Jeremy D. Cantlon

The Involvement of Proline-Rich 15 in Early Conceptus Development in Sheep

The ruminant conceptus undergoes a period of elongation that is required for maternal recognition of pregnancy, prior to attaching to the endometrium. The purpose of these studies was to investigate the role of proline-rich 15 (PRR15) in the sheep conceptus by examining mRNA expression, protein localization, and the effect of PRR15 mRNA degradation. Conceptuses were collected on Days 11, 13, 15, 16, 17, 21, and 30 after mating. Quantitative RT-PCR showed expression of PRR15 mRNA corresponded with the process of trophoblast elongation, with peak expression occurring on Days 15 and 16. A recombinant ovine PRR15 was generated and used to create polyclonal antibodies against PRR15. Immunohistochemistry of a Day 15 conceptus indicated that PRR15 was localized predominantly in the nucleus of the trophectoderm and extraembryonic primitive endoderm. To test whether PRR15 was required during early conceptus development, RNA interference was employed. Blastocysts collected on Day 8 after mating were infected with a lentivirus expressing a short-hairpin RNA (shRNA) that targeted PRR15 mRNA for degradation, an shRNA containing a three-nucleotide mismatch to PRR15 mRNA, or a lentivirus expressing no shRNA. After infection, blastocysts were transferred into recipient ewes and collected back on Day 15 of gestation. Although the majority of the control and mismatched shRNA-treated conceptuses elongated and survived to Day 15, none of the embryos treated with the lentivirus expressing shRNA against PRR15 mRNA elongated, and most died. In conclusion, expression of PRR15 mRNA occurred during a narrow window of conceptus development, and degradation of PRR15 mRNA led to conceptus demise or abnormal development.

Reduction in TRPC4 expression specifically attenuates G-protein coupled receptor-stimulated increases in intracellular calcium in human myometrial cells.

Canonical transient receptor potential (TRPC) proteins may play a role in regulating changes in intracellular calcium ([Ca(2+)](i)). Human myometrium expresses TRPC4, TRPC1 and TRPC6 mRNAs in greatest relative abundance. Contributions of TRPC4 to increases in [Ca(2+)](i) were assessed in PHM1-41 and primary human uterine smooth muscle (UtSMC) cells using short hairpin RNAs (shRNAs). Based on a reporter assay screen, one shRNA was selected to construct an adenoviral expression vector (TC4sh1). TC4sh1 induced both mRNA and protein TRPC4 knockdown in PHM1-41 cells without affecting expression of other TRPCs. Signal-regulated Ca(2+) entry (SRCE), defined as a stimulus- and extracellular Ca(2+)-dependent increase in [Ca(2+)](i), was measured in PHM1-41 cells treated with oxytocin (G-protein coupled receptor (GPCR)-stimulated), thapsigargin (store depletion-stimulated), and OAG (diacylglycerol-stimulated), using Fura-2. Cells infected with TC4sh1 exhibited attenuated oxytocin-, ATP- and PGF2alpha-mediated SRCE, but no change in thapsigargin- or OAG-stimulated SRCE. Similar results were obtained in primary uterine smooth muscle cells. Additionally, cells expressing TC4sh1 exhibited a significantly smaller increase in channel activity in response to oxytocin administration than did cells infected with empty virus. These data show that, in human myometrial cells, knockdown of endogenous TRPC4 specifically attenuates GPCR-stimulated, but not thapsigargin- or OAG-stimulated extracellular calcium-dependent increases in [Ca(2+)](i). These data imply that, in this cellular context, the mechanisms regulating extracellular Ca(2+)-dependent increases in [Ca(2+)](i) are differentially affected by different signaling pathways.

Role of LIN28A in Mouse and Human Trophoblast Cell Differentiation

ABSTRACT Proper regulation of trophoblast proliferation, differentiation, and function are critical for placenta development and function. The RNA-binding protein, LIN28A, has been well characterized as a potent regulator of differentiation in embryonic stem cells; however, little is known about the function of LIN28A in the placenta. We assessed LIN28A in vitro using mouse trophoblast stem (mTS) cells and human trophoblast cells (ACH-3P). We observed that LIN28A decreased and let-7 miRNA increased when mTS cells were induced to differentiate into mouse trophoblast giant cells (mTGCs) upon the removal of FGF4, heparin and conditioned medium. Similarly, we observed that LIN28A decreased in ACH-3P cells induced to syncytialize with forskolin treatment. To assess LIN28A in vivo we examined Embryonic Day 11.5 mouse placenta and observed abundant LIN28A in the chorioallantoic interface and labyrinth layer, with little LIN28A staining in spongiotrophoblast or differentiated mTGCs. Additionally, shRNA-mediated LIN28A knockdown in ACH-3P cells resulted in increased spontaneous syncytialization, and increased levels of syncytiotrophoblast markers hCG, LGALS13, and ERVW-1 mRNA. Additionally, targeted degradation of LIN28A mRNA increased responsiveness to forskolin-induced differentiation. In contrast, targeted degradation of Lin28a mRNA in mTS cells did not alter cell phenotype when maintained under proliferative culture conditions. Together, these data establish that LIN28A has a functional role in regulating trophoblast differentiation and function, and that loss of LIN28A in human trophoblast is sufficient to induce differentiation, but does not induce differentiation in the mouse.

Does a Nonclassical Signaling Mechanism Underlie an Increase of Estradiol-Mediated Gonadotropin-Releasing Hormone Receptor Binding in Ovine Pituitary Cells?

Estradiol-17beta (E2) is the major regulator of GnRH receptor (GnRHR) gene expression and number during the periovulatory period; however, the mechanisms underlying E2 regulation of the GNRHR gene remain undefined. Herein, we find that E2 conjugated to BSA (E2-BSA) mimics the stimulatory effect of E2 on GnRH binding in primary cultures of ovine pituitary cells. The time course for maximal GnRH analog binding was similar for both E2 and E2-BSA. The ability of E2 and E2-BSA to increase GnRH analog binding was blocked by the estrogen receptor (ER) antagonist ICI 182,780. Also, increased GnRH analog binding in response to E2 and the selective ESR1 agonist propylpyrazole triol was blocked by expression of a dominant-negative form of ESR1 (L540Q). Thus, membrane-associated ESR1 is the likely candidate for mediating E2 activation of the GNRHR gene. As cAMP response element binding protein (CREB) is an established target for E2 activation in gonadotrophs, we next explored a potential role for this protein as an intracellular mediator of the E2 signal. Consistent with this possibility, adenoviral-mediated expression of a dominant-negative form of CREB (A-CREB) completely abolished the ability of E2 to increase GnRH analog binding in primary cultures of ovine pituitary cells. Finally, the presence of membrane-associated E2 binding sites on ovine pituitary cells was demonstrated using a fluorescein isothiocyanate conjugate of E2-BSA. We suggest that E2 regulation of GnRHR number during the preovulatory period reflects a membrane site of action and may proceed through a nonclassical signaling mechanism, specifically a CREB-dependent pathway.

Effect of proline rich 15-deficiency on trophoblast viability and survival

Deviations from the normal program of gene expression during early pregnancy can lead to early embryonic loss as well as dysfunctional placentation, which can cause significant morbidity and mortality. Proline rich 15 (PRR15) is a low molecular weight nuclear protein expressed by the trophoblast during early gestation. Lentivirus-mediated knockdown of PRR15 mRNA in ovine trophectoderm led to demise of the embryo by gestational day 15, providing compelling evidence that PRR15 expression is critical during this precarious window of development. Our objective was to determine the effect of PRR15 knockdown on trophoblast gene expression, proliferation, and survival. The first-trimester human trophoblast cell line, ACH-3P, was infected with control lentivirus or a lentivirus expressing a short hairpin (sh)RNA to target PRR15 mRNA for degradation, resulting in a 68% reduction in PRR15 mRNA. Microarray analysis of these cell lines revealed differential expression of genes related to cancer, focal adhesion, and p53 signaling. These changes included significant up-regulation of GDF15, a cytokine increased in pregnancies with preeclampsia. Viability and proliferation decreased in PRR15-deficient cells, which was consistent with down-regulation of cell cycle-related genes CCND1 and CDK6 and an up-regulation of CCNG2 and CDKN1A in the PRR15-deficient cells. TNFSF10, a tumor necrosis factor superfamily member known to induce apoptosis increased significantly in the PRR15-deficient cells. Migration through a basement membrane matrix decreased and an increased population of apoptotic cells was present when treated with shRNA to target PRR15. These results suggest that PRR15 enhances trophoblast viability and survival during early implantation and placentation.