Zearalenone exposure differentially affects the ovarian proteome in prepubertal gilts during thermal neutral and heat stress conditions.

Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin, causes endocrine disruption and porcine reproductive dysfunction. Heat stress (HS) occurs when exogenous and metabolic heat accumulation exceeds heat dissipation. Independently, HS and ZEN both compromise swine reproduction; thus, the hypothesis investigated was two-pronged: that ZEN exposure would alter the ovarian proteome and that these effects would differ in thermal neutral and HS pigs. Pre-pubertal gilts (n = 38) were fed ad libitum and assigned to either thermal neutral (TN: 21.0 ± 0.1°C) or HS (12 h cyclic temperatures of 35.0 ± 0.2°C and 32.2 ± 0.1°C). Within the TN group, a subset of pigs were pair-fed (PF) to the amount of feed that the HS gilts consumed to eliminate the confounding effects of dissimilar nutrient intake. All gilts orally received a vehicle control (CT) or ZEN (40 µg/kg/BW) resulting in six treatment groups: thermoneutral (TN) vehicle control (TC; n = 6); TN ZEN (TZ; n = 6); pair-fed (PF) vehicle control (PC; n = 6); PF ZEN (PZ; n = 6); HS vehicle control (HC; n = 7); or HS ZEN (HZ; n = 7) for 7 d. When compared to the TC pigs, TZ pigs had 45 increased and 39 decreased proteins (P ≤ 0.05). In the HZ pigs, 47 proteins were increased and 61 were decreased (P ≤ 0.05). Exposure to ZEN during TN conditions altered sec61 translocon complex (40%), rough endoplasmic reticulum membrane (8.2%), and proteasome complex (5.4%), asparagine metabolic process (0.60%), aspartate family amino acid metabolic process (0.14%), and cellular amide metabolic process (0.02%) pathways. During HS, ZEN affected cellular pathways associated with proteasome core complex alpha subunit complex (0.23%), fibrillar collagen trimer (0.14%), proteasome complex (0.05%), and spliceosomal complex (0.03%). Thus, these data identify ovarian pathways altered by ZEN exposure and suggest that the molecular targets of ZEN differ in TN and HS pigs.

Heat stress alters the ovarian proteome in prepubertal gilts.

Heat stress (HS) occurs when exogenous and metabolic heat accumulation exceeds heat dissipation; a thermal imbalance that compromises female reproduction. This study investigated the hypothesis that HS alters the ovarian proteome and negatively impacts proteins engaged with insulin signaling, inflammation, and ovarian function. Prepubertal gilts (n = 19) were assigned to one of three environmental groups: thermal neutral with ad libitum feed intake (TN; n = 6), thermal neutral pair-fed (PF; n = 6), or HS (n = 7). For 7 d, HS gilts were exposed to 12-h cyclic temperatures of 35.0 ±â€…0.2 °C and 32.2 ±â€…0.1 °C, while TN and PF gilts were housed at 21.0 ±â€…0.1 °C. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on ovarian protein homogenates. Relative to TN gilts, 178 proteins were altered (P ≤ 0.05, log2foldchange ≥ 1) by HS, with 76 increased and 102 decreased. STRING gene ontology classified and identified 45 biological processes including those associated with chaperone protein refolding, cytoplasmic translational initiation, and immune activation; with a protein-protein interaction web network of 158 nodes and 563 edges connected based on protein function (FDR ≤ 0.05). Relative to PF, HS altered 330 proteins (P ≤ 0.05, log2foldchange ≥ 1), with 151 increased and 179 decreased. Fifty-seven biological pathways associated with protein function and assembly, RNA processing, and metabolic processes were identified, with a protein-protein interaction network of 303 nodes and 1,606 edges. Comparing HS with both the TN and PF treatments, 72 ovarian proteins were consistently altered by HS with 68 nodes and 104 edges, with biological pathways associated with translation and gene expression. This indicates that HS alters the ovarian proteome and multiple biological pathways and systems in prepubertal gilts; changes that potentially contribute to female infertility.

Heat stress impairs female fertility, yet the mechanisms underlying reduced fecundity remain unclear. This study investigated the ovarian proteomic changes resultant from heat stress in prepubertal gilts and discovered changes related to several important biological processes that could be responsible for reduced female fertility.

Phenotypic, endocrinological, and metabolic effects of zearalenone exposure and additive effect of heat stress in prepubertal female pigs.

Independently, both heat stress (HS) and zearalenone (ZEN) compromise female reproduction, thus the hypothesis that ZEN would affect phenotypic, endocrine, and metabolic parameters in pigs with a synergistic and/or additive impact of HS was investigated. Prepubertal gilts (n = 6-7) were assigned to: thermoneutral (TN) vehicle control (TC; n = 6); TN ZEN (40 µg/kg; TZ; n = 6); pair-fed (PF; n = 6) vehicle control (PC; n = 6); PF ZEN (40 µg/kg; PZ; n = 6); HS vehicle control (HC; n = 7); and HS ZEN (40 µg/kg; HZ; n = 7) and experienced either constant 21.0 ± 0.10 °C (TN and PF) or 35.0 ± 0.2 °C (12 h) and 32.2 ± 0.1 °C (12 h) to induce HS for 7 d. Elevated rectal temperature (P < 0.01) and respiration rate (P < 0.01) confirmed induction of HS. Rectal temperature was decreased (P = 0.03) by ZEN. Heat stress decreased (P < 0.01) feed intake, body weight, and average daily gain, with absence of a ZEN effect (P > 0.22). White blood cells, hematocrit, and lymphocytes decreased (P < 0.04) with HS. Prolactin increased (P < 0.01) in PC and PZ and increased in HZ females (P < 0.01). 17ß-estradiol reduced (P < 0.01) in HC and increased in TZ females (P = 0.03). Serum metabolites were altered by both HS and ZEN. Neither HS nor ZEN impacted ovary weight, uterus weight, teat size or vulva area in TN and PF treatments, although ZEN increased vulva area (P = 0.02) in HS females. Thus, ZEN and HS, independently and additively, altered blood composition, impacted the serum endocrine and metabolic profile and increased vulva size in prepubertal females, potentially contributing to infertility.

Scrambled eggs-Negative impacts of heat stress and chemical exposures on ovarian function in swine.

Exposure to environmental toxicants and hyperthermia can hamper reproduction in female mammals including swine. Phenotypic manifestations include poor quality oocytes, endocrine disruption, infertility, lengthened time to conceive, pregnancy loss, and embryonic defects. The ovary has the capacity for toxicant biotransformation, regulated in part by the phosphatidylinositol-3 kinase signaling pathway. The impacts of exposure to mycotoxins and pesticides on swine reproduction and the potential for an emerging chemical class of concern, the per- and polyfluoroalkylated substances, to hamper porcine reproduction are reviewed. The negative impairments of heat stress (HS) on swine reproductive outcomes are also described and the cumulative effect of environmental exposures, such as HS, when present in conjunction with a toxicant is considered.

The impact of Zearalenone on heat-stressed skeletal muscle in pigs.

Heat stress (HS) and Zearalenone (ZEN) exposure affect growth, production efficiency, and animal welfare; and, under extreme situations, both can be lethal. Given that both HS and ZEN independently cause oxidative stress, we hypothesized that simultaneous exposure to HS and ZEN would cause greater oxidative stress in porcine skeletal muscle than either condition, alone. To address this hypothesis, crossbred, prepubertal gilts were treated with either vehicle control (cookie dough) or ZEN (40 µg/kg) and exposed to either thermoneutral (TN; 21.0 °C) or 12-h diurnal HS conditions (night: 32.2 °C; day: 35.0 °C) for 7 d. Pigs were euthanized immediately following the environmental challenge and the glycolytic (STW) and oxidative (STR) portions of the semitendinosus muscle were collected for analysis. In STR, malondialdehyde (MDA) concentration, a marker of oxidative stress, tended to increase following ZEN exposure (P = 0.08). HS increased CAT (P = 0.019) and SOD1 (P = 0.049) protein abundance, while ZEN decreased GPX1 protein abundance (P = 0.064) and activity (P = 0.036). In STR, HS did not alter protein expression of HSP27, HSP70, or HSP90. Conversely, in STW, MDA-modified proteins remained similar between all groups. Consistent with STR, ZEN decreased GPX1 (P = 0.046) protein abundance in STW. In STW, ZEN decreased protein abundance of HSP27 (P = 0.032) and pHSP27 (P = 0.0068), while HS increased protein expression of HSP70 (P = 0.04) and HSP90 (P = 0.041). These data suggest a muscle fiber type-specific response to HS or ZEN exposure, potentially rendering STR more susceptible to HS- and/or ZEN-induced oxidative stress, however, the combination of HS and ZEN did not augment oxidative stress.

Heat stress (HS) and Zearalenone (ZEN), a toxic feed contaminant, affect growth, production efficiency, and animal welfare, and can cause death. As HS and ZEN independently increase oxidative stress, an imbalance of free radical production and clearance, and the likelihood of ZEN contamination during heat events, we hypothesized concomitant exposure would induce oxidative stress in pig skeletal muscle more than either agent alone. To address this, female pigs were treated with a placebo or low dose of ZEN and exposed to ambient temperature or a mild cyclic HS designed to mimic environmental conditions (hot days, cooler nights) for 7 d. Following these treatments, fast- and slow-twitch muscles were collected for analysis. In slow-twitch muscle, we observed increased markers of oxidative stress in pigs exposed to ZEN primarily driven by HS and ZEN treated pigs. Additionally, ZEN reduced antioxidant abundance and enzymatic activity regardless of the environment. Conversely, HS and/or ZEN did not cause oxidative stress in fast-twitch muscle, although ZEN altered antioxidant abundance. Although a mild HS and ZEN dose was used, oxidative stress markers were altered, suggesting that slow-twitch muscle is susceptible to HS- and ZEN-mediated changes. These data raise the possibility that more severe HS exposures and higher ZEN doses may compromise muscle health.

Impact of heat stress on prolactin-mediated ovarian JAK-STAT signaling in postpubertal gilts.

Heat stress (HS) compromises almost every aspect of animal agriculture including reproduction. In pigs, this infecundity is referred to as seasonal infertility (SI), a phenotype including ovarian dysfunction. In multiple species, HS-induced hyperprolactinemia has been described; hence, our study objectives were to characterize and compare HS effects on circulating prolactin (PRL) and ovarian Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling during the follicular (FOL) or luteal (LUT) phases of the estrous cycle in postpubertal gilts. Gilts were estrus synchronized using altrenogest and environmental treatments began immediately after altrenogest withdrawal. For the FOL study: postpubertal gilts were allocated to constant thermoneutral (TN; n = 6; 20 ± 1.2 °C) or cyclical HS (n = 6; 25 to 32 ± 1.2 °C) conditions for 5 d. In the LUT study: postpubertal gilts were assigned to either TN (n = 7; 20 ± 2.6 °C) or cyclical HS (n = 7; 32 to 35 ± 2.6 °C) conditions from 2 to 12 days postestrus (dpe). Blood was collected by jugular venipuncture for PRL quantification on day 5 in the FOL and on day 0 and day 12 in the LUT gilts. Ovaries and corpora lutea (CL) were obtained from euthanized FOL and LUT gilts on day 5 and day 12, respectively. Western blotting was performed to quantify prolactin receptor (PRLR) and JAK/STAT pathway protein abundance. In the FOL phase, no difference (P = 0.20) in circulating PRL between thermal groups was observed. There was no effect (P ≥ 0.34) of HS on PRLR, signal transducer and activator of transcription 3 (STAT3), signal transducer and activator of transcription 5α (STAT5α), and phosphorylated signal transducer and activator of transcription α/ß tyrosine 694/699 (pSTAT5α/ßTyr694/699) abundance and Janus kinase 2 (JAK2), phosphorylated janus kinase 2 tyrosine 1007/1008 (pJAK2Tyr1007/1008), STAT1, phosphorylated signal transducer and activator of transcription 1 tyrosine 701 (pSTAT1Tyr701), phosphorylated signal transducer and activator of transcription 1 serine 727 (pSTAT1Ser727), and phosphorylated signal transducer and activator of transcription 3 tyrosine 705 (pSTAT3Tyr705) were undetectable in FOL gilt ovaries. Ovarian pSTAT5α/ßTyr694/699 abundance tended to moderately increase (4%; P = 0.07) in FOL gilts by HS. In the LUT phase, circulating PRL increased progressively from 2 to 12 dpe, but no thermal treatment-induced difference (P = 0.37) was noted. There was no effect (P ≥ 0.16) of HS on CL abundance of PRLR, pJAK2Tyr1007/1008, JAK2, STAT1, pSTAT1Tyr701, pSTAT1Ser727, pSTAT3Tyr705, STAT5α, or pSTAT5α/ßTyr694/699. In LUT phase, CL STAT3 abundance was increased (11%; P < 0.03) by HS. There was no impact of HS (P ≥ 0.76) on levels of pJAK2Tyr1007/1008 and pSTAT5α/ßTyr694/699 in LUT gilts; however, the CL pSTAT3Tyr705:STAT3 ratio tended to be decreased (P = 0.10) due to HS. These results indicate an HS-induced estrous cycle-stage-dependent effect on the ovarian JAK/STAT pathway, establishing a potential role for this signaling pathway as a potential contributor to SI.

Heat stress (HS) negatively affects reproduction in pigs, though the precise mechanisms are not understood. This study determined if HS impacts the JAK-STAT signaling pathway in the ovary during two stages of the estrous cycle: follicular and luteal. While circulating prolactin hormone level was unchanged, there were changes to some aspects of ovarian JAK-STAT signaling that could be involved in infertility induced in pigs during HS.

Obesity alters the ovarian DNA damage response and apoptotic proteins.

In the overweight or obese female, reproductive complications include poor oocyte quality, decreased fecundity, gestational diabetes, and higher risk of reproductive cancers. Using lean and hyperphagia-induced obese female mice aged 10 weeks, we determined that the ovary from obese female mice had elevated (P < 0.10) levels of ataxia telangiectasia mutated (ATM) protein in oocytes of both small and large follicles. Phosphorylated ATM at serine 1981 was greater (P < 0.05) in large relative to small follicles with no additional impact of obesity. Obesity increased (P < 0.05) γH2AX in small follicles in obese relative to lean ovaries, while large follicles of both lean and obese mice had detectable levels of γH2AX. Cleaved caspase 3 was reduced (P < 0.05) in the small follicles of obese relative to lean ovaries. In large follicles of lean mice, cleaved caspase 3 was increased in large compared to small follicles (P < 0.05) but this pattern was absent in obese mice. Breast cancer type 1 susceptibility protein (BRCA1) or the phosphorylated BRCA1 proteins were observably altered by obesity. These data demonstrate that markers of DNA damage and repair have a follicle-dependent stage location and that obesity alters ATM and cleaved caspase 3 in a follicular stage dependent manner.