Temporal candidate gene expression in the sow placenta and embryo during early gestation and effect of maternal Progenos supplementation on embryonic and placental development.

The present study characterised gene expression associated with embryonic muscle development and placental vascularisation during early gestation in the pig and examined effects of Progenos supplementation in early pregnancy. Tissues were collected from commercial multiparous sows (n = 48) from Days 16 to 49 of gestation. In the placenta, qPCR revealed that vascular endothelial growth factor (VEGFA) expression did not change from Day 17 to 49 of gestation; however, KDR receptor and angiopoietin-1 and -2 expression were differentially regulated, with periods of high expression corresponding to two critical phases of angiogenesis in the pig. In the embryo, the pattern of myogenesis-related gene expression was consistent with available literature. A commercially available nutritional supplement Progenos (20 g day⁻¹ L-arginine) added to the diet of sows from either Day 15 to 29 (P15-29; n = 33), Day 30 to 44 (n = 29) or from Day 15 to 44 (n = 76) of gestation tended to increase (P = 0.058) embryonic growth rate compared with non-supplemented controls (n = 79) and angiogenin expression was higher (P = 0.028) at Day 30 of gestation in placentae from sows on the P15-29 Progenos treatment. These results are consistent with proposed beneficial effects of l-arginine on early embryonic development and placental vascularisation.

Restricted feed intake in lactating primiparous sows. II. Effects on subsequent litter sex ratio and embryonic gene expression.

Expression of panels of candidate genes controlling myogenesis, angiogenesis and gender-specific imprinting of development were analysed in embryonic, placental and endometrial tissues recovered at Day 30 of gestation from a subset of primiparous sows that were either feed restricted (Restrict; n=17) or fed to appetite (Control; n=15) during the last week of the previous lactation. Embryos were also sex typed to investigate gender bias in response to treatments. Average embryonic weight was lower in the subset of Restrict compared with Control litters (1.38±0.07vs 1.59±0.08g, respectively) and the male:female sex ratio was higher (P<0.05) in embryos (litters) recovered from Restrict sows. Treatment affected (P≤0.05) the expression of embryonic and placental genes involved in insulin-like growth factor (IGF) 2 signalling, including IGF2, INSR and IGF2R. Embryonic expression of ESR1 was also affected by treatment (P<0.03) and sex×treatment interactions were observed for the expression of embryonic ESR1 (P<0.05) and placental ANGPT2 (P<0.03). At the molecular level, these results support the suggestion that changes in placental function are not the primary mechanism mediating detrimental effects of previous sow catabolism on early embryonic development in the feed-restricted lactational sow model. However, perturbations in the IGF2 system are implicated as mediators of these effects.

Impact of genetic potential for residual feed intake and diet fed during early- to mid-gestation in beef heifers on carcass characteristics and meat quality attributes of their castrated male offspring.

Carcass attributes of steers were examined for influences of selection for residual feed intake (RFI), and exposure to different levels of prenatal nutrition. Heifers characterized for RFI corrected for backfat were mated to bulls with genetic potential for either High-RFI or Low-RFI, such that the progeny were expected to be H/H or L/L RFI (sire/dam). Pregnant heifers were assigned to a low diet (Ldiet; 0.40 kg/d ADG), or moderate diet (Mdiet; 0.57 kg/d ADG), from 30 to 150 days of gestation, after which all heifers were managed similarly. Steer offspring (n = 23) were also managed similarly until slaughter. Dressing percentage of steers from H-RFI dams/sires exposed to Ldiet during gestation was lower than all other groups (P = 0.02). Marbling was greater for steers from H-RFI parents, as was fat content of longissimus thoracis et lumborum and triceps brachii (P ≤ 0.02). Results suggest that parental selection for RFI and prenatal maternal diet can influence carcass characteristics of progeny.

Specific protein-protein interaction between basic helix-loop-helix transcription factors and homeoproteins of the Pitx family.

Homeoproteins and basic helix-loop-helix (bHLH) transcription factors are known for their critical role in development and cellular differentiation. The pituitary pro-opiomelanocortin (POMC) gene is a target for factors of both families. Indeed, pituitary-specific transcription of POMC depends on the action of the homeodomain-containing transcription factor Pitx1 and of bHLH heterodimers containing NeuroD1. We now show lineage-restricted expression of NeuroD1 in pituitary corticotroph cells and a direct physical interaction between bHLH heterodimers and Pitx1 that results in transcriptional synergism. The interaction between the bHLH and homeodomains is restricted to ubiquitous (class A) bHLH and to the Pitx subfamily. Since bHLH heterodimers interact with Pitx factors through their ubiquitous moiety, this mechanism may be implicated in other developmental processes involving bHLH factors, such as neurogenesis and myogenesis.

Ontogeny of metabolic effects on embryonic development in lactating and weaned primiparous sows.

Using an established experimental paradigm, feed restriction during the last week of lactation in primiparous sows reduces embryonic growth and development and produces female-specific embryonic mortality by Day 30 of gestation. Because this gender-specific loss of embryos at Day 30 was associated with changes in the variation of markers of epigenetic imprinting, the present study sought to establish the ontogeny of such epigenetic affects. Leucocyte DNA of restrict-fed sows exhibited decreased global methylation during the last week of lactation and during the return to oestrus (P < 0.05), but no associated changes in plasma folate and vitamin B(12). Furthermore, no changes in methylation of blastocyst DNA, embryonic sex ratios or development were evident at Day 6 of gestation that would characterise the underlying defects that reduced female embryo survival by Day 30. However, regardless of treatment, embryo recovery rates and synchrony in embryonic development were associated with the stage of development of the recovered embryos (r = 0.68; P < 0.001). The subset of sows classified as bearing litters with superior embryonic development had lower net energy balance over lactation (P < 0.01) and higher ovulation rates (P < 0.005) compared with sows classified as having poorer embryonic development. Collectively, these data suggest that a subset of litters within restrict-fed sows will be most sensitive to the latent epigenetic mechanisms that ultimately trigger gender-specific loss of embryos by Day 30 of gestation, but that these selective mechanisms are not evident by Day 6 of gestation.

Macroenvironment effects on oocytes and embryos in swine.

As in other domestic mammals, the interaction between genotype and environment in swine has profound effects on the ultimate phenotype of the individual born. Interactions within the litter in utero add an additional level of complexity in a litter-bearing species like the pig. Nutritional manipulations during the preovulatory period affect the maturity of the follicle and enclosed oocyte, and the metabolic and endocrine mechanisms potentially mediating these effects have been described. Extensive research on lactational catabolism in the first parity sow has established an association between the development of immature follicles and oocytes, and the reduced fertility of these sows when bred at the first postweaning estrus. This negative impact of lactational catabolism appears to be exaggerated in contemporary dam-lines by a minimal delay between weaning and first estrus, further limiting the maturity of the follicle and oocyte at the time of ovulation. Metabolic programming may induce gender-specific loss of embryos by Day 30 and affects embryonic development directly, without significant effects on placental size. In contrast, inadvertent crowding of embryos in utero, particularly evident in a sub-population of mature sows with high ovulation rates and moderate to high embryonic survival to Day 30, significantly limits placental development of crowded litters. However, even at Day 30, moderate crowding in utero also appears to affect myogenesis in the embryo in a gender-specific manner. In the absence of compensatory placental growth after Day 30, classic measures of IUGR are evident in surviving fetuses at Day 90 and at term.

Application of transcriptomic analyses to reproductive studies in contemporary commercial sows.

Genetic selection in sows has substantially increased the total number of pigs born, but there has been an associated decrease in litter birth weight. Furthermore, irrespective of the total number of pigs born, a repeatable low litter birth weight phenotype has evolved in a subpopulation of mature sows, in which a high ovulation rate drives extremes of intrauterine crowding in early gestation and limits placental development: placental development continues to be compromised in later gestation and is associated with intrauterine growth restriction and a low litter birth weight. Furthermore, although contemporary commercial sows are increasingly resilient to the challenges of lactational catabolism in terms of the traditional measures of reproductive performance, gender-specific effects on early embryonic development of the subsequent litter are apparent and are likely linked to effects of sow catabolism on folliculogenesis and oocyte maturation. Recent transcriptomic studies that have helped elucidate some of the regulatory mechanisms mediating the evolving changes in the reproductive biology of contemporary commercial sows are summarized in the present review. However, the lack of clearly differentiated reproductive phenotypes among commercial sow populations is a problem for ongoing studies. Access to an unselected "wild-type" population with which to make comparisons, or the application of more standardized experimental approaches to mechanistic studies, may help advance the field of sow reproductive biology.

Transcriptomic analysis by RNA sequencing reveals that hepatic interferon-induced genes may be associated with feed efficiency in beef heifers.

In beef cattle, production feedstuffs are the largest variable input cost. Beef cattle also have a large carbon footprint, raising concern about their environmental impact. Unfortunately, only a small proportion of dietary energy is directed toward protein deposition and muscle growth whereas the majority supports body maintenance. Improving feed efficiency would, therefore, have important consequences on productivity, profitability, and sustainability of the beef industry. Various measures of feed efficiency have been proposed to improve feed utilization, and currently, residual feed intake (RFI) is gaining popularity. However, the cost associated with measuring RFI and the limited knowledge of the biology underlying improved feed efficiency make its adoption prohibitive. Identifying molecular mechanisms explaining divergence in RFI in beef cattle would lead to the development of early detection methods for the selection of more efficient breeding stock. The objective of this study was to identify hepatic markers of metabolic feed efficiency in replacement beef heifers. A group of 87 heifers were tested for RFI adjusted for off-test backfat thickness (RFIfat). Preprandial liver biopsies were collected from 10 high- and 10 low-RFIfat heifers (7 Hereford­Aberdeen Angus and 3 Charolais­Red Angus­Main Anjou per group) and gene expression analysis was performed using RNA sequencing and quantitative real-time PCR. The heifers used in this study differed in RFIfat averaging 0.438 vs. ­0.584 kg DM/d in high- and low-RFIfat groups, respectively. As expected, DMI was correlated with RFIfat and ADG did not differ between high- and low-RFIfat heifers. Through a combination of whole transcriptome and candidate gene analyses, we identified differentially expressed genes involved in inflammatory processes including hemoglobin ß (HBB), myxovirus resistance 1 interferon-inducible protein p78 (MX1), ISG15 ubiquitin-like modifier (ISG15), hect domain and RLD 6 (HERC6), and interferon-induced protein 44 (IFI44) whose mRNA abundance was lower (HBB) or higher (MX1, ISG15, HERC6, and IFI44) in low-RFIfat heifers. These genes have been shown to be directly or indirectly modulated by interferon signaling and involved with innate immunity. Our results suggest that more efficient heifers respond differently to hepatic proinflammatory stimulus, potentially expending less energy toward combating systemic inflammation and redirecting nutrients toward growth and protein accretion.

The expression of Cellulomonas fimi cellulase genes in Brevibacterium lactofermentum.

The exoglucanase gene (cex) and the endoglucanase A gene (cenA) from Cellulomonas fimi were subcloned into the Escherichia coli/Brevibacterium lactofermentum shuttle vector pBK10. Both genes were expressed to five to ten times higher levels in B. lactofermentum than in E. coli, probably because these genes were expressed from C. fimi promoters. In B. lactofermentum virtually all of the enzyme activities were in the culture supernatant. This system will facilitate analysis of the expression of the C. fimi genes in and secretion of their products from a Gram-positive bacterium.

Biomarkers of in vivo fertility in sperm and seminal plasma of fertile stallions.

The global proteome of sperm and seminal plasma of fertile stallions was investigated to determine whether associations with relative in vivo fertility exist. Seven stallions at stud in a commercial breeding station were collected throughout the breeding season and bred to a total of 164 mares to determine conception rates. On three occasions during the breeding season, raw semen was obtained from a regular collection for proteomic analysis using two-dimensional electrophoresis and also assessed for routine semen quality end points. First cycle conception rate was negatively related to ejaculate volume (r = -0.43, P = 0.05) and total IGF1 content (ng) per ejaculate (r = -0.58, P = 0.006), whereas overall pregnancy rate was positively related to sperm concentration (r = 0.56, P = 0.01). The abundance of three proteins known to be involved in carbohydrate metabolism in sperm was positively related to fertility. Furthermore, the abundance of four seminal plasma proteins were identified as being negatively related to fertility; these were identified as kallikrein-1E2 (KLK2), clusterin, and seminal plasma proteins 1 (SP1) and 2 (SP2). Abundance of cysteine-rich secretory protein 3 (CRISP3) was positively related to first cycle conception rate (r = 0.495, P = 0.027) and may provide a good marker of fertility. Based on stepwise regression analysis, clusterin and SP1 in seminal plasma together with sperm citrate synthase were predictive of fertility (r = 0.77, P < 0.0001). This study identified proteins within sperm and seminal plasma that could serve as biomarkers of semen quality and fertility in stallions.

Intra-follicular regulatory mechanisms in the porcine ovary.

The mechanisms controlling the follicular growth continuum in the pig involve the interaction between local growth factors which are expressed throughout development and extra-follicular factors such as gonadotrophins. A large number of follicular growth factors, many belonging to the transforming growth factor-beta (TGF-beta) superfamily, have been identified in the somatic cells and in the oocyte. The relative importance of these intra-follicular factors varies with stage of development. The initiation of follicular growth and early preantral development is controlled locally (by factors including c-kit-kit ligand, members of the bone morphogenetic family (e.g BMP-15) and growth differentiation factor-9 (GDF-9)) and gonadotrophins are not thought to be involved until later. During antral follicle development, the oocyte secretes factors that stimulate porcine granulosa cell proliferation and differentiation, modulate apoptosis and suppress progesterone production, thereby preventing premature luteinisation. Likely candidates for mediating these effects include BMP-6, -15 and GDF-9 that are critical for fertility and ovulation rate in several mammals. There are also paracrine interactions between the somatic cells, with theca derived transforming growth factor beta (TGF-beta) playing a key role in regulating antral follicle maturation. Finally, during the periovulatory period, members of the EGF family from the granulosa cells stimulate cumulus expansion and oocyte maturation. Evidence indicates that some of these local factors may also influence oocyte developmental potential, emphasizing further the complexity, and importance, of these intra-follicular interactions.

Cloning and characterization of chromosomally encoded cephalosporinase gene of Enterobacter cloacae.

The cephalosporinase gene, cpa, which codes for an inducible class I chromosomal beta-lactamase in Enterobacter cloacae was cloned on a fragment of 6.05 kilobase pairs inserted into plasmid pACYC184 and transferred into Escherichia coli HB101 recipient cells. The constructed hybrid plasmid, designated pGGQ101, carried a genomic fragment which retained its parental inducibility characteristics, although its expression level in transformed E. coli cells fell to 40-65% of its initial level in E. cloacae. The localization of the cpa gene on pGGQ101 plasmid was determined by Bal31 exonuclease deletion mapping and further confirmed by subcloning HindIII-AvaI restriction fragment on pMB9 plasmid vector. Labeling with [35S]methionine of pGGQ101 specified proteins in a minicell system showed that six or seven proteins are encoded by the insert. Two proteins with apparent molecular mass of 42 000 and 39 500 daltons, respectively, most probably represent the premature and mature cephalosporinase forms.

Enzymatic specificities and modes of action of the two catalytic domains of the XynC xylanase from Fibrobacter succinogenes S85.

The xylanase XynC of Fibrobacter succinogenes S85 was recently shown to contain three distinct domains, A, B, and C (F. W. Paradis, H. Zhu, P. J. Krell, J. P. Phillips, and C. W. Forsberg, J. Bacteriol. 175:7666-7672, 1993). Domains A and B each bear an active site capable of hydrolyzing xylan, while domain C has no enzymatic activity. Two truncated proteins, each containing a single catalytic domain, named XynC-A and XynC-B were purified to homogeneity. The catalytic domains A and B had similar pH and temperature parameters of 6.0 and 50 degrees C for maximum hydrolytic activity and extensively degraded birch wood xylan to xylose and xylobiose. The Km and Vmax values, respectively, were 2.0 mg ml-1 and 6.1 U mg-1 for the intact enzyme, 1.83 mg ml-1 and 689 U mg-1 for domain A, and 2.38 mg ml-1 and 91.8 U mg-1 for domain B. Although domain A had a higher specific activity than domain B, domain B exhibited a broader substrate specificity and hydrolyzed rye arabinoxylan to a greater extent than domain A. Furthermore, domain B, but not domain A, was able to release xylose at the initial stage of the hydrolysis. Both catalytic domains cleaved xylotriose, xylotetraose, and xylopentaose but had no activity on xylobiose. Bond cleavage frequencies obtained from hydrolysis of xylo-alditol substrates suggest that while both domains have a strong preference for internal linkages of the xylan backbone, domain B has fewer subsites for substrate binding than domain A and cleaves arabinoxylan more efficiently. Chemical modification with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide methiodide and N-bromosuccinimide inactivated both XynC-A and XynC-B in the absence of xylan, indicating that carboxyl groups and tryptophan residues in the catalytic site of each domain have essential roles.

The xynC gene from Fibrobacter succinogenes S85 codes for a xylanase with two similar catalytic domains.

The xynC gene of Fibrobacter succinogenes S85 codes for a 66.4-kDa xylanase which consists of three distinct domains separated by two flexible regions rich in serine residues. Domains A and B of XynC code for catalytic domains with 56.5% identity and 9.6% similarity with each other, and both domains share homology with xylanases of Ruminococcus flavefaciens, Neocallimastix patriciarum, Clostridium acetobutylicum, Bacillus pumilus, Bacillus subtilis, and Bacillus circulans. More than 88% of the xylanase activity of Escherichia coli cells carrying the original 13-kb recombinant plasmid was released from intact cells by cold water washes. The major products of hydrolysis of xylan by both domains were xylose and xylobiose, indicating that the xynC gene product exhibits catalytic properties similar to those of the XynA xylanases from R. flavefaciens and N. patriciarum. So far, these features are not shared broadly with bacteria from other environments and may indicate specific selection for this domain structure in the highly competitive environment of the rumen.

Beta-mannanase of Streptomyces lividans 66: cloning and DNA sequence of the manA gene and characterization of the enzyme.

The gene coding for a beta-mannanase was cloned homologously from Streptomyces lividans and its DNA sequence was determined. The fully secreted enzyme was isolated and purified from culture filtrates of the hyperproducing clone S. lividans IAF36 grown in mineral salt media containing galactomannan as the main carbon source. It had a molecular mass of 36 kDa and a specific activity of 876 i.u./mg of protein. Under the assay conditions used, the optimal enzyme activity was obtained at 58 degrees C and a pH of 6.8. The pI was 3.5. The kinetic constants of this mannanase determined with galactomannan as substrate were a Vmax. of 205 i.u./mg of enzyme and a Km of 0.77 mg/ml. Data from SDS/PAGE and Western blotting show that the cloned enzyme was identical to that of the wild-type strain.

A novel shuttle vector for Streptomyces spp. and Escherichia coli as a tool in site-directed mutagenesis.

This paper describes the construction and utilization of a novel shuttle vector for Streptomyces spp. and Escherichia coli as a useful vector in site-directed mutagenesis. The shuttle vector pIAFS20 (6.7 kb) has the following features: a replicon for Streptomyces spp., isolated from plasmid pIJ702; the thiostrepton-resistance gene as a selective marker in Streptomyces; the ColE1 origin, allowing replication in E. coli; and the ampicillin-resistance gene as a selective marker in E. coli. Vector pIAFS20 also contains the phage f1 intergenic region, which permits production of single-stranded DNA in E. coli after superinfection with helper phage M13K07. Moreover, the lac promoter is located in front of the multiple cloning sites cassette, allowing eventual expression of the cloned genes in E. coli. After mutagenesis and screening of the mutants in E. coli, the plasmids can be readily used to transform Streptomyces spp. As a demonstration, a 3.2-kb DNA fragment containing the gene encoding the xylanase A from Streptomyces lividans 1326 was inserted into pIAFS20, and the promoter region of this gene served as a target for site-directed mutagenesis. The two deletions reported here confirm the efficiency of this new vector as a tool in mutagenesis.

Expression and secretion of beta-glucuronidase and Pertussis toxin S1 by Streptomyces lividans.

Streptomyces lividans IAF18, obtained by homologous cloning, is capable of over-producing XlnA. To investigate the possibility of the expression of foreign genes, various coding regions of the xylanase A gene (xlnA) were analysed. Expression/secretion vectors were constructed containing the regulatory elements of xlnA with the coding region of the leader peptide with or without the truncated structural gene encoding the first 310 amino acids of the XlnA. The genes coding for the Escherichia coli beta-glucuronidase and subunit 1 of the Bordetella pertussis toxin (S1) were used and their expression analysed. S. lividans transformants where the beta-glucuronidase gene was fused with the leader sequence produced up to 30 mg beta-glucuronidase/culture filtrate whereas only fused XlnA/S1 was detected and its yield was estimated to be 1 mg/1. The disappearance of the B. pertussis toxin S1 and beta-glucuronidase from the culture medium was due to the concomitant appearence of secreted proteases from S. lividans.

Overexpression of angiotensin II type I receptor in cardiomyocytes induces cardiac hypertrophy and remodeling.

Angiotensin II (AII) is a major determinant of arterial pressure and volume homeostasis, mainly because of its vascular action via the AII type 1 receptor (AT1R). AII has also been implicated in the development of cardiac hypertrophy because angiotensin I-converting enzyme inhibitors and AT1R antagonists prevent or regress ventricular hypertrophy in animal models and in human. However, because these treatments impede the action of AII at cardiac as well as vascular levels, and reduce blood pressure, it has been difficult to determine whether AII action on the heart is direct or a consequence of pressure-overload. To determine whether AII can induce cardiac hypertrophy directly via myocardial AT1R in the absence of vascular changes, transgenic mice overexpressing the human AT1R under the control of the mouse alpha-myosin heavy chain promoter were generated. Cardiomyocyte-specific overexpression of AT1R induced, in basal conditions, morphologic changes of myocytes and nonmyocytes that mimic those observed during the development of cardiac hypertrophy in human and in other mammals. These mice displayed significant cardiac hypertrophy and remodeling with increased expression of ventricular atrial natriuretic factor and interstitial collagen deposition and died prematurely of heart failure. Neither the systolic blood pressure nor the heart rate were changed. The data demonstrate a direct myocardial role for AII in the development of cardiac hypertrophy and failure and provide a useful model to elucidate the mechanisms of action of AII in the pathogenesis of cardiac diseases.