherichia coli. Then the recombinant protein was purified and injected into a female rabbit for the production of antiserum. The resulting antiserum was purified by affinity chromatography using chicken NENF-coupled column. PGCs were cultured by a cell culture system of blastoderm that was collected at stage X. The blastoderm was dissociated in a medium containing 15% knock out serum replacement and 100 ng/ml mouse LIF, and cell suspension was divided onto a 48-well gelatin-coated dish and cultured for 7 days. The neutralization effect of anti-chicken NENF antibody was investigated for estimation of PGC proliferation activity. Fluorescent immunocytochemistry using the anti-CVH, anti-cDAZL, anti-SSEA-1 and anti-chicken NENF antibodies was performed on PGCs collected from a vitelline artery of chicken embryos at stage 14 and a cell culture system of blastoderm. PGCs collected from blood samples were positively stained with anti-CVH, anti-SSEA-1 and anti-chicken NENF antibodies. After culture of the blastodermal cells for 7 days, PGCs greatly proliferated by approximate 1,500 times from 30 PGCs at stage X. Of interest, PGCs proliferated in cultured colonies were positively stained with anti-CVH, anti-SSEA-1 and anti-chicken NENF antibodies, indicating that the undifferentiated property of PGCs is maintained. However, the proliferation of PGCs was inhibited approximate 30% by addition of purified anti-chicken NENF antibody. Consequently, it is strongly suggested that chicken NENF is a novel proliferation factor of PGCs as autocrine fashion. Chicken NENF may be a useful tool for studies of molecular mechanism of PGC proliferation and differentiation.(poster)

The circadian rhythm is a fundamental biological system in mammals involved in the regulation of various physiological functions such as energy metabolism, endocrine system and reproduction. The molecular oscillations underlying the generation of circadian rhythmicity in mammals develop gradually during ontogenesis. However, the process of mammalian cellular circadian oscillator formation remains unknown in differentiated granulosa cells and apoptotic luteal cells. Two studies were conducted to detect the expression of clock genes and the related regulation mechanisms in different ovarian cell types during differentiation and apoptosis. Immature female Per2-dLuctransgenic rats (21-23 days) were used as experiment objects in the present study. In the first study, immature granulosa cells were collected from preantral follicles of diethylstilbestrol-injected rats. Using a bioluminescence system to monitor clock gene expression, the circadian expression of Per2was measured as the emblem for the maintenance of circadian rhythms. Granulosa cells were cultured with FSH in a serum-free medium containing BSA for 2 days. Then, the medium was replaced with the serum-free medium, dexamethasone or LH, together with luciferin for real-time monitoring examination during the next 3 days. Total RNA samples were collected around the second peak every 4 h interval and Bmal1and Per2transcripts were measured by qRT-PCR. Simultaneity, LH receptorwas measured as a differentiation marker of rat granulosa cells. A few oscillations of Per2promoter activity were detected and Bmal1transcript level was low in granulosa cells during FSH treatment. However, the generation of circadian oscillation with high amplitudes was induced in granulosa cells after treatment with dexamethasone and LH. Interestingly, a high expression of Bmal1was induced with reverse-phase to Per2gene. These findings imply that the circadian molecular oscillator develops during differentiation in vitro. On the other hand, Per2over-expression has been reported to induce apoptosis of cancer cells, suggesting the link between the circadian clock and apoptosis. In the second study, the circadian oscillations of clock genes were analyzed in the luteal cells during apoptosis. The luteal cells were prepared from rats primed with 50 IU eCG and followed by 25 IU hCG 60 h later. The cells were collected on day 5 after hCG injection and cultured for 2 days in a culture medium containing 10% FBS and then treated with progesterone receptor antagonist (mifepristone) for additional 2 days followed by addition of 100ng/ml anti-Fas antibody. Total RNA samples were collected around the second oscillation during treatment of anti-Fas antibody for measurements of Per2, Bmal1, Clock, Rev-erbalpha, Fas, caspase-3and other clock controlled or apoptosis related genes by qRT-PCR. In apoptotic cells, decreased expression of Bmal1was observed, whereas expression of Rev-erbalpha increased. The alteration of these genes might link to apoptosis of luteal cells. Collectively, the present results suggest that Bmal1especially contributes to switch-ON and switch-OFF upon initiation of differentiation and apoptosis.(poster)

Pregnancy is accompanied by dramatic tissue remodeling of uterus, but it is remains unclear because of its complicated processes. Steroid hormone, progesterone (P4) and estradiol critically regulate uterine receptivity that is needed to establish primary pregnant reaction, implantation and decidualization. P4-responsive gene, Indian hedgehog (Ihh) is one of mediators of it, however, it is unknown about its downstream components and physiological process. Ihh is one of Hedgehog ligands that play a crucial role in embryogenesis and tumorigenesis by cell proliferation, cell fate determination and polarization through epithelial-mesenchymal interaction. There are three ligands in mammal, called Desert hedgehog (Dhh), Sonic hedgehog (Shh) and Ihh. Not only Ihh, other hedgehog ligands also regulate organogenesis, germ cell migration and germ cell maturation in reproductive tract. For example, male mouse with Dhh null mutation causes abnormal testis development and is infertile, and Shh regulates angiogenesis and tumorigenesis in various tissues. Moreover three hedgehog genes are co-expressed in different manner and in different cell type of mouse ovary. Therefore we hypothesized that more hedgehog genes are expressed and function in uterus during pregnancy. In the present study, we firstly researched what hedgehog genes are expressed in rat uterus during early pregnancy and how their expressions are changed temporally by quantitative real-time RT-PCR. The results showed that all of three hedgehog mRNAs were detected in rat uterus at proestrus stage and early pregnant (1.5, 3.5, 5.5, 7.5 days postcoitus (dpc)). Ihh mRNA expression was variable and was peak at 3.5 dpc, while Dhh and Shh mRNA expressions remained unchanged, indicating that Ihh mainly regulates tissue remodeling in rat uterus during early pregnancy. Next, we detected cells expressed hedgehog genes by section in situhybridization. The results showed that Ihh mRNA was expressed in luminal and glandular epithelium, but Dhh and Shh mRNAs were not able to detected, indicating that Dhh and Shh expression levels were baseline or low. Finally, Dhh and Ihh CDS sequences were decided. Sequences got in this study corresponded with those analized in silico. Their amino acid sequences represent high homology with those of mouse or human, respectively. In summery, our results elucidates that Ihh is a predominant hedgehog protein in rat uterus during early pregnancy, while other hedgehog genes are potentially expressed and might play a role in other conditions such as cancer. This observation helps our understanding of basic molecular mechanism of uterine remodeling during early pregnancy, leads to woman reproductive health care. This research was supported by a grant from the Ministry of Education, Culture, Sport, Science, and Technology of Japan (Kiban-kenkyu C 18580282).

During embryo implantation in rodents and human, the endometrial stromal cells proliferate and differenciate into decidual cells. This process ultimately results in the formation of the maternal component of the placenta. Induction of decidualization of endometrial stromal cells is possible in an in vitro cell culture system. This could be a powerful tool for investigating the complex process of the decidualization in vitro. However, the methods to induce in vitro decidualization differ according to speicies or cell types and a more stable or universal system has not yet been developed. The purpose of present study is to establish an in vitro decidualization system in primary cultured rat endometrial stromal cells (RES). In order to find the most effective in vitro decidualization system, several decidual treatments were compared by their resulting gene expression and morphological change. Then, using the in vitro decidualization system, the effects of gonadotropins on decidualization, and the changes of MMP production in RES were investigated. The RES, derived from the uterus at implantation period (day 5 of pregnancy), were treated with 1) medroxyprogesterone acetate and dibutyryl-cyclic adenosine monophosphate (db-cAMP) (MPA treatment), 2) estradiol and progesterone (E+P) or 3) arachidonic acid (AA). Morphological changes in RES were introduced at 2 days (MPA) and 4 days (E+P and AA) after each treatment. After 24 h of the treatments, cells responded to all of the stimulations by expressing desmin mRNA. However, dPRP mRNA was only expressed in the MPA treated cells. Immunocytochemical study shows that desmin was expressed in MPA treated cells. We concluded that MPA treatment in RES was the most effective method for introducing decidualization. Therefore, we used the MPA treatment to induce in vitro decidualization of RES in further studies. Morphological changes introduced by decidual treatment occurred 2 days after treatment in mature rat derived RES, whilst, the changes in immature rat derived RES took a longer period (4 days). Furthermore, desmin and dPRP mRNA did not express in the RES derived from immature rat uteri. Similar to the RES from the implantation period, RES from the diestrous stage responded to stimulation by expressing desmin and dPRP mRNA. Injection of gonadotropins (eCG and hCG) to the immature rats (3 weeks) did not affect morphological changes of RES, before and after the decidual treatment. However, desmin and dPRP mRNA were expressed in RES derived from gonadotropin injected immature rats after the tratment with MPA. Expression of MMP-2 and -9 mRNA did not change after decidual treatment of RES examined by real-time PCR. However, the results of gelatinzymography showed that the active form of MMP-2 and -9 significantly increased after in vitro decidualization (P<0.05). We clarified that MPA + db-cAMP treatment is the most effective to induce in vitro decidualization in RES. Using this system, it was suggested that sexual maturation and gonadotropins are important in RES for their ability for decidualization. Furthermore, the activity of MMP-2 and -9 is suggested to be increased during decidualization without a corresponding increase of the expression of those genes. This research was supported by a grant from the Ministry of Education, Culture, Sport, Science, and Technology of Japan (Kiban-kenkyu C 18580282).

Circadian rhythm is generated by genetically determined biological clock with the 24-hour light-dark cycle. In mammals, the central clock is located in the suprachiasmatic nucleus of the hypothalamus. At molecular level, the clock system is composed of interlocked transcriptional and translational feedback loops. The CLOCK and BMAL1, associated as heterodimers, bind to the E-box enhancer and positively drive the expression of Period genes, Cryptochrome genes and Rev-erbA genes whose proteins, in turn, form multimeric complexes and feed back to repress the transactivation by CLOCK/BMAL1 in the nucleus. Circadian rhythms also exist in a variety of peripheral tissues, and are synchronized by the central pacemaker through complex interaction of neural, humoral, and behavioral cues. A variety of factors including cAMP analog, forskolin, phorbol-12-myristate-13-acetate, endothelin, prostaglandin E2, fibroblast growth factor, calcium ionophors, retinoic acid, glucocorticoids, and parathyroid hormone have been unraveled having the potentials to reset circadian rhythms in peripheral cells. The uterus is regulated under both estrogen and progesterone, but very little is currently known about the molecular mechanism underlying the sex steroidal effects on circadian clockwork. In the present study, the real-time monitoring system was employed to evaluate the clockwork in the uterus stromal cells obtained from transgenic rats that have been constructed with Per2 promoter (chr1 (-):93289505-93293019)-destabilized luciferase reporter gene. The uteri were obtained from eCG and/or hCG-treated immature rats and pregnant rats (day 5 gestation) and the stromal cells prepared were cultured to be confluent in DMEM/F12 supplemented with 2% FBS. Cultured cells were treated for 2 h with or without dexamethasone or forskolin in serum-free medium, then the medium was replaced with 2 ml of DMEM/F12 containing 5% FBS, 15 mM HEPES, and 0.1 mM luciferin. Real-time monitoring of bioluminescence was performed at 37°C using Kronos AB-2500 interfaced to computer for continuous data acquisition. The photon counts were integrated for 1 min at intervals of 8 min. The cells prepared from eCG-treated immature rats (high estrogen level) showed irregular Per2 oscillation after treatment with dexamethasone or forskolin, although Per2 expression was elevated. In contrast, persistent oscillation was observed in the cells prepared from eCG/hCG-treated immature rats (high progesterone level). Interestingly, the irregular Per2 oscillation was converted to persistent oscillation with about 24-h cycle in the presence of progesterone. In pregnant rat uterus cells, also irregular Per2 oscillation was observed with estradiol, whereas the oscillation was converted to persistent oscillation with progesterone. Collectively, it is concluded that both estradiol and progesterone are involved in modification of circadian rhythm via direct regulation of the expression of clock genes.

Chicken primordial germ cells (PGCs) dynamically migrate towards prospective gonadal area through germinal crescent region and bloodstream at early embryonic stages. So far, chicken PGC has been mainly identified by histochemical and immunohistochemical methods or its morphological characteristics. However, its origin, migration and differentiation are not fully understood, because of a lack of specific PGC molecular markers and difficulty in detection of chicken primordial germ cells. Recently, dead end, a novel vertebrate germ plasm component coding RNA binding protein was discovered in zebrafish and its expression was proved to be highly specific to germ cells. In the present study, we have isolated the chicken dead end homologue (CDH) to clone its full-length cDNA by Rapid amplification of cDNA ends (RACE)-PCR. Whole-mount in situ hybridization (WISH) analysis detecting CDH mRNA was also performed both in vivo and in vitro. The nucleotide sequence (1,465 bp) of full-length-cDNA revealed an open reading frame for 329 amino acids. The RNA binding motif (Prosite database profile PS50102) in N terminal half present in CDH at 54–133 was highly homologous to those in human (65%), mouse (66%), and Xenopus (66%) Dead end proteins, whereas its similarity to zebrafish Dead end protein was low (55%). There are also great variations in both the N-terminal and C-terminal amino acid sequences among these vertebrates. Our WISH analysis showed that CDH mRNA specifically expressed in primordial germ cells. CDH mRNA positive cells were also detected in embryonic stem cell (ES)-like colonies in vitro derived from blastderm at stage X. Consequently, detection of CDH expression is useful to identify chicken PGCs both in vivo and in vitro. This reseach was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (No. 09744) (to S.A.). (poster)