The process of implantation is mediated by a complex network of signaling and adhesive factors. In the pig, latent and active transforming growth factor beta (TGFB), TGFB receptors (TGFBR), and integrins (ITGs) are present during the peri-implantation period. TGFB signals via TGFBR and activates downstream effector SMAD proteins 2 and 3 (p-SMAD2/3). Latency-associated peptide (LAP), part of the latent TGFB complex, is known to bind to ITG heterodimers and activate TGFB. We hypothesize that active TGFBs and TGFBRs along with LAP and ITGs functionally interact at the conceptus-maternal interface to mediate events essential for conceptus development and attachment in pigs. Uteri and conceptuses from days 10, 12, 16, 20, and 24 pregnant gilts were immunostained for TGFB, LAP, and ITG subunits (ITGAV, ITGB1, ITGB3, ITGB5, ITGB6, and ITGB8). Activation of TGFBRs was evaluated by the presence of phosphorylated downstream effector SMAD2/3. Binding of LAP to ITGs was also evaluated using porcine trophectoderm cells. Abundant active TGFB was detected at the apical surfaces of epithelia at the conceptus-maternal interface, and p-SMAD2/3 was detected at both conceptus attachment and nonattachment sites during implantation. Separate aggregates of LAP, ITGB1, ITGB5, and later ITGB3 were detected at the porcine conceptus-maternal interface, and binding of LAP to ITGs on apical surfaces was demonstrated. Results suggest that functional LAP-ITG adhesion complexes support conceptus attachment and promote TGFB activation leading to TGFB interaction with TGFBR supporting events of porcine implantation.

Estradiol up-regulates expression of the estrogen receptor ␣ gene in the uterus by stabilizing estrogen receptor ␣ mRNA. Previously, we defined two discrete minimal estradiol-modulated stability sequences (MEMSS) within the extensive 3-untranslated region of estrogen receptor ␣ mRNA with an in vitro stability assay using cytosolic extracts from sheep uterus. We report here that excess MEMSS RNA inhibited the enhanced stability of estrogen receptor ␣ mRNA in extracts from estradiol-treated ewes compared with those from control ewes. Several estradiol-induced MEMSS-binding proteins were characterized by UV cross-linking in uterine extracts from ewes in a time course study (0, 8, 16, and 24 h after estradiol injection). The pattern of binding proteins changed at 16 h post-injection, concurrent with enhanced estrogen receptor ␣ mRNA stability and the highest rate of accumulation of estrogen receptor ␣ mRNA. The predominant MEMSS-binding protein induced by estradiol treatment was identified as AUF1 (A ؉ U-rich RNA-binding factor 1) protein isoform p45 (a product of the heterogeneous nuclear ribonucleoprotein D gene). Immunoblot analysis indicated that only two of four AUF1 protein isoforms were present in the uterine cytosolic extracts and that estradiol treatment strongly increased the ratio of AUF1 isoforms p45 to p37. Nonphosphorylated recombinant AUF1p45 protected estrogen receptor ␣ mRNA in vitro in a dose-dependent manner. These studies describe estrogenic induction of AUF1p45 binding to the estrogen receptor ␣ mRNA as a molecular mechanism for post-transcriptional up-regulation of gene expression.Estrogens are a family of hormones with important roles in reproduction as well as in cardiovascular, bone, and brain function. Estrogens potently regulate physiology by altering gene expression in sensitive tissues (1). The estrogen responsiveness of a tissue can be determined by its expression of the two genes encoding estrogen receptors, ER␣ 2 and ER␤ (2). Estrogens are best known for their ability to activate transcription of genes (1). However, nontranscriptional mechanisms of estrogen regulation of gene expression have been elucidated more recently, and many of them involve the actions of kinases and phosphatases (1, 3). Expression of the ER␣ gene is tightly regulated by steroid hormones from the ovary in responsive tissues. In the uterus, expression of the ER␣ gene is 10-fold greater than that of ER␤ (4). Studies with knock-out mice and with ER␣-and ER␤-specific ligands indicate that the uterine effects of estradiol (E2) depend primarily upon expression of the ER␣ gene (5, 6). Therefore, the up-regulation of ER␣ gene expression in the uterus was the focus of our studies. Both mRNA and protein products of the ER␣ gene are short-lived, so that changes in their rates of synthesis and degradation rapidly alter tissue concentrations of ER␣ (7-9). The preovulatory surge of E2 up-regulates concentrations of ER␣ mRNA and protein in the uterus. This regulation occurs in several E2-sensitive tissues across diverse specie...

In the pig, transforming growth factor beta (TGFB), TGFB receptors (TGFBRs), and integrins are present during the peri-implantation period. Latency-associated peptide (LAP), a part of latent TGFB, can bind to integrin heterodimers via its Arg-Gly-Asp (RGD) sequence; therefore, ligand-receptor interactions between TGFB and TGFBRs, along with LAP and integrin heterodimers, may be functional in mediating events supporting conceptus elongation and attachment. With the use of surgically implantable osmotic pumps, we were able to maintain pregnancy with the aim of mechanistically altering in vivo receptor-ligand interactions involving TGFB with TGFBRs and LAP with integrins during porcine pregnancy. Day 9 pregnant gilts received intrauterine infusions of LAP-RGD, a recombinant mutant of LAP (LAP-RGE), or vehicle control and were ovariohysterectomized on Day 13 or 24 of pregnancy. We hypothesized that intrauterine infusion of LAP-RGD would decrease downstream signaling of TGFB while increasing LAP-integrin interactions and that net effect would enhance conceptus survival and attachment early in the peri-implantation period but possibly increase the chance of abnormal placentation later in pregnancy. Additionally, we hypothesized that infusion of LAP-RGE would disrupt TGFB signals but not alter integrin signaling, and thus the net result would be decreased conceptus survival and abnormal development. Unexpectedly, LAP-RGD intrauterine infusions resulted in a reduction of conceptus elongation, whereas infusions of LAP-RGE permitted implantation and placentation but resulted in larger fetal weight, allantois length, and allantoic fluid volume. Results suggest TGFB and integrins are contributing factors in the regulation of conceptus elongation and placental and fetal size.

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