Jennifer L. Farmer

Endogenous retroviruses regulate periimplantation placental growth and differentiation

Endogenous retroviruses (ERVs) are fixed and abundant in the genomes of vertebrates. Circumstantial evidence suggests that ERVs play a role in mammalian reproduction, particularly placental morphogenesis, because intact ERV envelope genes were found to be expressed in the syncytiotrophoblasts of human and mouse placenta and to elicit fusion of cells in vitro. We report here in vivo and in vitro experiments finding that the envelope of a particular class of ERVs of sheep, endogenous Jaagsiekte sheep retroviruses (enJSRVs), regulates trophectoderm growth and differentiation in the periimplantation conceptus (embryo/fetus and associated extraembryonic membranes). The enJSRV envelope gene is expressed in the trophectoderm of the elongating ovine conceptus after day 12 of pregnancy. Loss-of-function experiments were conducted in utero by injecting morpholino antisense oligonucleotides on day 8 of pregnancy that blocked enJSRV envelope protein production in the conceptus trophectoderm. This approach retarded trophectoderm outgrowth during conceptus elongation and inhibited trophoblast giant binucleate cell differentiation as observed on day 16. Pregnancy loss was observed by day 20 in sheep receiving morpholino antisense oligonucleotides. In vitro inhibition of the enJSRV envelope reduced the proliferation of mononuclear trophectoderm cells isolated from day 15 conceptuses. Consequently, these results demonstrate that the enJSRV envelope regulates trophectoderm growth and differentiation in the periimplantation ovine conceptus. This work supports the hypothesis that ERVs play fundamental roles in placental morphogenesis and mammalian reproduction.

Identification of Endometrial Genes Regulated by Early Pregnancy, Progesterone, and Interferon Tau in the Ovine Uterus

Abstract During early pregnancy in ruminants, progesterone (P4) from the corpus luteum and interferon tau (IFNT) from the conceptus act on the endometrium to regulate genes important for uterine receptivity and conceptus growth. The use of the uterine gland knockout (UGKO) ewe has demonstrated the critical role of epithelial secretions in regulation of conceptus survival and growth. A custom ovine cDNA array was used to identify alterations in gene expression of endometria from Day 14 cyclic, pregnant, and UGKO ewes (study 1) and from cyclic ewes treated with P4 or P4 with ZK 136,317 antiprogestin and control proteins or IFNT (study 2). In study 1, expression of 47 genes was more than 2-fold different between Day 14 pregnant and cyclic endometria, whereas 23 genes was different between Day 14 cyclic and UGKO endometria. In study 2, 70 genes were different due to P4 alone, 74 genes were affected by IFNT in a P4-dependent manner, and 180 genes were regulated by IFNT in a P4-independent manner. In each study, an approximately equal number of genes were found to be activated or repressed in each group. Endometrial genes increased by pregnancy and P4 and/or IFNT include B2M, CTSL, CXCL10, G1P3, GRP, IFI27, IFIT1, IFITM3, LGALS15, MX1, POSTN, RSAD2, and STAT5A. Transcripts decreased by pregnancy and P4 and/or IFNT include COL3A1, LUM, PTMA, PUM1, RPL9, SPARC, and VIM. Identification and analysis of these hormonally responsive genes will help define endometrial pathways critical for uterine support of peri-implantation conceptus survival, growth, and implantation.

Galectin 15 (LGALS15) functions in trophectoderm migration and attachment

Galectin 15 (LGALS15) is expressed specifically by the endometrial luminal epithelium (LE) of the ovine uterus in concert with blastocyst growth, elongation, and implantation. LGALS15 contains a predicted carbohydrate recognition domain (CRD) as well as LDV and RGD recognition sequences for integrin binding. Studies tested the hypothesis that LGALS15 is a secreted regulator of blastocyst development, as well as growth, migration, adhesion, and apoptosis of tro‐phoblast. Bovine embryos were produced in vitro by standard conditions, and putative zygotes were cultured in the presence of recombinant ovine LGALS15. Rates of embryo cleavage and blastocyst formation were not affected by LGALS15. LGALS15 moderately increased proliferation of ovine trophectoderm (oTr) cells. Staurosporine elicited apoptosis of oTr cells, which could be partially inhibited by LGALS15. Migration of oTr cells was stimulated by LGALS15 that was dependent on Jun N‐terminal kinase (JNK). A dose‐dependent increase in oTr cell attachment to LGALS15 was found that could be inhibited by cyclic GRGDS, but not GRADS, peptides. Mutation of the LDVRGD integrin binding sequence of LGALS15 to LADRAD decreased its ability to promote oTr cell attachment, whereas mutation of the CRD had little effect. LGALS15 induced formation of robust focal adhesions in oTr cells that was abolished by mutation of the LDVRGD sequence. Collectively, these results support the hypothesis that LGALS15 stimulates trophectoderm cell migration and attachment via integrin binding and activation which are critical to blastocyst elongation and implantation.—Farmer, J. L., Burghardt, R. C., Jousan, F. D., Hansen, P. J., Bazer, F. W., Spencer, T. E. Galectin 15 (LGALS15) functions in trophectoderm migration and attachment. FASEB J. 22, 548–560 (2008)

Insulin-Like Growth Factor II Activates Phosphatidylinositol 3-Kinase-Protooncogenic Protein Kinase 1 and Mitogen-Activated Protein Kinase Cell Signaling Pathways, and Stimulates Migration of Ovine Trophectoderm Cells

IGF-II, a potent stimulator of cellular proliferation, differentiation, and development, regulates uterine function and conceptus growth in several species. In situ hybridization analyses found that IGF-II mRNA was most abundant in the caruncular endometrial stroma of both cyclical and pregnant ewes. In the intercaruncular endometrium, IGF-II mRNA transitioned from stroma to luminal epithelium between d 14 and 20 of pregnancy. IGF-II mRNA was present in all cells of the conceptus but was particularly abundant in the yolk sac. Immunohistochemical analyses revealed that phosphorylated (p)-protooncogenic protein kinase 1, p-ribosomal protein S6 kinase, p-ERK1/2, and p-P38 MAPK proteins were present at low levels in a majority of endometrial cells but were most abundant in the nuclei of endometrial luminal epithelium and conceptus trophectoderm of pregnant ewes. In mononuclear trophectoderm cells isolated from d-15 conceptuses, IGF-II increased the abundance of p-pyruvate dehydrogenase kinase 1, p-protooncogenic protein kinase 1, p-glycogen synthase kinase 3B, p-FK506 binding protein 12-rapamycin associated protein 1, and p-ribosomal protein S6 kinase protein within 15 min, and the increase was maintained for 90 min. IGF-II also elicited a rapid increase in p-ERK1/2 and p-P38 MAPK proteins that was maximal at 15 or 30 min posttreatment. Moreover, IGF-II increased migration of trophectoderm cells. Collectively, these results support the hypothesis that IGF-II coordinately activates multiple cell signaling pathways critical to survival, growth, and differentiation of the ovine conceptus during early pregnancy.

Galectin 15 (LGALS15): a gene uniquely expressed in the uteri of sheep and goats that functions in trophoblast attachment.

Abstract Galectins are a family of secreted animal lectins with biological roles in cell adhesion and migration. In sheep, galectin 15 (LGALS15) is expressed specifically in the endometrial luminal (LE) and superficial glandular (sGE) epithelia of the uterus in concert with blastocyst elongation during the peri-implantation period. The present study examined LGALS15 expression in the uterus of cattle, goats, and pigs. Although the bovine genome contains an LGALS15-like gene, expressed sequence tags encoding LGALS15 mRNA were found only for sheep, and full-length LGALS15 cDNAs were cloned only from endometrial total RNA isolated from pregnant sheep and goats, but not pregnant cattle or pigs. Ovine and caprine LGALS15 were highly homologous at the mRNA (95%) and protein (91%) levels, and all contained a conserved carbohydrate recognition domain and RGD recognition sequence for integrin binding. Endometrial LGALS15 mRNA levels increased after Day 11 of both the estrous cycle and pregnancy, and were considerably increased after Day 15 of pregnancy in goats. In situ hybridization detected abundant LGALS15 mRNA in endometrial LE and sGE of early pregnant goats, but not in cattle or pigs. Immunoreactive LGALS15 protein was present in endometrial epithelia and conceptus trophectoderm of goat uteri and detected within intracellular crystal structures in trophectoderm and LE. Recombinant ovine and caprine LGALS15 proteins elicited a dose-dependent increase in ovine trophectoderm cell attachment in vitro that was comparable to bovine fibronectin. These results support the hypothesis that LGALS15 is uniquely expressed in Caprinae endometria and functions as an attachment factor important for peri-implantation blastocyst elongation.

Environmental Gestagens Activate Fathead Minnow (Pimephales promelas) Nuclear Progesterone and Androgen Receptors in Vitro

Gestagen is a collective term for endogenous and synthetic progesterone receptor (PR) ligands. In teleost fishes, 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S) are the predominant progestogens, whereas in other vertebrates the major progestogen is progesterone (P4). Progestins are components of human contraceptives and hormone replacement pharmaceuticals and, with P4, can enter the environment and alter fish and amphibian reproductive health. In this study, our primary objectives were to clone the fathead minnow (FHM) nuclear PR (nPR), to develop an in vitro assay for FHM nPR transactivation, and to screen eight gestagens for their ability to transactivate FHM nPR. We also investigated the ability of these gestagens to transactivate FHM androgen receptor (AR). Fish progestogens activated FHM nPR, with DHP being more potent than 20β-S. The progestin drospirenone and P4 transactivated the FHM nPR, whereas five progestins and P4 transactivated FHM AR, all at environmentally relevant concentrations. Progestins are designed to activate human PR, but older generation progestins have unwanted androgenic side effects in humans. In FHMs, several progestins proved to be strong agonists of AR. Here, we present the first mechanistic evidence that environmental gestagens can activate FHM nPR and AR, suggesting that gestagens may affect phenotype through nPR- and AR-mediated pathways.

Progesterone and Placentation Increase Secreted Phosphoprotein One (SPP1 or Osteopontin) in Uterine Glands and Stroma for Histotrophic and Hematotrophic Support of Ovine Pregnancy

Abstract Secreted phosphoprotein one (SPP1, osteopontin) may regulate conceptus implantation and placentation. We investigated effects of progesterone (P4) and the conceptus on expression and localization of SPP1 in the ovine uterus. Steady-state levels of SPP1 mRNA in the endometrium of unilaterally pregnant ewes did not differ significantly between nongravid and gravid horns within their respective days of pregnancy; however, levels did increase as pregnancy progressed. SPP1 mRNA was detectable in the glandular epithelium (GE) of both nongravid and gravid horns via in situ hybridization. SPP1 protein was localized to the apical surface of the luminal epithelium of both nongravid and gravid uterine horns. Gravid horns exhibited extensive stromal SPP1 on Days 40 through 120, whereas SPP1 was markedly lower in the stroma of nongravid uterine horns through Day 80 of pregnancy. By Day 120, stromal expression of SPP1 between nongravid and gravid horns was similar. Long-term P4 treatment of ovariectomized ewes induced SPP1 in the uterine stroma and GE. A bioactive 45-kDa SPP1 fragment was purified from uterine secretions and promoted ovine trophectoderm cell attachment in vitro. Interestingly, increased stromal cell expression of SPP1 was positively associated with vascularization as assessed by von Willebrand factor staining. Finally, ovine uterine artery endothelial cells produced SPP1 during outgrowth into three-dimensional collagen matrices in an in vitro model system that recapitulates angiogenesis. Collectively, P4 induces and the conceptus further stimulates SPP1 in uterine GE and stroma, where SPP1 likely influences histotrophic and hematotrophic support of conceptus development..

Progesterone and Interferon Tau Regulated Genes in the Ovine Uterine Endometrium: Identification of Periostin as a Potential Mediator of Conceptus Elongation

During early pregnancy in ruminants, progesterone (P(4)) and interferon tau (IFNT) act on the endometrium to regulate genes hypothesized to be important for conceptus development and implantation. The present study was conducted to verify several candidate genes (actin alpha-2, smooth muscle, aorta (ACTA2), collagen, type III, alpha-1 (COL3A1), periostin (POSTN), secreted protein acidic cysteine-rich (SPARC), S100 calcium-binding protein A2 (S100A2), STAT5A and transgelin (TAGLN)) regulated by pregnancy, P(4), and/or IFNT in the endometrium determined using a custom ovine cDNA array. S100A2 mRNA was detected primarily in endometrial epithelia and conceptuses. S100A2 mRNA increased in endometrial epithelia from days 10 to 16 in cyclic ewes and from days 10 to 14 in pregnant ewes and declined thereafter. The abundance of S100A2 mRNA was less in endometrial luminal epithelium of IFNT-infused ewes receiving P(4). Expression of COL3A1, SPARC, ACTA2, and TAGLN was independent of pregnancy, P(4), or IFNT. POSTN mRNA was detected primarily in compact stroma of intercaruncular and caruncular endometria, but not in the conceptus. Endometrial POSTN mRNA increased between days 12 and 14 in pregnant but not cyclic ewes, and POSTN mRNA was more abundant in uterine stroma of ewes treated with P(4). POSTN protein was detected in uterine flushings of pregnant ewes and found to mediate attachment and stimulate migration of ovine trophectoderm cells in vitro. These results support the ideas that POSTN and S100A2 are regulated by P(4) and IFNT respectively, and that POSTN is involved in conceptus elongation during early pregnancy.

Friend Flips: A Story Activity about Relationships.

Adolescents are influenced by the type, length and quality of the connections shared with different people throughout their lifespan. Relationships with peers, friends, and adults help to shape knowledge, attitudes, and beliefs related to health. Recognizing healthy or unhealthy characteristics allow youth to strengthen relationships and interactions with others. In this interactive teaching strategy, students identify and rank characteristics of friend and romantic relationships. Students are challenged to generate authentic stories about relationships incorporating these traits. National Health Education Standards and National Sexuality Education Standards provide the base of the teaching strategy.