A novel swine sex-linked marker and its application across different mammalian species.

Advances in genome editing tools have reduced barriers to the creation of animal models. Due to their anatomical and physiological similarities to humans, there has been a growing need for pig models to study human diseases, for xenotransplantation and translational research. The ability to determine the sex of genetically modified embryos, cells or fetuses is beneficial for every project involving the production of transgenic animals. This strategy can improve the time-efficiency and lower the production costs. Additionally, sex assessment is very useful for wildlife studies to understand population behavior and structure. Thus, we developed a simple and fast PCR-based protocol for sex determination in pigs by using a unique primer set to amplify either the DDX3X or DDX3Y gene. The sex was 100% correctly assigned when tail genomic DNA, Day-35 fetus and hair samples from pigs were used. For both blastocysts and oocytes (84.6% and 96.5% of efficacy, respectively) the unidentified samples were potentially due to a limitation in sample size. Our assay also worked for domestic sheep (Ovis aries), American bison (Bison bison) and European cattle (Bos taurus) samples and by in silico analysis we confirmed X-Y amplicon length polymorphisms for the DDX3 gene in 12 other mammalian species. This PCR protocol for determining sex in pig tissues and cells showed to be simple, specific, highly reproducible and less time consuming as well as an important tool for other livestock species and wildlife studies.

Quantification and cellular localization of ovine placental lactogen messenger ribonucleic acid expression during mid- and late gestation.

Ovine placental lactogen (oPL) is structurally similar to PRL, is a product of the chorionic epithelium, and has been implicated in playing a supportive role in fetal growth. This study examined the concentration and cellular location of oPL mRNA at five stages of pregnancy (days 60, 90, 105, 120, and 135) in 21 cross-bred ewes, and results were compared to maternal and fetal serum oPL concentrations, cotyledonary DNA and actin mRNA concentrations, and total fetal weight. The concentration of oPL mRNA in fetal cotyledonary tissue increased (P < or = 0.05) from day 60 (15.4 pg/micrograms total cellular RNA) to day 120 (73.7 pg/micrograms total cellular RNA) of gestation and then plateaued, whereas no significant changes occurred in the concentration of actin mRNA over the gestational ages examined. The concentration of DNA in cotyledonary tissue (micrograms per mg wet tissue) increased (P < or = 0.05) from days 60 through 120 and remained constant through day 135, such that when oPL mRNA was expressed on a picogram per microgram DNA basis, no stage of gestation effect (P > or = 0.10) was observed. The maternal serum oPL concentration increased (P < or = 0.05) from day 60 (7.1 ng/ml) to day 105 (417.7 ng/ml), followed by a large but nonsignificant (P > or = 0.10) increase in maternal serum oPL occurring on day 135 (902.0 ng/ml). Fetal serum oPL concentrations increased (P < or = 0.05) from day 60 (11.0 ng/ml) to day 90 (29.0 ng/ml) and then remained relatively constant. Maternal serum oPL (r = 0.68; P < or = 0.01) and cotyledonary oPL mRNA levels (r = 0.61; P < or = 0.05) were correlated with total fetal weight when adjusted for fetal number and gestational age, and together accounted for 80.6% (r2 value) of the variation found in total fetal weight. The correlation between fetal serum oPL concentrations and total fetal weight was nonsignificant (P < or = 0.10). Examination of placentome cross-sections by immunocytochemistry and in situ hybridization at the five gestational ages indicated that the chorionic binucleate cell was the sole source of oPL. These data provide evidence that, like maternal serum concentrations of oPL, oPL mRNA expression by chorionic binucleate cells increases until late gestation, whereas fetal serum concentrations of oPL plateau during midgestation.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning of the cDNAs coding for cat growth hormone and prolactin.

Characterization of the prolactin (PRL) amino acid (aa) or cDNA sequences has not been reported for any member of the Felidae family. We cloned cat growth hormone (cGH) and cat PRL (cPRL) cDNA sequences from a feline pituitary cDNA library. High homology between species allowed bovine PRL(bPRL) and bGH cDNA clones to be used to identify clones encoding the 229-aa cPRL and 216-aa cGH sequences. The cGH protein is most homologous to pig and dog GH. Similarly, cPRL shares the most aa identity to pig PRL (pPRL). Northern blot analysis revealed the mRNA size for cGH and cPRL to be approx. 1 and 1.1 kb, respectively. These results reveal that GH and PRL from the Felidae family are highly conserved to other families of GH and PRL.

Increased production of peptide deformylase eliminates retention of formylmethionine in bovine somatotropin overproduced in Escherichia coli.

In Escherichia coli and most other microorganisms, peptide synthesis is started at methionine start codons which are read only by N-formyl-methionine-tRNA. The formyl group is normally removed from the N-terminal Met residue of the peptide by peptide deformylase (PDF). However, it has been observed that overproduction of proteins in recombinant bacteria often yields protein products which are incompletely deformylated. Certain proteins could be poor substrates for PDF and exhibit incomplete deformylation, particularly when they are overproduced. Strains of E. coli which overproduce bovine somatotropin (BST) have a significant fraction of the BST with the formyl group retained. The PDF gene was isolated and positioned into a BST production vector in such a way that the BST and PDF genes were coexpressed. In strains containing this coexpression vector, the levels of PDF were increased and formylated BST was undetectable.

Purification and structural characterization of ovine placental lactogen.

Discrepancies exist in the reported purity and biological activity of ovine placental lactogen (oPL), and little structural characterization has been reported. Ovine PL was purified from fetal cotyledonary tissue (day 100 of gestation) by monitoring activity with a recombinant bovine GH (bGH) liver radioreceptor assay. Two hundred grams of tissue yielded 4.2 mg of oPL, with an approximately 1000-fold purification of oPL specific activity following initial tissue extraction. The oPL was radioiodinated and used in an ovine fetal liver (day 100 of gestation) radioreceptor assay to examine competitive displacement of oPL, ovine GH (oGH) and ovine prolactin (oPRL). The potency of oPL (ED50 = 0.18 nmol/l; ED50 is the quantity of ligand necessary to displace 50% of specifically bound 125I-labelled oPL) was greater than that of oGH (ED50 = 4.1 nmol/l) and oPRL (ED50 = 1.1 mumol/l) in competing for 125I-labelled oPL-binding sites. Attempts to sequence the NH2 terminus of oPL by vapour-phase sequencing indicated that the NH2 terminus was blocked. Purified oPL was subjected to trypsin and CnBr digestion, and two CnBr and six tryptic peptides were sequenced. The peptide sequences were compared with other PLs, oPRL and bGH for sequence similarity, and found to be most similar to bovine PL (bPL; 68% overall identity) and oPRL (47% overall identity). Complementary DNA (cDNA) clones were isolated for oPL by screening a lambda ZAP cDNA library with a cDNA coding for bPL. Three cDNAs were nucleotide sequenced, and their combined sequence included 41 nucleotides of 5'-untranslated region, the complete coding region of pre-oPL (708 nucleotides) and a portion of the 3' untranslated region (158 nucleotides).(ABSTRACT TRUNCATED AT 250 WORDS)

Spi-1: an hepatic serine protease inhibitor regulated by GH and other hormones.

A sensitive RNAse protection method was used to show that serine protease inhibitor-1 (Spi-1) is expressed in rat liver and heart, but not in kidney or brain. Bovine somatotropin (bGH) and placental lactogen (bPL) induced rat hepatocyte cultures to express both Spi-1 and IGF-1 mRNA, with bPL approximately 100-fold more potent than bGH. Bovine prolactin (bPrL) did not induce hepatocyte Spi-1 mRNA, demonstrating lack of involvement of lactogenic receptors. Albumin mRNA levels were stable during hepatocyte culturing and were unaffected by growth hormone (GH) treatment, showing that neither culture conditions nor GH treatment affected cellular differentiation. Eliminating serum-free medium hormone supplements one at a time, estradiol, testosterone and T3 were shown to be unnecessary for GH induction of Spi-1, while dexamethasone removal decreased Spi-1 mRNA levels to 10% of GH-stimulated controls. bGH induction of Spi-1 mRNA in the presence of only dexamethasone and glucagon was 75% higher (p < 0.01) than levels seen with insulin also present.

Evaluation of the somatogenic activity of bovine placental lactogen with cell lines transfected with the bovine somatotropin receptor.

Studies have shown that bovine placental lactogen (bPL) has partial somatogenic activity in vivo even though binding results clearly indicate bPL does not cause homodimerization of the bovine somatotropin receptor (bST-R). To help understand the receptor binding versus biological activity of bovine somatotropin (bST) and bPL we have developed a homologous model system. Full length bST-R was stably transfected into a murine lymphoid cell line, Ba/F3 and a hamster kidney cell line, BHK. From both transfected cell lines, clones were isolated (Ba/F3-C1 and BHK-24) which demonstrated specific binding of bST and, or bPL. Bovine ST stimulated proliferation of the Ba/F3-C1 clonal line over a dose range of 10 to 3000 pM with an EC50 of 100 pM. A bST variant (des 1-4 bST) and porcine ST (pST) which both have approximately 10% of the binding affinity for bST-R as native bST were 1 and 10% as potent as bST in this bioassay, respectively. This suggests that affinity and biological activity are correlated for this system. Proliferation was initiated through the bST-R because addition of a monoclonal antibody which recognizes the extracellular domain of bST-R and inhibits binding of bST to its receptor, inhibited bST-stimulated mitosis. However, even though the affinity of bPL for the bST-R is similar to that of bST, bPL antagonized the proliferative action of bST with an IC50 of 1 nM. Components of the somatogenic signal transduction pathway were also evaluated in both cell lines. Addition of bST to the cell cultures increased phosphorylation of JAK2 in Ba/F3-C1 and BHK-24 cells in a dose-responsive manner but bPL failed to increase phosphorylation of JAK2 in either cell line. In summary, these data support the hypothesis that ST-R homodimerization is necessary for bioactivity in this model system but fail to explain apparent somatogenic activity of bPL in vivo.

Synthesis and secretion of ovine placental lactogen and its biochemical properties.

The biochemical properties of ovine placental lactogen (oPL) have been previously determined following purification, which has yielded various results. To clarify the properties of oPL prior to purification, oPL was examined in solubilized fetal cotyledonary tissue (d 100 of gestation) or conditioned culture medium by electrophoretic, immunoblotting and immunoprecipitation techniques. In cotyledonary tissue or conditioned culture medium, oPL has an apparent molecular weight (Mr) of 22,000 with an isoelectric point (pI) of 9.2. Incorporation of [3H]-glucosamine or [3H]-mannose into immunoreactive oPL could not be detected, nor did the presence of tunicamycin in explant culture medium alter the apparent Mr of oPL. In vitro translation of d 100 fetal cotyledonary mRNA, followed by immunoprecipitation, provided evidence that pre-oPL has an apparent Mr of 25,000. The size of oPL mRNA was determined to be approximately 1,350 base pairs by Northern hybridization procedures using an oligonucleotide probe which was generated from oPL amino acid sequence data. These experiments suggest that the only intracellular processing oPL undergoes is removal of a amino-terminal signal sequence. We conclude that oPL is synthesized and secreted as a single nonglycosylated-basic protein, at a time during gestation when circulating oPL is elevated.

Beef cow reproduction as affected by postpartum nutrition and temporary calf removal.

Data were collected on 306 multiparous cows over a 3-yr period (1984-1986) to evaluate the effect of postpartum nutrition and temporary calf removal on reproductive performance. All cows calved in a body condition score (BCS) of >/= 5 and were randomly allotted at parturition to an experiment with a 2 x 2 factorial arrangement of treatments. The factors consisted of high or low-flush levels of postpartum nutritional management, with or without temporary calf removal for 48 h prior to the breeding season. The high diet contained 26.0 Mcal metabolizable energy. The low regimen of the low-flush diet contained 14.7 Mcal metabolizable energy, with the flushing regime consisting of a diet of 36 Mcal metabolizable energy. The flushing diet was offered 2 wk prior to breeding through the first 30 d of breeding. Interval to estrus was 45 and 44 d for high and low-flush, respectively (P>0.05), while interval to estrus with temporary calf removal and without temporary calf removal was 47 and 42 d, respectively (P<0.05). Means for the cumulative percent in estrus at 20, 40 and 60 d of breeding, respectively, were high, 93, 97 and 98; low-flush, 95, 96 and 98; with temporary calf removal, 92, 96 and 99; and without temporary calf removal, 96, 97 and 98 (P>0.05). Interval to pregnancy was 81 and 82 d for high and low-flush, respectively (P>0.05), while interval to pregnancy with temporary calf removal and without temporary calf removal was 82 and 81 d, respectively (P>0.05). Cumulative percentages of animals pregnant at 20, 40 and 60 d of breeding, respectively, were high, 59, 83 and 91; low-flush, 59, 80 and 90; with temporary calf removal, 58, 83 and 91; and without temporary calf removal, 60, 80 and 90 (P>0.05). Calf performance in response to dam nutrition and temporary calf removal was not affected at weaning or at intervals prior to weaning (P>0.05). Interactions between nutrition level and removal of calf factors were not significant (P>0.05) for any of the parameters measured. The data suggest cows calving in BCS >/= 5 have equivalent reproductive performance whether receiving high levels of energy postpartum or low levels of energy early postpartum, followed by a flushing diet fed immediately proceeding and continuing through the first 30 d of breeding. No beneficial effect of temporary calf removal was observed for cows calving with BCS >/= 5.

Ruminant placental lactogens: structure and biology.

Ruminant placental lactogens (PL) are members of the somatotropin, prolactin gene family that are synthesized by trophectodermal binucleate cells. The structure and biology of PL has been studied in the cow, sheep, and goat. Ruminant PL have greater structural identity to prolactin than somatotropin, although they bind to both lactogenic and somatogenic receptors. The molecular weights of ovine and caprine PL are approximately 23,000, whereas bovine PL is larger (31,000 to 34,000) due to glycosylation. Placental lactogen is secreted into both the fetal and maternal circulations. The concentration of PL in the fetus decreases with advancing gestation, whereas PL concentration peaks in the maternal circulation during the last third of pregnancy then reaches a plateau. Furthermore, the maternal concentration of PL is 100- to 1,000-fold higher in sheep and goats than in cows. The precise factors that modulate secretion of PL are unknown, although placental mass and nutrition seem to play a role. Ruminant PL have both lactogenic and somatogenic biological activities and may also have unique activities mediated through a specific receptor. There is circumstantial evidence to suggest that PL plays a role in stimulating mammogenesis. Placental lactogen secreted into the fetal compartment may also help regulate fetal growth. Direct experimental data indicate that PL can regulate maternal intermediary metabolism. Thus, it may act as a partitioning agent to regulate nutrient supply for fetal growth. The precise biological function of PL in ruminants, therefore, still needs to be defined.

Tissue repair by thrombin-derived peptides in the rat.

An ordered sequence of events occurs following tissue damage to bring about healing. One molecule involved in this process is thrombin. Utilizing rat linear incision and full dermal excision models, we have investigated the ability of two thrombin-derived RGD-containing peptides, p517 and p508, to enhance tissue repair under normal and healing-impaired conditions. p508, at 0.5 micrograms peptide per wound, produced a significant .23% improvement in wound strength in a dose-dependent manner. Similarly, a single application of 0.5 micrograms p517 per 6-cm linear incision would significantly increased wound-breaking strength approximately 18% at nine days postsurgery (control: 3.95 +/- 0.13 Newtons, vs. p517: 4.68 +/- 0.13 Newtons; P < 0.001). However, in glucocorticoid-stressed rats, the application of 0.5 micrograms per wound p508 or 517 did not significantly influence steroid-impaired healing. In the full dermal skin excision wound model, a single application of 0.5 micrograms p508 per wound at the time of surgery significantly reduced average wound area at days 3 and 5, when healing was impaired by glucocorticoid administration. Wound area was also reduced by p508 treatment at day 3 in the normal animal, but this effect was not statistically significant. We suggest wound-healing benefits of p508 and p517 may activate wound fibroblast proliferation or stimulate other cell types in the wound site through an RGD-mediated interaction.

Development of microsatellite markers and comparative mapping for bovine chromosome 19.

Previous research has mapped an ovulation rate quantitative trait locus (QTL) to bovine chromosome 19. In an effort to enhance comparative mapping information and develop additional markers for refined QTL mapping, microsatellite markers were developed in a targeted approach. A bovine bacterial artificial chromosome (BAC) library was screened for loci with either known or predicted locations on bovine chromosome 19. An average of 6.4 positive BAC were identified per screened locus. A total of 10 microsatellite markers were developed for five targeted loci with heterozygosity of 7-83% in a sample of reference family parents. The newly developed markers were typed on reference families along with four previously mapped marker loci and used to create a linkage map. Comparison of locus order between human and cattle provides support for previously observed rearrangement. One of the mapped loci myotubularin related protein 4 (MTMR4) potentially extends the proximal boundary of a conserved linkage group.

Mapping microsatellite markers identified in porcine EST sequences.

A sequence search of swine expressed sequence tags (EST) data in GenBank identified over 100 sequence files which contained a microsatellite repeat or simple sequence repeat (SSR). Most of these repeat motifs were dinucleotide (CA/GT) repeats; however, a number of tri-, tetra-, penta- and hexa-nucleotide repeats were also detected. An initial assessment of six dinucleotide and 14 higher-order repeat markers indicated that only dinucleotide markers yielded a sufficient number of informative markers (100% vs. 14% for dinucleotide and higher order repeats, respectively). Primers were designed for an additional 50 di- and one tri-nucleotide SSRs. Overall, 42 markers were polymorphic in the US Meat Animal Research Center (MARC) reference population, 17 markers were uninformative and 12 primer pairs failed to satisfactorily amplify genomic DNA. A comparison of di-nucleotide repeat vs. markers with repeat motifs of three to six bases demonstrated that 72% of dinucleotide markers were informative relative to only 7% of other repeat motifs. The difference was the result of a much higher percentage of monomorphic markers in the three to six base repeat motif markers than in the dinucleotide markers (64% vs. 14%). Either higher order repeat motifs are less polymorphic in the porcine genome or our selection criteria for repeat length of more than 17 contiguous bases was too low. The mapped microsatellite markers add to the porcine genetic map and provide valuable links between the porcine and human genome.