Mouse models of NADK2 deficiency analyzed for metabolic and gene expression changes to elucidate pathophysiology.

NADK2 encodes the mitochondrial form of nicotinamide adenine dinucleotide (NAD) kinase, which phosphorylates NAD. Rare recessive mutations in human NADK2 are associated with a syndromic neurological mitochondrial disease that includes metabolic changes, such as hyperlysinemia and 2,4 dienoyl CoA reductase (DECR) deficiency. However, the full pathophysiology resulting from NADK2 deficiency is not known. Here, we describe two chemically induced mouse mutations in Nadk2-S326L and S330P-which cause severe neuromuscular disease and shorten lifespan. The S330P allele was characterized in detail and shown to have marked denervation of neuromuscular junctions by 5 weeks of age and muscle atrophy by 11 weeks of age. Cerebellar Purkinje cells also showed progressive degeneration in this model. Transcriptome profiling on brain and muscle was performed at early and late disease stages. In addition, metabolomic profiling was performed on the brain, muscle, liver and spinal cord at the same ages and on plasma at 5 weeks. Combined transcriptomic and metabolomic analyses identified hyperlysinemia, DECR deficiency and generalized metabolic dysfunction in Nadk2 mutant mice, indicating relevance to the human disease. We compared findings from the Nadk model to equivalent RNA sequencing and metabolomic datasets from a mouse model of infantile neuroaxonal dystrophy, caused by recessive mutations in Pla2g6. This enabled us to identify disrupted biological processes that are common between these mouse models of neurological disease, as well as those processes that are gene-specific. These findings improve our understanding of the pathophysiology of neuromuscular diseases and describe mouse models that will be useful for future preclinical studies.

Expression of microRNA in male reproductive tissues and their role in male fertility.

MicroRNA (miRNA) are small non-coding RNA, approximately 22 nucleotides in length, that regulate gene expression through their ability to bind to mRNA. The role of miRNA in cellular and tissue development is well documented and their importance in male reproductive tissue development is actively being evaluated. They are present in spermatogonia, Sertoli and Leydig cells within the testis and are present in mature spermatozoa, indicating roles in normal testicular development, function and spermatogenesis. Their presence in spermatozoa has led to postulations about the roles of male miRNA during early embryonic development after fertilisation, including chromatin restructuring and possible epigenetic effects on embryo development. MiRNAs are also present in body fluids, such as blood serum, milk, ovarian follicular fluid and seminal fluid. Circulating miRNAs are stable, and aberrant expression of cellular or extracellular miRNA has been associated with multiple pathophysiological conditions, the most studied being numerous forms of cancer. Considering that miRNAs are present in spermatozoa and in seminal fluid, their stability and the relatively non-invasive procedures required to obtain these samples make miRNAs excellent candidates for use as biomarkers of male reproduction and fertility. Biomarkers, such as miRNAs, identifying fertile males would be of financial interest to the animal production industry.

Effects of maternal nutrient restriction during gestation on bovine serum microRNA abundance.

The objective was to determine the effects of maternal nutrient restriction during gestation on serum microRNA (miRNA) abundance in cattle. Primiparous Angus-cross cows (n=22) were fed either control (CON; to gain 1 Kg/week) or nutrient restricted (NR; 0.55% NEm) diets based on National Research Council requirements. On day 30 of gestation, cows were blocked by body condition and randomly assigned to one of three diets: CON (n=8) days 30-190; NR (n=7) days 30-110 followed by CON days 110-190 (NR/C); or CON (n=7) days 30-110 followed by NR days 110-190 (C/NR). At 190 days of gestation, maternal serum was collected for RNA isolation and analyzed using a miRNA microarray of known Bos taurus sequences. Data were normalized using LOWESS and analyzed via ANOVA. At 190 days of gestation, 16 miRNAs exhibited differential abundance (P<0.05) between treatments. Cows that underwent NR, irrespective of when the insult occurred, had downregulated bta-miR-126-3p compared to CON cows. Bta-miR-16b was downregulated and three miRNAs upregulated in NR/C compared to C/NR and CON cows. Additionally, seven miRNAs were downregulated and four miRNAs upregulated in C/NR compared to NR/C and CON cows. Comparison of NR/C and C/NR cows revealed three differentially abundant (P<0.04) miRNAs (bta-miR-2487_L-2R-3_1ss15CT, bta-miR-215, and bta-miR-760-5p). Top KEGG pathway enrichment of target genes included: pathways in cancer, PI3K-Akt signaling, focal adhesion, Ras signaling, proteoglycans in cancer, and MAPK signaling. In summary, maternal nutrient restriction altered serum miRNA abundance profiles irrespective of the time at which the nutritional insult was induced.

Does lesion type or severity predict outcome of therapy for horses with equine glandular gastric disease? - A retrospective study.

BACKGROUND: Equine glandular gastric disease (EGGD) is a common condition of the horse. Misoprostol is reported to be superior to oral omeprazole and sucralfate for treatment. Long-acting intramuscular injectable omeprazole (LAIOMEP) is a novel treatment shown to be effective in a small population. OBJECTIVES: This study aimed to determine LAIOMEP efficacy compared to misoprostol and oral omeprazole and identify characteristics that predict treatment outcome. METHODS: All horses that underwent gastroscopy between 2012 and 2019 were reviewed. Lesions were characterised by 4 blinded observers, all of whom are diplomates in equine internal medicine, using established descriptors from the ECEIM consensus statement and subjective severity. Treatment outcome was ranked as worsened, improved or healed. Consensus lesion type, lesion severity and treatment choice were compared to outcome and data screened using univariate analysis (chi-squared) to determine whether each predicted outcome. Lesion types where univariate analysis predicted a trend (p<0.2) were included in a multiple-regression analysis to identify predictors of outcome irrespective of treatment. RESULTS: Only severity significantly predicted final outcome (p = 0.025) with severe lesions being more likely to improve. Treatment choice did not significantly predict outcome. Overall healing rate was 29% (24 horses), and 43% (44 horses) improved. Treatment healing rates were 23% (10), 12% (7) and 27% (7) for LAIOMEP, misoprostol and oral omeprazole, respectively, with improvement in 69% (14), 76% (21) and 61% (9). 64% of the latter group received sucralfate. Worsening occurred in 7% (6). Treatment length varied with a median of 4 weeks (range 4-20 weeks). CONCLUSIONS: This study showed poorer therapy outcome compared to previous studies. The only initial lesion descriptor to predict outcome was severity and treatment choice did not affect outcome.

Myoepithelial cell differentiation markers in prepubertal bovine mammary gland: effect of ovariectomy.

We reported previously that ovariectomy alters prepubertal development of mammary myoepithelial cells (MC) by mechanisms that are not well understood. Therefore, in the present study, we analyzed expression of 2 myoepithelial differentiation markers, α-smooth muscle actin (SMA) and the common acute lymphoblastic leukemia antigen (CD10), in mammary parenchymal tissue from intact (INT) and ovariectomized (OVX) heifers. On d 40, Holstein heifers underwent either an ovariectomy (OVX; n=16) or a sham (INT; n=21) operation. At 55, 70, 85, 100, 130, and 160 d of age, tissues were collected, and multispectral imaging was used to quantify immunofluorescent staining for myoepithelial cell (MC) markers. Fluorescent intensity (FI) of the markers was normalized against a control sample. In the basal epithelial layer, CD10 FI was less and SMA FI was greater in OVX than INT. The ratio of SMA to CD10 FI, as a proxy indicator for MC differentiation, was greater in tissue from OVX compared with INT heifers after 55 d of age. The staining for SMA was frequently more intense along the basal aspect of cells, whereas CD10 expression was localized on the apical surface of the MC. In mammary tissue from both INT and OVX heifers, we observed basal cells that were negative for both CD10 and SMA, some of which appeared to span the distance from basement membrane to the ductal lumen. Interestingly, we also observed CD10+ cells adjacent to the ductal lumen, a situation that was more prevalent in OVX than in INT heifers. Also, ovariectomy affects MC expression of both SMA and CD10, as well as the pattern of MC development. Myoepithelial cells are known to limit parenchymal growth in other species. Involvement of MC as regulators of prepubertal bovine mammary development is worthy of further investigation.

Effects of nutrient restriction during early or mid-gestation in bovine on placental development and miRNA expression in the cotyledon.

The objective of this study was to determine effects of maternal nutrient restriction (NR) during early or mid-gestation on uterine composition and miRNA expression in cotyledons. Primiparous Angus-cross cows (n = 38) were synchronized and inseminated using male sexed semen, blocked by body condition score and body weight (BW), and assigned to treatments. Animals were fed either: control (CON; gain 1 kg/week) or NR (55% maintenance energy and crude protein requirements) based on BW. An initial set of animals were fed either NR (n = 8) or CON (n = 8) from day 30-110 of gestation. A second set of animals were fed CON (n = 8) d 30-190 (CON/CON); NR (n = 7) day 30-110 followed by CON day 110-190 (NR/CON); or CON (n = 7) day 30-110 followed by NR day 110-190 (CON/NR). Cows were harvested on day 110 or 190 of gestation to collect placental tissues. RNA was isolated from cotyledon samples (3 animals/group) prior to microarray analysis using known Bos taurus microRNA sequences. Relative microRNA abundance was analyzed via ANOVA. Maternal NR increased (P < 0.05) cotyledon weight and total placentome surface area irrespective of gestational day. At day 110 of gestation, 51 microRNAs were reduced while 91 microRNAs observed greater abundance (P < 0.05) in NR verses CON cotyledons. At day 190 of gestation, 40 microRNAs were reduced and 26 microRNAs were increased (P < 0.05) in both NR/CON and CON/NR verses CON cotyledons. Top KEGG pathway analysis included: axon guidance, endocytosis, neuroactive ligand receptor interaction, and MAPK signaling pathway. Early-gestation maternal NR altered microRNA abundance to a greater extent than mid-gestation NR.

The integrated stress response contributes to tRNA synthetase-associated peripheral neuropathy.

Dominant mutations in ubiquitously expressed transfer RNA (tRNA) synthetase genes cause axonal peripheral neuropathy, accounting for at least six forms of Charcot-Marie-Tooth (CMT) disease. Genetic evidence in mouse and Drosophila models suggests a gain-of-function mechanism. In this study, we used in vivo, cell type­specific transcriptional and translational profiling to show that mutant tRNA synthetases activate the integrated stress response (ISR) through the sensor kinase GCN2 (general control nonderepressible 2). The chronic activation of the ISR contributed to the pathophysiology, and genetic deletion or pharmacological inhibition of Gcn2 alleviated the peripheral neuropathy. The activation of GCN2 suggests that the aberrant activity of the mutant tRNA synthetases is still related to translation and that inhibiting GCN2 or the ISR may represent a therapeutic strategy in CMT.

Bovine dopamine type-2 receptor SNP has no effect on growth, semen characteristics and prolactin concentrations in beef bulls treated with a dopamine agonist.

A dopamine type-2 receptor (DRD2) SNP, previously found to be correlated with serum prolactin (PRL) concentrations in cattle, was evaluated for impact on growth traits, serum prolactin concentration, and semen quality. Over a four-year period, yearling beef bulls were allowed diets containing or lacking ergot alkaloids (EA). Every 21 or 28 d semen was collected for semen motility and morphology assessment and blood samples were collected to measure serum PRL concentrations. In addition, body condition score and scrotal circumference were evaluated. Serum PRL concentrations were assessed using a radioimmunoassay. In the first year, all bulls were sacrificed at the end of a 126-day study. Testicles and epididymis were collected at the end of the study or 60 days after removal from treatment. Immunohistochemistry was performed on testis, epididymis, and sperm cells, incubated with or without a primary antibody for DRD2 and counterstained with DAPI. Isolation of DNA was performed on sperm pellets using DNAzol (Thermo Fisher Scientific, Waltham, MA, USA) methods. Polymerase chain reaction was performed to amplify the region of the DRD2 gene containing the SNP of interest. The products were subjected to restriction fragment length polymorphism analysis. Further, all samples were subjected to genotyping using a custom Taqman genotyping assay (Applied Biosystems, Foster city, CA, USA). The presence of DRD2 was detected in the testis, epididymis, and sperm cells. The DRD2 genotype was not associated with semen quality, serum PRL, or growth traits. Consumption of EA resulted in lesser PRL serum concentrations but had no effect on values for other variable examined.

Hot topic: Prepubertal ovariectomy alters the development of myoepithelial cells in the bovine mammary gland.

Prepubertal ovariectomy can dramatically inhibit mammary development, but the mechanism of inhibition is not well characterized. Holstein heifers were ovariectomized (OVX) or sham operated but left intact (INT) at d 40 and then sacrificed at d 55, 70, 85, 100, 130, or 160 to provide tissues for histologic analysis of cell proliferation. Our histologic analyses unexpectedly revealed a pronounced effect of ovariectomy on myoepithelial cell development. Myoepithelial cells were identified on the basis of location, morphology, and immunohistochemical staining for alpha-smooth muscle actin (SMA). Vascular smooth muscle staining served as an internal positive control for all immunohistochemical analyses. Mammary tissues from d 40 heifers had an abundance of SMA+ cells associated with the ductal parenchyma. In INT heifers, the frequency of SMA+ cells decreased as development progressed. Only a limited number of isolated SMA+ cells were observed in d 70 to d 160 INT heifers. In OVX heifers, SMA+ cells were abundant, had elongated morphology, and frequently stained more intensively than vascular smooth muscle cells. The intense SMA staining and altered morphology was most prominent in older heifers. Limited analysis of gene expression revealed that maspin, a protease inhibitor expressed by myoepithelial cells, was expressed in parenchyma from both INT and OVX heifers. Our hypothesis is that ovarian secretions stimulate epithelial proliferation, and block myoepithelial differentiation. Myoepithelial cells are known to limit parenchymal cell proliferation. Ovariectomy may thus remove an estrogenic growth stimulus and permit the emergence of inhibitory cell populations that further limit parenchymal expansion. Our observation has important implications for control mechanisms that regulate parenchymal development.

Assessment of semen quality and fertility in young growing beef bulls exposed to ergot alkaloids.

The objective of this study was to evaluate semen quality and fertility in beef bulls grazing the ergot alkaloid (EA) producing tall fescue cultivar, Kentucky 31 (KY31), compared to a novel endophyte (NE) cultivar lacking EA. Two studies were conducted over a 3-year period. In studies 1 (n = 10; ages ≥ 24 mo) and 2 (n = 53 over two years; ages 12-16 mo), Angus (AN) bulls were stratified by body weight (BW), body condition score (BCS), and scrotal circumference (SC), and then allotted to graze either KY31 or NE for 56 days. Semen samples were collected, and BW, BCS, and SC were evaluated at the start of treatment (TRT) on day (d) 0 and every 28 days to the end of each study. In addition, blood samples were collected on d 0 and every 28 days for assessment of circulating prolactin (PRL) levels in study 2. On d 56, for both studies, semen from bulls (n = 2 per treatment in study 1 and n = 4 per treatment in study 2) with similar and acceptable quality were extended, kept at 19° C, and used for timed artificial insemination (TAI) of primi- and multiparous AN and AN- crossbred females. Pregnancy was evaluated at 35 and 90 days post-TAI via transrectal ultrasonography to determine pregnancy rates. Serum PRL concentrations showed a TRT by d effect (P ≤ 0.05), with values for bulls grazing KY31 decreased on d 28 and d 56 of grazing compared to NE. In studies 1 and 2, bull BW and BCS were affected by d (P ≤ 0.05), but not by TRT. No TRT or TRT by d effect on semen quality was observed in either study; however, d impacted both velocity and concentration in study 2 (P ≤ 0.05). In study 1, TAI pregnancy rates at 35 days post-TAI were lower (P ≤ 0.05) in the group inseminated with semen from bulls grazing KY31; however, in study 2, pregnancy rates did not differ due to treatment 35 post-TAI (P > 0.05). Grazing KY31 negatively impacted serum PRL concentrations, supporting previous observations; however, consumption of KY31 had no effect on growth or semen quality of AN bulls ranging from 12 to ≥24 mo of age. Furthermore, fertility data is inconsistent between studies and requires further investigation.

Comparison of meat composition from offspring of cloned and conventionally produced boars.

This study compares the meat composition of the offspring from boars produced by somatic cell nuclear transfer (n=4) to that of the offspring from conventionally produced boars (n=3). In total, 89 commercial gilts were artificially inseminated and 61 progressed to term and farrowed. All of the resulting piglets were housed and raised identically under standard commercial settings and slaughtered upon reaching market weight. Loin samples were taken from each slaughtered animal and shipped offsite for meat composition analysis. In total, loin samples from 404 animals (242 from offspring of clones and 162 from controls) were analyzed for 58 different parameters generating 14,036 and 9396 data points from offspring of clones and the controls, respectively. Values for controls were used to establish a range for each parameter. Ten percent was then added to the maximum and subtracted from the minimum of the control range, and all results within this range were considered clinically irrelevant. Of the 14,036 data points from the offspring of clones, only three points were found outside the clinically irrelevant range, two of which were within the range established by the USDA National Nutrient Database for Standard Reference, Release 18, 2005; website: (www.nal.usda.gov/fnic/foodcomp/search/). The only outlier was the presence of Eicosadienoic acid (C20:2) in one sample which is typically present in minute quantities in pork; no reference data were found regarding this fatty acid in the USDA National Nutrient Database. In conclusion, these data indicated that meat from the offspring of clones was not chemically different than meat from controls and therefore supported the case for the safety of meat from the offspring of clones.

Effects of grazing tall fescue containing ergot alkaloids on bull sperm cryopreservation.

There are many positive agronomic traits that make tall fescue a desirable forage, however, reduced fertility rates are reported for beef cattle grazing pasture containing the ergot alkaloid-producing endophyte, Epichloë coenophiala. The objective of this study was to assess the influence of consuming tall fescue containing ergot alkaloids on sperm physiology, as measured by survival of sperm following cryopreservation. Yearling Angus bulls (n=25), having passed a breeding soundness exam (BSE), were assigned to one of two treatments accounting for body weight (BW) and body condition score (BCS). Bulls were allotted to one of two treatments on day (d) 0, grazing toxic Kentucky 31 (KY31) or a novel endophyte-containing cultivar, Texoma Max Q II (NE; AR584 Ag Research) that does not produce ergot alkaloids, for 112 days. On d 112, all bulls were placed on NE pasture to the end of test (d 168) to evaluate recovery from grazing KY31. Blood, urine, and semen samples were collected every 28 days. Semen collected on d 28, 84, 112, 140, and 168 was extended, frozen, thawed 48h later, and subjected to analyses. There were significant treatment by day interactions for serum prolactin (PRL) concentrations, verifying the effectiveness of treatment (P<0.05). Serum PRL concentrations were less in the bulls pastured on KY31 compared to NE on d 28, 84, and 112. Urinary alkaloid concentrations were affected by treatment by day interactions, confirming ergot alkaloids were present in animal systems (P<0.05). Bulls in the NE treatment group had lesser urinary alkaloid concentrations than those pastured on KY31 on d 28, 84, and 112. Post-thaw sperm analyses revealed that the percentage of progressively motile sperm was less in bulls pastured on KY31 when compared to NE (P<0.05). There were treatment by day interactions for sperm concentration, percent motile sperm, percent motile sperm concentration, and percent progressively motile sperm concentration post-thawing (P<0.05). The KY31 treatment group had a lesser sperm 1) concentration than the NE group on d 84; 2) percent motility on d 28, 84, and 168; 3) motile concentration on d 28, 84, and 168; and 4) progressively motile concentration on d 28 and 84. Sperm motility was affected post-thawing for at least 56 d following removal from the KY31 pasture.

A comparison of reproductive characteristics of boars generated by somatic cell nuclear transfer to highly related conventionally produced boars.

This study compares the reproductive performance of boars produced by somatic cell nuclear transfer versus conventional breeding. Two different genotypes were selected for comparison: terminal cross line 1 (TX1) and terminal cross line 2 (TX2). The boars selected for comparison from TX1 were three cloned boars, produced by somatic cell nuclear transfer and the conventionally produced progenitor of the clones. The boars selected for comparison from TX2 were a cloned boar produced by somatic cell nuclear transfer and two conventionally produced half sibling boars that were offspring of the progenitor of the clone. Semen from each boar was collected, extended, evaluated and shipped offsite. Upon arrival, the semen was reevaluated and utilized for artificial insemination of 89 commercial gilts, at least 12 gilts per boar, producing 625 piglets. Pregnancy rates were determined at day 30 and 110 of gestation; and farrowing rate and gestation length were recorded. Differences were observed in some of the semen characteristics analyzed with the clones usually possessing superior semen quality to the control, this likely being a result of age differences amongst the clones and controls. Additionally no differences were noted between the clones and controls (progenitor) or between individual boars within genetic line for pregnancy rates, gestation length or any of the litter parameters examined between the clones and controls. These data further support previous reports with limited numbers that the reproductive capabilities of cloned boars are equal to that of conventionally produced boars.

Prolactin gene-disruption arrests mammary gland development and retards T-antigen-induced tumor growth.

Prolactin (PRL), interacting with other hormones from the pituitary, gonad, and placenta, activates specific signals that drive the appropriately timed morphological and functional development of the mammary gland. A mouse model of isolated PRL deficiency (PRL-/-) was created by gene disruption in an effort to further understand the molecular basis of mammary gland development and breast cancer. Whereas primary ductal growth was normal in PRL-/- mice, ductal arborization was minimal (branches/mm2=1.5+/-0.5), and lobular budding was absent. Replacement therapy with PRL injections stimulated a modest degree of lobular budding and ductal arborization (3.75+/-0.9). Pituitary transplants to the kidney capsule of PRL-/- mice restored lobular budding and ductal arborization, to the full extent of that seen in control animals (20. 3+/-5.5). Pregnancy, established by mating progesterone-treated PRL-/- females with PRL-/- males, led to complete morphological development of the mammary gland, appropriate to the gestational stage. PRL treatment stimulated tyrosine phosphorylation and DNA binding activity of Stat5a, but not Stat1 in PRL-/- or PRL+/- females, and Stat5a, but not Stat1, was elevated by estradiol within 24 h. PRL-deficient mice were crossed with mice expressing a dominant oncogene (polyoma middle-T antigen driven by the MMTV promoter, PyVT mice). Palpable (1 mm3) tumors were detected an average of 9 days earlier in hormonally normal females (PRL+/-:PyVT) compared with littermates that were PRL-deficient (PRL-/-:PyVT). The growth rate of PyVT-induced tumors was 30% faster in PRL+/-, than in PRL-/- females.

BILL E. KUNKLE INTERDISCIPLINARY BEEF SYMPOSIUM: Does tall fescue toxicosis negatively impact bull growth and breeding potential?

The predominant cool-season forage in the southeastern United States is the tall fescue cultivar Kentucky 31 (KY31). Kentucky 31 possesses an endophyte (), which produces a family of toxins called ergot alkaloids. These toxins negatively affect the physiology of animals on consumption and result in the syndrome known as fescue toxicosis. Currently, the United States annually produces approximately 11.4 billion kg of beef, of which 25% originates in the southeastern region of the United States where forage systems frequently are tall fescue based. Cattle within this forage system exhibit reduced gains and reproductive performance. The result is a reduction in the nation's beef supply with annual revenue losses recently estimated at approximately US$1 billion. Our hypothesis is that exposure to these ergot alkaloids in conjunction with limited availability of nutrients decreases bull semen quality and fertility. Although the literature is clear that these toxins affect BW, body temperature, blood flow, hair growth, and female reproduction in cattle, their effect on bull reproduction and the mechanisms through which the toxins act are not well defined. Six studies published from 2004 to 2015 assessed bull growth, body composition, and semen quality of young beef bulls exposed to ergot alkaloids. If semen quality or fertility is altered, the mechanisms involved may be either direct effects of ergot alkaloids through neurotransmitter receptors or indirect effects such as inhibiting the release of prolactin (PRL). The possible effects of ergot alkaloids or PRL require establishing the presence or absence of dopamine, adrenergic, serotonin, or PRL receptors in the testis, epididymis, and sperm cell of the bull. The objective of this review is to relate our findings to the few previous studies conducted that evaluated the impact of fescue toxicosis on bull reproduction and to propose possible mechanisms of action for lowered semen quality.

Identification of bovine prolactin in seminal fluid, and expression and localization of the prolactin receptor and prolactin-inducible protein in the testis and epididymis of bulls exposed to ergot alkaloids.

The objectives of this study were to determine (1) the presence and expression levels of bovine prolactin receptor (PRLR) and prolactin-inducible protein (PIP) in bovine testis and epididymis, and (2) the presence and concentrations of prolactin (PRL) present in seminiferous fluid in bulls consuming diets with (E+) or without (E-) ergot alkaloids. Bulls (n = 8) were sacrificed after 126 days (group A) of E+ or E- treatment or 60 days after all bulls (n = 6) were switched to the E- ration (group B). End point and real-time quantitative reverse transcription-polymerase chain reaction and immunohistochemistry were conducted on testis and epididymis samples to establish the presence and relative expression of PRLR and PIP. Seminal fluid samples obtained from bulls consuming E- and E+ diets were subjected to RIA for PRL. Both PIP and PRLR were present in testis and epididymis as determined by reverse transcription-polymerase chain reaction and immunohistochemistry. Prolactin-inducible protein mRNA abundance was affected by time of slaughter in testis and epididymis head, respectively (P < 0.05). Prolactin receptor mRNA expression was affected by time of slaughter in the epididymis (P < 0.05) and differed in testis samples because of treatment (P < 0.05). Radioimmunoassay establishes the presence of PRL in seminal fluid; however, differences in the concentration of PRL over two separate studies were inconsistent, possibly because of differences in diet. The presence and localization of the PRLR are consistent with expression data reported for other species, and the presence of PIP and PRL in seminal fluid is consistent with data generated in humans.

Bulls grazing Kentucky 31 tall fescue exhibit impaired growth, semen quality, and decreased semen freezing potential.

Serum prolactin (PRL) and testosterone concentrations, body weight, body composition, semen quality, and semen freezing potential for bulls grazing the toxic tall fescue (Lolium arundinaceum [Schreb.] Darbysh. = Schedonorous arundinaceum [Schreb.] Dumort.) cultivar Kentucky 31 (E+) compared with a novel endophyte cultivar lacking ergot alkaloids (E-) were evaluated. Angus bulls were allotted to treatment (Day 0) and grazed E+ or E- for 155 days. Treatment-by-day interaction was significant (P < 0.05) for serum PRL concentrations with E+treated bulls exhibiting reduced PRL values compared with E- control bulls, but no differences were observed for serum testosterone concentrations (P > 0.05). Further, bulls on the E+ treatment exhibited decreased total gain, average daily gain, and body weight by Day 140 (P < 0.05) compared with the E- bulls. Rump muscle depth was lower because the treatment in bulls grazing E+ compared with E- (P < 0.05) and intramuscular fat in the E- bulls compared with the E+ group was higher by Day 155 (P < 0.05). Analysis of ejaculates showed significant treatment × day effects for sperm concentration with lower values observed for bulls on the E+ treatment (P < 0.05). The percent normal morphology was reduced in ejaculates from E+ bulls compared with E- bulls (P < 0.05), and the difference was due to an increase in abnormal sperm present in the E+ ejaculates from Day 84 to 140 (P < 0.05). In addition, spermatozoa motility and progressive motility were decreased on thawing in semen samples from E+ bulls compared with E- bulls (P < 0.05).

The growth hormone receptor messenger ribonucleic acid present in ovine fetal liver is a variant form.

Specific binding of ovine GH (oGH) to microsomal membranes isolated from fetal sheep liver is slight to nonexistent. The complementary DNA sequence encoding the oGH receptor (oGHR) has been reported, and Northern blot analysis has indicated that oGHR messenger RNA (mRNA) is present in fetal liver and skeletal muscle from mid- to late gestation. In human tissues, the GHR mRNA exists in multiple forms, including the deletion of exon 3 and variable 5'-untranslated regions. In rodents, the GHR mRNA exists in two forms, one encoding the membrane-bound receptor and the other encoding the soluble GH-binding protein. To further characterize the oGHR mRNA transcript present in ovine fetal liver during gestation, we designed a series of primers to be used in reverse transcriptase-polymerase chain reactions (RT-PCR), which generate products that span from the 5'-untranslated region through the coding region of the oGHR mRNA. Nucleotide sequences of the resulting complementary DNAs revealed that an oGHR mRNA is present from mid- to late gestation (days 60-135) which contains the region analogous to exon 3 of the human GHR gene. However, the 5'-untranslated region previously reported in adult tissues was not present until day 135 of gestation in fetal liver, nor was it present in day 100 fetal skeletal muscle. Northern hybridization analysis indicates that the major oGHR transcript in day 105 fetal liver is 5.8 kilobases (kb) in size, with minor transcripts observed at 4.7 kb and three transcripts greater than 6.5 kb. By day 135 of gestation, the transcript size is the same as that observed in day 100 pregnant ewe liver (5.5 kb). We conclude that the oGHR mRNA present in midgestation fetal liver differs structurally from the transcript present in late gestation fetal liver and adult liver, and this difference may explain the lack of specific GH binding to ovine fetal liver membranes. Furthermore, our results suggest that there is a developmental switch in the structure of oGHR mRNA that occurs shortly before term, potentially preparing the fetus to respond to GH postnatally.

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)

Identification of two Y-box binding proteins that interact with the promoters of columbid annexin I genes.

Two annexin I (anxI) genes, called cp35 and cp37, are expressed from the pigeon (Columba livia) genome, but they are regulated differently at both the transcriptional and post-transcriptional levels. The proximal promoter elements of these two genes are very similar. A conserved sequence from the cp35 and cp37 promoters bound specifically with proteins present in cropsac cell extracts. This sequence of DNA was used to screen a lambdagt11 cDNA expression library. Clones encoding two pigeon Y-box binding proteins (YB) were isolated. One of the pigeon YB cDNAs was found to be most similar to YB1 from other species, and the other was most similar to chicken YB2. Each YB is encoded by a single-copy gene in the pigeon, and their mRNAs are expressed in many tissues. On Northern blots, the sizes of the mRNAs encoding pigeon YB1 (pYB1) and pigeon YB2 (pYB2) were 1.8 and 1.7kb, respectively. The sequences of both pYB1 and pYB2 diverge from their previously identified relatives in the N-terminal domain 'A'. Antisera were developed to unique peptide epitopes in YB1 or 2. Affinity-purified anti-YB1 and anti-YB2 detected immunoreactive proteins in extracts from a variety of pigeon tissues, including the cropsac. To confirm that pYB1 and pYB2 interact with the cp35 promoter, electrophoretic gel mobility shift reactions were carried out in the presence or absence of YB antibodies. Binding to the cp35 promoter was specifically neutralized by either anti-pYB1 or anti-pYB2. These results are the first evidence that two YB proteins simultaneously bind to a promoter element, and thereby may interact during regulation of gene expression.