Enrichment of ovine gonadotropes via adenovirus gene targeting enhances assessment of transcriptional changes in response to estradiol-17 beta†.

Gonadotropes represent approximately 5-15% of the total endocrine cell population in the mammalian anterior pituitary. Therefore, assessing the effects of experimental manipulation on virtually any parameter of gonadotrope biology is difficult to detect and parse from background noise. In non-rodent species, applying techniques such as high-throughput ribonucleic acid (RNA) sequencing is problematic due to difficulty in isolating and analyzing individual endocrine cell populations.

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.

Development of ovine chorionic somatomammotropin hormone-deficient pregnancies.

Intrauterine growth restriction (IUGR) is a leading cause of neonatal mortality and morbidity. Chorionic somatomammotropin hormone (CSH), a placenta-specific secretory product found at high concentrations in maternal and fetal circulation throughout gestation, is significantly reduced in human and sheep IUGR pregnancies. The objective of this study was to knock down ovine CSH (oCSH) expression in vivo using lentiviral-mediated short-hairpin RNA to test the hypothesis that oCSH deficiency would result in IUGR of near-term fetal lambs.

Role of LIN28A in mouse and human trophoblast cell differentiation.

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).

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 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.

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).

Activin responsiveness of the murine gonadotropin-releasing hormone receptor gene is mediated by a composite enhancer containing spatially distinct regulatory elements.

The promoters of mouse and rat GnRH receptor (GnRHR) genes differ markedly in regard to activin regulation. Activin stimulates the mouse GnRHR promoter, although it has no impact on that of the rat. To test whether this difference was due to a single nucleotide change in the rat GnRHR activating sequence (GRAS) homolog, we tested a mouse promoter with the rat GRAS homolog and a rat promoter with intact mouse GRAS.