Specific microRNAs in stallion spermatozoa are potential biomarkers of high functionality.

Males of some species, from horses to humans, require medical help for subfertility problems. There is an urgent need for novel molecular assays that reflect spermatozoal function. In the last 25 years, studies examined RNAs in spermatozoa as a window into gene expression during their development and, more recently, for their functions in early embryo development. In clinics, more dense spermatozoa are isolated by density gradient centrifugation before use in artificial insemination to increase pregnancy rates. The objectives of the current study were to discover and quantify the microRNAs in stallion spermatozoa and identify those with differential expression levels in more dense versus less dense spermatozoa. First, spermatozoa from seven stallions were separated into more dense and less dense populations by density gradient centrifugation. Next, small RNAs were sequenced from each of the 14 RNA samples. We identified 287 different mature microRNAs within the 11,824,720 total mature miRNA reads from stallion spermatozoa. The most prevalent was miR-10a/b-5p. The less dense spermatozoa had fewer mature microRNAs and more microRNA precursor sequences than more dense spermatozoa, perhaps indicating that less dense spermatozoa are less mature. Two of the most prevalent microRNAs in more dense stallion spermatozoa were predicted to target mRNAs that encode proteins that accelerate mRNA decay. Nine microRNAs were more highly expressed in more dense spermatozoa. Three of those microRNAs were predicted to target mRNAs that encode proteins involved in protein decay. Both mRNA and protein decay are very active in late spermiogenesis but not in mature spermatozoa. The identified microRNAs may be part of the mechanism to shut down those processes. The microRNAs with greater expression in more dense spermatozoa may be useful biomarkers for spermatozoa with greater functional capabilities.

The Use of Diets in the Diagnosis and Treatment of Common Gastrointestinal Diseases in Dogs and Cats.

The nutritional health of dogs and cats is important to pet owners around the world. Nutrition is inextricably linked to the health of the gastrointestinal system and vice versa. Gastrointestinal signs, such as vomiting, diarrhea, anorexia, or weight loss, are one of the most common reasons that dog and cat owners make non-routine appointments with veterinarians. Those patients are evaluated systematically to identify and/or rule out the causes of the symptoms. Some causes of chronic diarrhea are within the gastrointestinal tract while others are secondary to pathogenic factors outside the digestive system. Some useful biomarkers of chronic intestinal disease (enteropathy) exist in serum and feces. After determination that the clinical signs are due to primary gastrointestinal disease and that there is no parasitism, specific diets are used for at least two weeks. There are several types of diets for pets with chronic enteropathies. There are limited ingredient diets and hydrolyzed protein diets with reduced levels of allergens. There are also highly digestible and fiber-enhanced diets. Some diets contain probiotics and/or prebiotics. If symptoms do not improve and the patient is stable, a diet from a different class may be tried. For chronic enteropathies, the prognosis is generally good for symptom resolution or at least improvement. However, if interventions with novel diets do not ameliorate the symptoms of chronic enteropathy, then antibiotic, anti-inflammatory, or immunosuppressant therapy or further, more invasive diagnostics such as taking an intestinal biopsy, may be indicated. Pancreatitis is a common gastrointestinal disease in dogs and cats and patients may present with mild to severe disease. Many patients with mild to moderate disease can be successfully treated with early supportive care, including feeding a low-fat diet. A novel pharmaceutical, fuzapladib (Panoquell-CA1) looks very promising for treating more severe forms of acute pancreatitis in dogs. Maintenance on a low-fat diet may prevent pancreatitis in at-risk dogs. Future advances in medicine will allow pet owners and veterinarians to use dietary management to maximize the health of their dogs and cats.

Conservation of Glutathione Transferase mRNA and Protein Sequences Similar to Human and Horse Alpha Class GST A3-3 across Dog, Goat, and Opossum Species.

The glutathione transferase A3-3 (GST A3-3) homodimeric enzyme is the most efficient enzyme that catalyzes isomerization of the precursors of testosterone, estradiol, and progesterone in the gonads of humans and horses. However, the presence of GST A3-3 orthologs with equally high ketosteroid isomerase activity has not been verified in other mammalian species, even though pig and cattle homologs have been cloned and studied. Identifying GSTA3 genes is a challenge because of multiple GSTA gene duplications (e.g., 12 in the human genome); consequently, the GSTA3 gene is not annotated in most genomes. To improve our understanding of GSTA3 gene products and their functions across diverse mammalian species, we cloned homologs of the horse and human GSTA3 mRNAs from the testes of a dog, goat, and gray short-tailed opossum, the genomes of which all currently lack GSTA3 gene annotations. The resultant novel GSTA3 mRNA and inferred protein sequences had a high level of conservation with human GSTA3 mRNA and protein sequences (≥70% and ≥64% identities, respectively). Sequence conservation was also apparent for the 12 residues of the "H-site" in the 222 amino acid GSTA3 protein that is known to interact with the steroid substrates. Modeling predicted that the dog GSTA3-3 may be a more active ketosteroid isomerase than the corresponding goat or opossum enzymes. However, expression of the GSTA3 gene was higher in liver than in other dog tissue. Our results improve understanding of the active sites of mammalian GST A3-3 enzymes, inhibitors of which might be useful for reducing steroidogenesis for medical purposes, such as fertility control or treatment of steroid-dependent diseases.

Glutathione Transferases as Efficient Ketosteroid Isomerases.

In addition to their well-established role in detoxication, glutathione transferases (GSTs) have other biological functions. We are focusing on the ketosteroid isomerase activity, which appears to contribute to steroid hormone biosynthesis in mammalian tissues. A highly efficient GST A3-3 is present in some, but not all, mammals. The alpha class enzyme GST A3-3 in humans and the horse shows the highest catalytic efficiency with kcat/Km values of approximately 107 M-1s-1, ranking close to the most active enzymes known. The expression of GST A3-3 in steroidogenic tissues suggests that the enzyme has evolved to support the activity of 3ß-hydroxysteroid dehydrogenase, which catalyzes the formation of 5-androsten-3,17-dione and 5-pregnen-3,20-dione that are substrates for the double-bond isomerization catalyzed by GST A3-3. The dehydrogenase also catalyzes the isomerization, but its kcat of approximately 1 s-1 is 200-fold lower than the kcat values of human and equine GST A3-3. Inhibition of GST A3-3 in progesterone-producing human cells suppress the formation of the hormone. Glutathione serves as a coenzyme contributing a thiolate as a base in the isomerase mechanism, which also involves the active-site Tyr9 and Arg15. These conserved residues are necessary but not sufficient for the ketosteroid isomerase activity. A proper assortment of H-site residues is crucial to efficient catalysis by forming the cavity binding the hydrophobic substrate. It remains to elucidate why some mammals, such as rats and mice, lack GSTs with the prominent ketosteroid isomerase activity found in certain other species. Remarkably, the fruit fly Drosophila melanogaster, expresses a GSTE14 with notable steroid isomerase activity, even though Ser14 has evolved as the active-site residue corresponding to Tyr9 in the mammalian alpha class.

Transcriptomic analysis of the honey bee (Apis mellifera) queen spermathecae reveals genes that may be involved in sperm storage after mating.

Honey bee (Apis mellifera) queens have a remarkable organ, the spermatheca, which successfully stores sperm for years after a virgin queen mates. This study uniquely characterized and quantified the transcriptomes of the spermathecae from mated and virgin honey bee queens via RNA sequencing to identify differences in mRNA levels based on a queen's mating status. The transcriptome of drone semen was analyzed for comparison. Samples from three individual bees were independently analyzed for mated queen spermathecae and virgin queen spermathecae, and three pools of semen from ten drones each were collected from three separate colonies. In total, the expression of 11,233 genes was identified in mated queen spermathecae, 10,521 in virgin queen spermathecae, and 10,407 in drone semen. Using a cutoff log2 fold-change value of 2.0, we identified 212 differentially expressed genes between mated and virgin spermathecal queen tissues: 129 (1.4% of total) were up-regulated and 83 (0.9% of total) were down-regulated in mated queen spermathecae. Three genes in mated queen spermathecae, three genes in virgin queen spermathecae and four genes in drone semen that were more highly expressed in those tissues from the RNA sequencing data were further validated by real time quantitative PCR. Among others, expression of Kielin/chordin-like and Trehalase mRNAs was highest in the spermathecae of mated queens compared to virgin queen spermathecae and drone semen. Expression of the mRNA encoding Alpha glucosidase 2 was higher in the spermathecae of virgin queens. Finally, expression of Facilitated trehalose transporter 1 mRNA was greatest in drone semen. This is the first characterization of gene expression in the spermathecae of honey bee queens revealing the alterations in mRNA levels within them after mating. Future studies will extend to other reproductive tissues with the purpose of relating levels of specific mRNAs to the functional competence of honey bee queens and the colonies they head.

Identification and quantification of coding and long non-coding RNAs in stallion spermatozoa separated by density.

BACKGROUND: It is not unusual for stallions to have fertility problems. For many, artificial insemination with more dense spermatozoa (isolated by density gradient centrifugation) results in greater pregnancy rates compared with the rates when using unfractionated spermatozoa. RNAs in spermatozoa delivered to the oocyte at conception are required for embryo development. Novel molecular assays of spermatozoa that reflect function are needed to predict the fertility of stallions. OBJECTIVES: To describe and compare the RNA populations in more dense and less dense spermatozoa from stallions. MATERIALS AND METHODS: Spermatozoa from five stallions were separated into more dense and less dense populations by density gradient centrifugation. Complementary DNA libraries were made from each of the ten total RNA samples after ribosomal RNA removal. Next-generation sequencing characterized the RNA populations in more and less dense spermatozoa. Quantitative reverse transcription-PCR was used to confirm differential expression of selected RNAs. RESULTS: Stallion spermatozoa contain 11 215 RNAs, with the most prevalent RNA being a 1492 base long non-coding RNA. The levels of 159 RNAs were greater in more dense spermatozoa, while levels of seven other RNAs were greater in less dense spermatozoa. Quantitative reverse transcription-PCR confirmed the threefold greater levels of solute carrier family 26 member 8 (SLC26A8) mRNA in less dense spermatozoa, and sixfold and threefold greater expression levels of the SCP2 sterol binding domain containing 1 (SCP2D1) and spermatogenesis-associated protein 31D1 (SPATA31D1) mRNAs in more dense spermatozoa, respectively. DISCUSSION AND CONCLUSION: We identified 11 215 RNAs in stallion spermatozoa and 166 with differential expression between more dense and less dense fractions. Many prevalent RNAs were also found in bull, boar, and human spermatozoa. Many differentially expressed RNAs are known to be testis- or spermatozoa-specific. Our results may lead to identification of an RNA population in spermatozoa that is optimal for establishing successful pregnancies.

Dexamethasone downregulates expression of several genes encoding orphan nuclear receptors that are important to steroidogenesis in stallion testes.

Glucocorticoids impair testosterone synthesis by an unknown mechanism. Stallions treated with the synthetic glucocorticoid dexamethasone had testes collected at 6 or 12 hours postinjection. The testicular expression of selected genes encoding nuclear receptors and steroidogenic enzymes was measured. At 6 hours, dexamethasone treatment decreased levels of NR0B2, NR4A1, NR5A1, and NR5A2 messenger RNAs (mRNAs) and NR5A2 mRNA levels remained depressed at 12 hours. In contrast, dexamethasone increased levels of NFKBIA mRNA at both time points. At 6 hours, dexamethasone did not alter levels of NR0B1, NR2F1, NR2F2, NR3C1, CYP11A1, CYP17A1, CYP19A1, DHCR24, GSTA3, HSD3B2, HSD17B3, LHCGR, or STAR mRNAs. In primary cultures of Leydig cells, 10 -9 and 10 -7 M dexamethasone decreased levels of NR4A1 and NR5A1 mRNAs and increased those of NFKBIA mRNA. Our discovery that dexamethasone downregulates NR4A1, NR5A1, and NR5A2 genes, known to be important for testicular functions, may be part of the mechanism by which glucocorticoids acutely decreases testosterone.

Characterization of equine GST A3-3 as a steroid isomerase.

Glutathione transferases (GSTs) comprise a superfamily of enzymes prominently involved in detoxication by making toxic electrophiles more polar and therefore more easily excretable. However some GSTs have developed alternative functions. Thus, a member of the Alpha class GSTs in pig and human tissues is involved in steroid hormone biosynthesis, catalyzing the obligatory double-bond isomerization of Δ5-androstene-3,17-dione to Δ4-androstene-3,17-dione and of Δ5-pregnene-3,20-dione to Δ4-pregnene-3,20-dione on the biosynthetic pathways to testosterone and progesterone. The human GST A3-3 is the most efficient steroid double-bond isomerase known so far in mammals. The current work extends discoveries of GST enzymes that act in the steroidogenic pathways in large mammals. The mRNA encoding the steroid isomerase GST A3-3 was cloned from testis of the horse (Equus ferus caballus). The concentrations of GSTA3 mRNA were highest in hormone-producing organs such as ovary, testis and adrenal gland. EcaGST A3-3 produced in E. coli has been characterized and shown to have highly efficient steroid double-bond isomerase activity, exceeding its activities with conventional GST substrates. The enzyme now ranks as one of the most efficient steroid isomerases known in mammals and approaches the activity of the bacterial ketosteroid isomerase, one of the most efficient enzymes of all categories known today. The high efficiency and the tissue distribution of EcaGST A3-3 support the view that the enzyme plays a physiologically significant role in the biosynthesis of steroid hormones.

Dexamethasone acutely down-regulates genes involved in steroidogenesis in stallion testes.

In rodents, livestock and primate species, a single dose of the synthetic glucocorticoid dexamethasone acutely lowers testosterone biosynthesis. To determine the mechanism of decreased testosterone biosynthesis, stallions were treated with 0.1mg/kg dexamethasone 12h prior to castration. Dexamethasone decreased serum concentrations of testosterone by 60% compared to saline-treated control stallions. Transcriptome analyses (microarrays, northern blots and quantitative PCR) of testes discovered that dexamethasone treatment decreased concentrations of glucocorticoid receptor alpha (NR3C1), alpha actinin 4 (ACTN4), luteinizing hormone receptor (LHCGR), squalene epoxidase (SQLE), 24-dehydrocholesterol reductase (DHCR24), glutathione S-transferase A3 (GSTA3) and aromatase (CYP19A1) mRNAs. Dexamethasone increased concentrations of NFkB inhibitor A (NFKBIA) mRNA in testes. SQLE, DHCR24 and GSTA3 mRNAs were predominantly expressed by Leydig cells. In man and livestock, the GSTA3 protein provides a major 3-ketosteroid isomerase activity: conversion of Δ(5)-androstenedione to Δ(4)-androstenedione, the immediate precursor of testosterone. Consistent with the decrease in GSTA3 mRNA, dexamethasone decreased the 3-ketosteroid isomerase activity in testicular extracts. In conclusion, dexamethasone acutely decreased the expression of genes involved in hormone signaling (NR3C1, ACTN4 and LHCGR), cholesterol synthesis (SQLE and DHCR24) and steroidogenesis (GSTA3 and CYP19A1) along with testosterone production. This is the first report of dexamethasone down-regulating expression of the GSTA3 gene and a very late step in testosterone biosynthesis. Elucidation of the molecular mechanisms involved may lead to new approaches to modulate androgen regulation of the physiology of humans and livestock in health and disease.

Marinobufagenin regulates permeability and gene expression of brain endothelial cells.

Marinobufagenin (MBG) is a cardiotonic steroid that increases in the circulation in preeclampsia. Preeclampsia and eclampsia are associated with cerebral edema. Therefore, we examined the effects of MBG on human brain microvascular endothelial cells (HBMEC) in vitro. MBG enhanced the permeability of HBMEC monolayers at 1-, 10-, and 100-nM doses, but had no effect at 0.1 nM. Agilent Human Gene Expression microarrays were utilized in these studies. MBG treatment (10 nM for 12 h) downregulated concentrations of the soluble VEGFR transcript sFLT by 59% but did not alter those of FLTv3 mRNA (determined by quantitative PCR). When treated and control HBMEC transcriptomes were interrogated on microarrays, 1,069 genes appeared to be regulated by MBG. Quantitative RT-PCR confirmed that MBG treatment upregulated ENKUR mRNA concentrations by 57%. Its protein product interacts with calmodulin and calcium channel proteins. MBG treatment downregulated several genes whose protein products are involved in cell adhesion (ITGA2B, FERMT1, CLDN16, and TMEM207) and cell signaling (GRIN2C, SLC8A1, and ESR1). The level of downregulation ranged from 22 to 66%. Altogether, MBG actively enhanced the permeability of HBMEC monolayers while downregulating genes involved in adhesion. MBG treatment had variable effects on ENKUR, GRIN2C, and SLC8A1 genes, all associated with calcium transport. These studies provide the basis for future investigations of MBG actions in normal physiology and disease.

CatSper and the relationship of hyperactivated motility to intracellular calcium and pH kinetics in equine sperm.

In vitro fertilization does not occur readily in the horse. This may be related to failure of equine sperm to initiate hyperactivated motility, as treating with procaine to induce hyperactivation increases fertilization rates. In mice, hyperactivated motility requires a sperm-specific pH-gated calcium channel (CatSper); therefore, we investigated this channel in equine sperm. Motility was assessed by computer-assisted sperm motility analysis and changes in intracellular pH and calcium were assessed using fluorescent probes. Increasing intracellular pH induced a rise in intracellular calcium, which was inhibited by the known CatSper blocker mibefradil, supporting the presence of a pH-gated calcium channel, presumably CatSper. Hyperactivation was associated with moderately increased intracellular pH, but appeared inversely related to increases in intracellular calcium. In calcium-deficient medium, high-pH treatment induced motility loss, consistent with influx of sodium through open CatSper channels in the absence of environmental calcium. However, sperm treated with procaine in calcium-deficient medium both maintained motility and underwent hyperactivation, suggesting that procaine did not act via opening of the CatSper channel. CATSPER1 mRNA was identified in equine sperm by PCR, and CATSPER1 protein was localized to the principal piece on immunocytochemistry. Analysis of the predicted equine CATSPER1 protein revealed species-specific differences in structure in the pH-sensor region. We conclude that the CatSper channel is present in equine sperm but that the relationship of hyperactivated motility to calcium influx is weak. Procaine does not appear to act via CatSper in equine sperm, and its initial hyperactivating action is not dependent upon external calcium influx.

Estradiol up-regulates expression of the A + U-rich binding factor 1 (AUF1) gene in the sheep uterus.

The A+U-rich binding factor 1 (AUF1 or HNRPD) gene produces predominant RNA binding proteins. The AUF1 transcript is alternatively spliced to produce four protein isoforms that stabilize or destabilize hundreds of mRNAs. Previously, we discovered that estradiol (E2) treatment of ovariectomized sheep increased concentrations of AUF1p45 protein which stabilized estrogen receptor alpha (ER) mRNA in the uterus. This study examined E2 regulation of AUF1 mRNAs in the sheep uterus. Northern analysis determined that E2 treatment increased concentrations of total AUF1 mRNAs twofold in endometrial and myometrial tissue compartments. In situ hybridization indicated that the increase was most intense in the glandular epithelium of endometrium. In a well characterized in vitro RNA stability assay, AUF1 3'UTR sequences were much more stable in uterine extracts from E2-treated ewes compared to extracts from control ewes. AUF1 mRNAs with alternative splicing of exons 2 and 7 (in the coding sequence) and exon 9 (in the 3'UTR) were identified. The only effect of E2 treatment on alternative splicing was that it reduced the percentage of AUF1 mRNAs containing exon 9-derived sequences. These data indicate that E2 up-regulates AUF1 and ER genes coordinately by a post-transcriptional mechanism.

Production and characterization of agonistic monoclonal antibodies against chicken CD40.

CD40 is mainly expressed by professional antigen-presenting cells (APCs). Its ligand, CD40L, is transiently expressed on activated CD4(+) T-cells. CD40-CD40L interactions mediate T-cell help to APCs and provide crucial signals for affinity maturation and B-cell class switching. In mammals, agonistic monoclonal anti-CD40 antibodies (mAbs) mimic the effects of CD40L on APCs, leading to enhanced T-cell priming and expansion, increased antibody production and class switching. In this study, we describe agonistic anti-chicken CD40 mAb 2C5. This mAb detected CD40 on primary chicken B-cells and macrophages, DT40 B-cells, and HD11 macrophages, induced NO synthesis in HD11 macrophages, and stimulated DT40 B-cell proliferation. These observations demonstrated at least partial functional equivalence of 2C5 to chicken CD154. This mAb may therefore constitute a new tool to study the role of CD40 in the chicken immune system, and its agonistic effects suggest that it could also be used as an adjuvant.

Intrauterine infusion of latency-associated peptide (LAP) during early porcine pregnancy affects conceptus elongation and placental size.

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.

In vitro culture of precision-cut testicular tissue as a novel tool for the study of responses to LH.

In vitro culture systems are valuable tools for investigating reproductive mechanisms in the testis. Here, we report the use of the precision-cut in vitro system using equine testicular slices. Testes were collected from immature light breed stallions (n=3) and cut into slices (mean slice weight= 13.85 ± 0.20 mg; mean slice thickness=515.00 ± 2.33 µm) using the precision-cut tissue-slicing method. Four tissue slices were placed on a grid floating on medium in individual vials. After a 1-h preincubation, they were exposed to medium containing ovine luteinizing hormone (oLH) at concentrations of 0, 5, 50, and 500 ng/ml for 6 h at 32 °C. Viability of the tissue was maintained based on histological integrity and lack of appreciable lactate dehydrogenase in the medium. The production and release of testosterone (T) and estradiol-17ß (E2) into the medium was measured following in vitro culture. The addition of oLH increased T and E2 at least 400% and 120%, respectively, over the 0-ng oLH control cultures. Testicular gene expression was assessed with in situ hybridization methodology for steroidogenic acute regulatory protein (StAR protein), phosphodiesterase 3B (PDE3B), and outer dense fiber of sperm tails 2 (ODF2) mRNAs. In situ hybridization revealed an oLH concentration-dependent increase in the concentration of StAR protein mRNA in Leydig cells. No differences were observed for the expression of PDE3B or ODF2 genes in seminiferous tubules among treatment groups as expected. These results demonstrate the value of in vitro culture of the precision-cut tissue slices for studies of the regulation of steroidogenesis and gene expression in the stallion testes.

Transforming growth factor beta (TGFB) signaling is activated during porcine implantation: proposed role for latency-associated peptide interactions with integrins at the conceptus-maternal interface.

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 AUF1p45 binding to stabilizing regions within the 3'-untranslated region of estrogen receptor alpha mRNA.

Estradiol up-regulates expression of the estrogen receptor alpha gene in the uterus by stabilizing estrogen receptor alpha mRNA. Previously, we defined two discrete minimal estradiol-modulated stability sequences (MEMSS) within the extensive 3'-untranslated region of estrogen receptor alpha 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 alpha 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 alpha mRNA stability and the highest rate of accumulation of estrogen receptor alpha 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 alpha mRNA in vitro in a dose-dependent manner. These studies describe estrogenic induction of AUF1p45 binding to the estrogen receptor alpha mRNA as a molecular mechanism for post-transcriptional up-regulation of gene expression.

Steroid hormones acutely regulate expression of a Nudix protein-encoding gene in the endometrial epithelium of sheep.

Steroid hormones regulate endometrial gene expression to meet the needs of developing embryos. Our hypothesis is that steroid hormones transiently induce expression of genes in the endometrial epithelium to make the uterine environment different between the earliest days of pregnancy. We identified one such gene product using differential display-polymerase chain reactions. The gene product that was strongly induced in ewes between day 3 and 6 of the estrous cycle was cloned and sequenced to identify it as encoding a member of the Nudix family of hydrolase enzymes. Northern blot analyses indicated that NUDT16 mRNA concentrations were elevated 10-fold in the endometrium of sheep from day 5 to 9 of the estrous cycle and returned to basal levels by day 11. In assays of RNA samples from 15 different tissues from an adult ewe, the concentrations of NUDT16 mRNA were greatest in endometrium. In situ hybridization localized NUDT16 mRNA exclusively to the endometrial epithelial cells of the glands and uterine lumen. In ovariectomized ewes, NUDT16 mRNA was induced by a regimen of alternating estrogen and progesterone therapy designed to mimic the hormonal experiences of a ewe at day 6 of the estrous cycle. The final estrogen treatment in the regimen was critical to the expression of NUDT16 as well as progesterone receptor and estrogen receptor-beta genes. Characterization of the NUDT16 gene identified putative steroid hormone response elements, which can now be investigated to understand its unique pattern of regulation in the earliest days of pregnancy.

Functional effects of transforming growth factor beta on adhesive properties of porcine trophectoderm.

In pigs, expression and amounts of biologically active TGFbetas at the conceptus-maternal interface increase significantly as conceptuses elongate and begin the implantation process. Before their activation, secreted TGFbetas are noncovalently associated with their respective, isoform-specific latency-associated peptides (LAPs), which contain the Arg-Gly-Asp (RGD) amino acid sequence that serves as a ligand for numerous integrins. Objectives of this study were to determine whether TGFbeta1 increases production of fibronectin by porcine trophectoderm, whether porcine trophectoderm adheres specifically to fibronectin and LAP, and whether functional interactions between porcine trophectoderm and the two TGFbeta-associated proteins, fibronectin and LAP, are integrin mediated. Porcine trophectoderm cells (pTr2) were cultured in presence of TGFbeta1, LAP, or pan-neutralizing anti-TGFbeta antibody; TGFbeta specifically increased (P < 0.05) fibronectin mRNA levels, as determined by Northern and slot blot analyses. Immunofluorescence microscopy demonstrated a TGFbeta-induced increase in fibronectin in pTr2 cells. In dispersed cell adhesion assays, adhesion of pTr2 cells to fibronectin was inhibited by an RGD-containing peptide (P < 0.05) and pTr2 cells attached to recombinant LAP but not to an LAP mutant, which contained an RGE sequence rather than the RGD site (P < 0.05). Fibronectin- and LAP-coated microbeads induced integrin activation at apical surfaces of both trophectoderm and uterine luminal epithelial cells, as indicated by aggregation and transmembrane accumulation of talin detected with immunofluorescence microscopy. Cell surface biotinylation and immunoprecipitation revealed integrin subunits alphav and beta1 on apical membranes of pTr2 cells. These results suggest multiple effects of TGFbeta at the porcine conceptus-maternal interface, including integrin-mediated conceptus-maternal communication through LAP.

Steroid hormones regulate gene expression posttranscriptionally by altering the stabilities of messenger RNAs.

Hormones exert powerful effects on reproductive physiology by regulating gene expression. Recent discoveries in hormone action emphasize that regulation of gene expression is not restricted to their alterations of the rate of gene transcription. On the contrary, hormonal effects on the stability of a specific mRNA can profoundly alter its steady-state concentration. The mRNAs encoding hormone receptors are commonly regulated by their own hormones to create autoregulatory feedback loops. Negative and positive autoregulatory feedback loops serve to limit or augment hormonal responses, respectively. After introducing the topics of mRNA degradation and regulated stability, this review focuses on steroid hormone effects on mRNA stabilities. Autoregulation of the mRNAs encoding estrogen, progesterone, androgen, and glucocorticoid receptors by the steroid hormones in reproductive tissues is discussed. In addition, steroid hormone effects on the stabilities of many other mRNAs that are important to reproductive biology are reviewed. These include mRNAs that encode gonadotropin hormones, integrins, growth factors, and inflammatory response proteins. Through these posttranscriptional effects, steroid hormones impact the expression of a large population of genes. Studies of the molecular mechanisms of hormonally regulated mRNA stabilities continue to identify critical mRNA sequence elements and their interactions with proteins. Increased understanding of how hormones affect mRNA stability may yield novel approaches to the therapeutic control of hormone effects, including those essential to reproductive physiology in animals.