BackgroundLong-term maintenance of avian primordial germ cells (PGCs) in vitro has tremendous potential because it can be used to deepen our understanding of the biology of PGCs. A transgenic bioreactor based on the unique migration of PGCs toward the recipients' sex cord via the bloodstream and thereby creating a germline chimeric bird has many potential applications. However, the growth factors and the signaling pathway essential for inducing proliferation of chicken PGCs are unknown.Methodology/Principal FindingsTherefore, we conducted this study to investigate the effects of various combinations of growth factors on the survival and proliferation of PGCs under feeder-free conditions. We observed proliferation of PGCs in media containing bFGF. Subsequent characterization confirmed that the cultured PGCs maintained expression of PGC-specific markers, telomerase activity, normal migrational activity, and germline transmission. We also found that bFGF activates the mitogen-activated protein kinase kinase/extracellular-signal regulated kinase (MEK/ERK) signaling. Also, the expression of 133 transcripts was reversibly altered by bFGF withdrawal.Conclusions/SignificanceOur results demonstrate that chicken PGCs can be maintained in vitro without any differentiation or dedifferentiation in feeder free culture conditions, and subsequent analysis revealed that bFGF is one of the key factors that enable proliferation of chicken PGCs via MEK/ERK signaling regulating downstream genes that may be important for PGC proliferation and survival.

Cathepsins (CTS) are peptidases that have biological roles in degrading extracellular matrix, catabolism of intracellular proteins, and processing of prohormones. Expression of CTSB, CTSD, CTSH, CTSK, CTSL, CTSS, and CTSZ genes was detected in the endometria of cyclic and early pregnant ewes with distinct temporal and spatial expression patterns. In the d 18 and 20 conceptus, expression of CTSB, CTSD, CTSL, and CTSZ mRNA was detected in the trophectoderm. Of particular note, CTSL mRNA was the most abundant CTS mRNA in the ovine endometrium and detected only in the luminal epithelium and superficial glandular epithelium of cyclic and pregnant ewes. CTSL mRNA increased 8-fold between d 10 and 18 in endometria of pregnant ewes, whereas it declined between d 14 and 16 in cyclic ewes. CTSL protein was also detected in conceptus trophectoderm, and pro-CTSL was detected in uterine flushings from ewes between d 12 and 16 of pregnancy. In ovariectomized and catheterized ewes, CTSL mRNA in the endometrium was increased by progesterone and intrauterine injections of ovine interferon (IFN)tau. Other endometrial CTS genes were also regulated by progesterone alone (CTSB, CTSK, CTSS, and CTSZ) or progesterone and IFNtau (CTSH, CTSK, CTSS, and CTSZ). These results indicate that CTS of endometrial and conceptus origin may regulate endometrial remodeling and conceptus implantation, endometrial CTS genes are regulated by ovarian and placental hormones, and CTSL is a novel IFNtau-stimulated gene expressed only in luminal epithelium and superficial glandular epithelium of the endometrium.

There is a dialogue between the developing conceptus (embryo-fetus and associated placental membranes) and maternal uterus which must be established during the peri-implantation period for pregnancy recognition signaling, implantation, regulation of gene expression by uterine epithelial and stromal cells, placentation and exchange of nutrients and gases. The uterus provide a microenvironment in which molecules secreted by uterine epithelia or transported into the uterine lumen represent histotroph required for growth and development of the conceptus and receptivity of the uterus to implantation. Pregnancy recognition signaling mechanisms sustain the functional lifespan of the corpora lutea (CL) which produce progesterone, the hormone of pregnancy essential for uterine functions that support implantation and placentation required for a successful outcome of pregnancy. It is within the peri-implantation period that most embryonic deaths occur due to deficiencies attributed to uterine functions or failure of the conceptus to develop appropriately, signal pregnancy recognition and/or undergo implantation and placentation. With proper placentation, the fetal fluids and fetal membranes each have unique functions to ensure hematotrophic and histotrophic nutrition in support of growth and development of the fetus. The endocrine status of the pregnant female and her nutritional status are critical for successful establishment and maintenance of pregnancy. This review addresses the complexity of key mechanisms that are characteristic of successful reproduction in sheep and pigs and gaps in knowledge that must be the subject of research in order to enhance fertility and reproductive health of livestock species.

The objective of this study was to determine the temporal and spatial expression patterns of genes encoding transporters, as well as selected secreted proteins that may be regulated by progesterone (P4) and/or the presence of the conceptus in the bovine endometrium. Estrus-synchronized beef heifers were randomly assigned to either: 1) pregnant, high P4; 2) pregnant, normal P4; 3) cyclic, high P4; or 4) cyclic, normal P4. Uteri were collected on days 5, 7, 13, and 16 of the estrous cycle or pregnancy. Localization of mRNAs for ANPEP, CTGF, LPL, LTF, and SLC5A1 in the uteri was determined by radioactive in situ hybridization, and expression quantified in the endometria by quantitative real-time PCR. ANPEP localized to luminal (LE) and superficial glandular (sGE) epithelia of all heifers on days 5 and 7 only. SLC5A1 mRNA was detected in the LE and sGE on days 13 and 16 in all heifers, and expression increased on day 16 in pregnant groups. CTGF localized weakly to the LE and GE on days 5 and 7 but increased on days 13 and 16 with an increase (P < 0.05) in CTGF expression in high P4 (day 7) and pregnant heifers (day 16). Both LPL and LTF localized to the GE only on days 5 and 7. In conclusion we have characterized the temporal expression pattern of these genes and modulation of their transcript abundance by P4 (CTGF, LPL) and/or the conceptus (CTGF, SLC5A1) likely modifies the uterine microenvironment, enhancing histotroph composition and contributing to advanced conceptus elongation.

MicroRNAs (miRNAs) play a critical role in determining the differentiation fate of pluripotent stem cells and germ cells in mammals. However, the mechanism(s) of miRNA-mediated posttranscriptional regulation with regard to lineage specification and differentiation in chick development require further investigation. Therefore, we conducted miRNA expression profiling to explore specific miRNA signatures in undifferentiated blastoderm and primordial germ cells (PGCs). We identified seven miRNAs that are highly expressed in blastoderm and 10 that are highly expressed in PGCs. In this study, miR-302a and miR-456 for blastoderm and miR-181a* for PGCs were analyzed further for their target transcripts and regulatory pathways. Both miR-302a and miR-456 bound directly to the sex-determining region Y box 11 transcript and could act as posttranscriptional coregulators to maintain the undifferentiated state of the chicken blastoderm through the suppression of somatic gene expression and differentiation. Moreover, miR-181a* showed a bifunctional role in PGCs by binding to two different transcripts. miR-181a* inhibited the somatic differentiation of PGCs by silencing homeobox A1 expression. Additionally, miR-181a* prevented PGCs from entering meiosis through the repression of the nuclear receptor subfamily 6, group A, member 1 transcript. Collectively, our data demonstrate that in chickens miRNAs intrinsically regulate the differentiation fate of blastoderms and PGCs and that the specific timing of germ cell meiosis is controlled through miRNA expression.A t stage X, the chicken blastoderm consists of 40,000-60,000 undifferentiated embryonic cells and is able to develop pluripotent stem cells through in vitro culture (1). During chicken germline development, primordial germ cells (PGCs) first appear from the epiblast in the blastoderm and translocate to the hypoblast area of the pellucida (2, 3). During gastrulation, PGCs circulate through the vascular system and settle down in the gonadal anlagen. Such a differentiation pathway, including germ cell lineage during chicken embryo development, is a systematic process, governed by the concerted action of multiple unknown regulatory mechanisms (4-6).MicroRNAs (miRNAs) are small, noncoding RNAs ranging from 18 to 23 nucleotides that posttranscriptionally regulate gene expression in various tissues and cell types. Typically, miRNAs act as specific regulators of gene expression and are capable of controlling the fate of cells in a time-and tissue-specific manner (7, 8) through regulation of cellular differentiation, in addition to developmental patterning and morphogenesis (9-11). To date, several miRNA profiles have been classified as ESC-specific miRNAs, including miR-290-295 and miR-302-367 clusters (12, 13). However, both the miRNA expression profiling and posttranscriptional gene regulation for lineage specification, commitment, and differentiation during chicken embryo development remain largely uninvestigated. It has been shown recently that miRNA biogenesis and specific expr...

Chrysophanol is an anthraquinone compound, mainly isolated from rhubarb, with anti-cancer effects on some types of cancer cells. However, effects of chrysophanol on human choriocarcinoma cells are not known. Therefore, the objective of this study was to determine effects of chrysophanol on choriocarcinoma cells (JAR and JEG-3) and identify signal transduction cascades activated by chrysophanol. Results of present study, showed that chrysophanol decreased cell viability and induced apoptosis of JEG-3, but not JAR cells, in a dose-dependent manner. Chrysophanol also increased oxidative stress in JEG-3 cells by inducing ROS generation followed by mitochondrial dysfunction including depolarization of mitochondrial inner membrane potential. Western blot analysis revealed that ERK1/2, P90RSK, AKT, and P70S6K were increased significantly in JEG-3 cells by chrysophanol. Next, we investigated chrysophanol-mediated effects on proliferation of JEG-3 cells using pharmacological inhibitors of PI3K/AKT (LY294002) and ERK1/2 (U0126). Inhibition of AKT and ERK1/2 prevented chrysophanol-induced stimulation of proliferation of JEG-3 cells. In addition, the phosphorylation of AKT and ERK1/2 was suppressed by LY294002 and U0126 in JEG-3 cells treated with chrysophanol, whereas, the AKT protein was activated by pre-treatment of JEG-3 cells with U0126. Furthermore, we compared therapeutic effects of chrysophanol with cisplatin and paclitaxel which are conventional salvage regimens for choriocarcinoma. Our results verified that chrysophanol has synergistic effects with traditional therapy to increase apoptosis of JEG-3 cells. Collectively, these results indicate that chrysophanol is a potential effective chometherapeutic agent for treatment of choriocarcinoma therapy, and minimizing side effects of conventional treatment regimens. J. Cell. Physiol. 232: 331-339, 2017. © 2016 Wiley Periodicals, Inc.

Cystatin C (CST3) is a secreted inhibitor of lysosomal cysteine proteases cathepsins B (CTSB) and CTSL, which are abundant in the ovine endometrium and conceptus. In mice, cathepsins and cystatins play important roles in implantation and placentation. This study determined effects of the estrous cycle, pregnancy, progesterone (P4), and interferon-tau (IFNT) on CST3 in the ovine uterus. In cyclic ewes, CST3 mRNA was low on d 10, increased about 12-fold by d 12, and declined thereafter. In early pregnant ewes, CST3 mRNA was low on d 10 and increased about 130-fold from d 10 to d 20. CST3 mRNA and protein were abundant in the endometrial luminal epithelium (LE) and glandular epithelium and also in conceptus trophectoderm. In uterine flushes from pregnant ewes, CST3 protein was not detected on d 10 but was abundant on d 12, 14, and 16. In another study, treatment of ovariectomized, cyclic ewes with P4 induced a 14-fold increase in endometrial CST3 mRNA, and IFNT stimulated an additional 2-fold increase in CST3 mRNA in P4-treated ewes but not in ewes treated with P4 and the antiprogestin ZK 136,317. CST3 mRNA and protein were abundant in the endometrial luminal epithelium and superficial glandular epithelium of P4-treated ewes but were very low or not detectable in endometria of P4- and ZK-treated ewes. These results indicate that CST3 is a novel P4-induced and IFNT-stimulated gene expressed only in the epithelial cells of the ovine endometrium and implicate CST3 in regulation of uterine cathepsin activity during conceptus implantation.

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