Identification of measures predictive of age of puberty onset in gilts

A potential indicator of female lifetime productivity in swine is age of puberty, when a gilt achieves her first behavioral estrus. Follicular activity, as determined by tertiary follicle development, in prepubertal gilts begins during postnatal day (PND) 75-115. The central hypothesis of this study is that gilts demonstrating tertiary follicle development earlier in life, assessed using vulva size as a proxy, achieve puberty earlier in life compared to counterparts of a similar age and weight that lack tertiary follicle development. The objectives of this project were to identify a developmental time point when variation in ovarian development exists and to determine if a relationship between the age prepubertal ovarian development and the age at onset of puberty exists. To accomplish this, 155 gilts of similar age (± 2 days) were weighed and vulva size measured on PND 75, 85, 95, 105 and 115. Vulva measures, including vulva width (VW), length (VL) and area (VA) were utilized as developmental proxies for follicular activity. At each time point, gilts (n = 10) were sacrificed and ovarian follicular activity recorded. In a subset of gilts (n = 105), estrus detection was conducted daily on PND days 126 to 200. Mean vulva area (VA) on PND 75, 85, 95, 105 and 115 was 596 ± 206, 683 ± 190, 864 ± 212, 1014 ± 228 and 1265 ± 252 mm2, respectively. Of the gilts demonstrating behavioral estrus, 28 were within PND 140-160, 36 between PND 161-180, 15 between PND 181-200, and 26 did not demonstrate estrus behavior within 200 days of age. All gilts euthanized at PND 75 lacked follicular activity as defined by having a minimum of two antral follicles per ovary, while 60%, 80%, 90% and 100% demonstrated follicular activity on PND 85, 95, 105, and 115, respectively. Body weight at PND 75 and VW at PND 115 were correlated to age at first estrus (P \u3c 0.05). Of the gilts whose VA was less than one standard deviation from the mean on PND 95 (i.e. \u3c 652 mm2), 31% and 50% demonstrated their first behavioral estrus by PND 180 and 200, respectively. However, of gilts whose VA was within or greater than one standard deviation of the mean (i.e. ≥ 652 mm2), 66% and 79% exhibited estrus prior to PND 180 and 200, respectively. These data support utilization of VA changes between 95 and 115 days of age as a useful tool to identify replacement gilts prior to puberty for inclusion into the sow herd

Pharmacologic treatment with CPI-613 and PS48 decreases mitochondrial membrane potential and increases quantity of autolysosomes in porcine fibroblasts

A metabolic phenomenon known as the Warburg effect has been characterized in certain cancerous cells, embryonic stem cells, and other rapidly proliferative cell types. Previously, our attempts to induce a Warburg-like state pharmaceutically via CPI-613 and PS48 treatment did augment metabolite production and gene expression; however, this treatment demonstrated a Reverse Warburg effect phenotype observed in cancer-associated stroma. In the current study, we inquired whether the mitochondria were affected by the aforementioned pharmaceutical treatment as observed in cancerous stromal fibroblasts. While the pharmaceutical agents decreased mitochondrial membrane potential in porcine fetal fibroblasts, the number and size of mitochondria were similar, as was the overall cell size. Moreover, the fibroblasts that were treated with CPI-613 and PS48 for a week had increased numbers of large autolysosome vesicles. This coincided with increased intensity of LysoTracker staining in treated cells as observed by flow cytometry. Treated fibroblasts thus may utilize changes in metabolism and autophagy to mitigate the damage of treatment with pharmaceutical agents. These findings shed light on how these pharmaceutical agents interact and how treated cells augment metabolism to sustain viability. c2019, The Author(s).Includes bibliographical references

Improvement of in vitro and early in utero porcine clone development after somatic donor cells are cultured under hypoxia

Genetically engineered pigs serve as excellent biomedical and agricultural models. To date, the most reliable way to generate genetically engineered pigs is via somatic cell nuclear transfer (SCNT), however, the efficiency of cloning in pigs is low (1–3 percent). Somatic cells such as fibroblasts frequently used in nuclear transfer utilize the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation for efficient energy production. The metabolism of somatic cells contrasts with cells within the early embryo, which predominately use glycolysis. We hypothesized that fibroblast cells could become blastomere-like if mitochondrial oxidative phosphorylation was inhibited by hypoxia and that this would result in improved in vitro embryonic development after SCNT. In a previous study, we demonstrated that fibroblasts cultured under hypoxic conditions had changes in gene expression consistent with increased glycolytic/gluconeogenic metabolism. The goal of this pilot study was to determine if subsequent in vitro embryo development is impacted by cloning porcine embryonic fibroblasts cultured in hypoxia. Here we demonstrate that in vitro measures such as early cleavage, blastocyst development, and blastocyst cell number are improved (4.4 percent, 5.5 percent, and 17.6 cells, respectively) when donor cells are cultured in hypoxia before nuclear transfer. Survival probability was increased in clones from hypoxic cultured donors compared to controls (8.5 vs. 4.0 [plus or minus] 0.2). These results suggest that the clones from donor cells cultured in hypoxia are more developmentally competent and this may be due to improved nuclear reprogramming during somatic cell nuclear transfer

The association of homeobox gene expression with stem cell formation and morphogenesis in cultured Medicago truncatula

Somatic embryogenesis (SE) is induced in vitro in Medicago truncatula 2HA by auxin and cytokinin but rarely in wild type Jemalong. The putative WUSCHEL (MtWUS), CLAVATA3 (MtCLV3) and the WUSCHEL-related homeobox gene WOX5 (MtWOX5) were investigated in M. truncatula (Mt) and identified by the similarity to Arabidopsis WUS, CLV3 and WOX5 in amino acid sequence, phylogeny and in planta and in vitro expression patterns. MtWUS was induced throughout embryogenic cultures by cytokinin after 24–48 h and maximum expression occurred after 1 week, which coincides with the induction of totipotent stem cells. During this period there was no MtCLV3 expression to suppress MtWUS. MtWUS expression, as illustrated by promoter-GUS studies, subsequently localised to the embryo, and there was then the onset of MtCLV3 expression. This suggests that the expression of the putative MtCLV3 coincides with the WUS-CLAVATA feedback loop becoming operational. RNAi studies showed that MtWUS expression is essential for callus and somatic embryo production. Based on the presence of MtWUS promoter binding sites, MtWUS may be required for the induction of MtSERF1, postulated to have a key role in the signalling required for SE induced in 2HA. MtWOX5 expressed in auxin-induced root primordia and root meristems and appears to be involved in pluripotent stem cell induction. The evidence is discussed that the homeobox genes MtWUS and MtWOX5 are “hijacked” for stem cell induction, which is key to somatic embryo and de novo root induction. In relation to SE, a role for WUS in the signalling involved in induction is discussed

The GimA Locus of Extraintestinal Pathogenic E. coli: Does Reductive Evolution Correlate with Habitat and Pathotype?

IbeA (invasion of brain endothelium), which is located on a genomic island termed GimA, is involved in the pathogenesis of several extraintestinal pathogenic E. coli (ExPEC) pathotypes, including newborn meningitic E. coli (NMEC) and avian pathogenic E. coli (APEC). To unravel the phylogeny of GimA and to investigate its island character, the putative insertion locus of GimA was determined via Long Range PCR and DNA-DNA hybridization in 410 E. coli isolates, including APEC, NMEC, uropathogenic (UPEC), septicemia-associated E. coli (SEPEC), and human and animal fecal isolates as well as in 72 strains of the E. coli reference (ECOR) collection. In addition to a complete GimA (∼20.3 kb) and a locus lacking GimA we found a third pattern containing a 342 bp remnant of GimA in this strain collection. The presence of GimA was almost exclusively detected in strains belonging to phylogenetic group B2. In addition, the complete GimA was significantly more frequent in APEC and NMEC strains while the GimA remnant showed a higher association with UPEC strains. A detailed analysis of the ibeA sequences revealed the phylogeny of this gene to be consistent with that obtained by Multi Locus Sequence Typing of the strains. Although common criteria for genomic islands are partially fulfilled, GimA rather seems to be an ancestral part of phylogenetic group B2, and it would therefore be more appropriate to term this genomic region GimA locus instead of genomic island. The existence of two other patterns reflects a genomic rearrangement in a reductive evolution-like manner

Identification of measures predictive of age of puberty onset in gilts

A potential indicator of female lifetime productivity in swine is age of puberty, when a gilt achieves her first behavioral estrus. Follicular activity, as determined by tertiary follicle development, in prepubertal gilts begins during postnatal day (PND) 75-115. The central hypothesis of this study is that gilts demonstrating tertiary follicle development earlier in life, assessed using vulva size as a proxy, achieve puberty earlier in life compared to counterparts of a similar age and weight that lack tertiary follicle development. The objectives of this project were to identify a developmental time point when variation in ovarian development exists and to determine if a relationship between the age prepubertal ovarian development and the age at onset of puberty exists. To accomplish this, 155 gilts of similar age (± 2 days) were weighed and vulva size measured on PND 75, 85, 95, 105 and 115. Vulva measures, including vulva width (VW), length (VL) and area (VA) were utilized as developmental proxies for follicular activity. At each time point, gilts (n = 10) were sacrificed and ovarian follicular activity recorded. In a subset of gilts (n = 105), estrus detection was conducted daily on PND days 126 to 200. Mean vulva area (VA) on PND 75, 85, 95, 105 and 115 was 596 ± 206, 683 ± 190, 864 ± 212, 1014 ± 228 and 1265 ± 252 mm2, respectively. Of the gilts demonstrating behavioral estrus, 28 were within PND 140-160, 36 between PND 161-180, 15 between PND 181-200, and 26 did not demonstrate estrus behavior within 200 days of age. All gilts euthanized at PND 75 lacked follicular activity as defined by having a minimum of two antral follicles per ovary, while 60%, 80%, 90% and 100% demonstrated follicular activity on PND 85, 95, 105, and 115, respectively. Body weight at PND 75 and VW at PND 115 were correlated to age at first estrus (P This is a manuscript of an article published as Graves, K. L., B. R. Mordhorst, E. C. Wright, B. J. Hale, K. J. Stalder, A. F. Keating, and J. W. Ross. "Identification of measures predictive of age of puberty onset in gilts." Translational Animal Science (2019). doi: 10.1093/tas/txz173.</p

Arabidopsis LEAFY COTYLEDON2 induces maturation traits and auxin activity: Implications for somatic embryogenesis

LEAFY COTYLEDON2 (LEC2) is a central regulator of embryogenesis sufficient to induce somatic cells to form embryos when expressed ectopically. Here, we analyze the cellular processes induced by LEC2, a B3 domain transcription factor, that may underlie its ability to promote somatic embryogenesis. We show auxin-responsive genes are induced after LEC2 activation in seedlings. Genes encoding enzymes involved in auxin biosynthesis, YUC2 and YUC4, are activated within 1 h after induction of LEC2 activity, and YUC4 appears to be a direct transcriptional target of LEC2. We also show ectopic LEC2 expression induces accumulation of seed storage protein and oil bodies in vegetative and reproductive organs, events that normally occur during the maturation phase of embryogenesis. Furthermore, LEC2 activates seed protein genes before an increase in RNAs encoding LEC1 or FUS3 is observed. Thus, LEC2 causes rapid changes in auxin responses and induces cellular differentiation characteristic of the maturation phase. The relevance of these changes to the ability of LEC2 to promote somatic embryogenesis is discussed