Exposure to 7,12-dimethylbenz[a]anthracene impacts ovarian DNA damage sensing and repair proteins differently in lean and obese female mice and weight loss may mitigate obesity-induced ovarian dysfunction.

Obesity impairs oocyte quality, fertility, pregnancy maintenance, and is associated with offspring birth defects. The model ovotoxicant, 7,12-dimethylbenz[a]anthracene (DMBA), causes ovarian DNA damage and follicle loss. Both DMBA-induced chemical biotransformation and the DNA damage response are partially attenuated in obese relative to lean female mice but whether weight loss could improve the DNA damage response to DMBA exposure has not been explored. Thus, at six weeks of age, C57BL/6 J female mice were divided in three groups: 1) Lean (L; n = 20) fed a chow diet for 12 weeks, 2) obese (O; n = 20) fed a high fat high sugar (HFHS) diet for 12 weeks and, 3) slim-down (S; n = 20). The S group was fed with HFHS diet for 7 weeks until attaining a higher body relative to L mice on week 7.5 and switched to a chow diet for 5 weeks to achieve weight loss. Mice then received either corn oil (CT) or DMBA (D; 1 mg/kg) for 7 d via intraperitoneal injection (n = 10/treatment). Obesity increased (P < 0.05) kidney and spleen weight, and DMBA decreased uterine weight (P < 0.05). Ovarian weight was reduced (P < 0.05) in S mice, but DMBA exposure increased ovary weight in the S mice. LC-MS/MS identified 18, 64, and 7 ovarian proteins as altered (P < 0.05) by DMBA in the L, S and O groups, respectively. In S and O mice, 24 and 8 proteins differed, respectively, from L mice. These findings support weight loss as a strategy to modulate the ovarian genotoxicant response.

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.

Trajectory of primordial follicle depletion is accelerated in obese mice in response to 7,12-dimethylbenz[a]anthracene exposure.

Both obesity and exposure to environmental genotoxicants, such as 7,12-dimethylbenz[a]anthracene (DMBA), negatively impair female reproductive health. Hyperphagic lean KK.Cg-a/a (n = 8) and obese KK.Cg-Ay/J (n = 10) mice were exposed to corn oil as vehicle control (CT) or DMBA (1 mg/kg/day) for 7d intraperitoneally, followed by a recovery period. Obesity increased liver and spleen weight (P < 0.05), and DMBA exposure decreased uterine weight (P < 0.05) in obese mice. Primordial follicle loss (P < 0.05) caused by DMBA exposure was observed in obese mice only. Primary (lean P < 0.1; obese P < 0.05) and secondary (lean P < 0.05, obese P < 0.1) follicle loss initiated by DMBA exposure continued across recovery. Reduced pre-antral follicle number in lean mice (P < 0.05), regardless of DMBA exposure, was evident with no effect on antral follicles or corpora lutea number. Immunofluorescence staining of DNA damage marker, γH2AX, did not indicate ongoing DNA damage but TRP53 abundance was decreased in follicles (P < 0.05) of DMBA-exposed obese mice. In contrast, increased (P < 0.05) superoxide dismutase was observed in the corpora lutea of DMBA-exposed obese mice and reduced (P < 0.05) TRP53 abundance was noted in preantral and antral follicles of DMBA-exposed obese mice. This study indicates that obesity influences ovotoxicity caused by a genotoxicant, potentially involving accelerated primordial follicle activation and hampering normal follicular dynamics.

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.

Biological sex differences in hepatic response to in utero dimethylbenz(a)anthracene exposure.

Fetal hepatic dimethylbenz(a)anthracene (DMBA) biotransformation is not defined, thus, this study investigated whether the fetal liver metabolizes DMBA and differs with biological sex. KK.Cg-a/a (lean; n = 20) or KK.Cg-Ay/J (obese; n = 20) pregnant mice were exposed to corn oil (CT) or DMBA (1 mg/kg bw/day) by intraperitoneal injection (n = 10/treatment) from gestation day 7-14. Postnatal day 2 male or female offspring livers were collected. Total RNA (n = 6) and protein (n = 6) were analyzed via a PCR-based array or LC-MS/MS, respectively. The level of Mgst3 was lower (P < 0.05) in livers of female compared to male offspring. Furthermore, in utero DMBA exposure increased (P < 0.1) Cyp2c29 and Gpx3 levels (P < 0.05) in female offspring. In male offspring, the abundance of Ahr, Comt (P < 0.1), Alox5, and Asna1 (P < 0.05) decreased due to DMBA exposure. Female and male offspring had 34 and 21 hepatic proteins altered (P < 0.05) by in utero DMBA exposure, respectively. Opposing patterns for hepatic CD81 and KRT78 occurred, being decreased in females but increased in males, while YWHAG was decreased by DMBA exposure in both. Functional KEGG pathway analysis identified enrichment of 26 and 13 hepatic metabolic proteins in male and female offspring, respectively, due to in utero DMBA exposure. In silico transcription factor analysis of differentially expressed proteins predicted involvement of female NRF1 but male AHR. Thus, hepatic biological sex differences and capacity to respond to toxicants in utero are supported.

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.

Altered histone abundance as a mode of ovotoxicity during 7,12-dimethylbenz[a]anthracene exposure with additive influence of obesity†.

Histones are slowly evolving chromatin components and chromatin remodeling can incorporate histone variants differing from canonical histones as an epigenetic modification. Several identified histone variants are involved with the environmental stress-induced DNA damage response (DDR). Mechanisms of DDR in transcriptionally inactive, prophase-arrested oocytes and epigenetic regulation are under-explored in ovarian toxicology. The study objective was to identify ovarian proteomic and histone modifications induced by DMBA exposure and an influence of obesity. Post-pubertal wildtype (KK.Cg-a/a; lean) and agouti (KK.Cg-Ay/J; obese) female mice, were exposed to either corn oil (control; CT) or DMBA (1 mg/kg) for 7d via intraperitoneal injection (n = 10/treatment). Ovarian proteome analysis (LC-MS/MS) determined that obesity altered 225 proteins (P < 0.05) with histone 3 being the second least abundant (FC = -5.98, P < 0.05). Histone 4 decreased by 3.33-fold, histone variant H3.3 decreased by 3.05-fold, and H1.2, H1.4 and H1.1(alpha) variants increased by 1.59, 1.90 and 2.01-fold, respectively (P < 0.05). DMBA exposure altered 48 proteins in lean mice with no observed alterations in histones or histone variants. In obese mice, DMBA exposure altered 120 proteins and histone 2B abundance increased by 0.30-fold (P < 0.05). In DMBA-exposed mice, obesity altered the abundance of 634 proteins. Histones 4, 3 and 2A type 1-F decreased by 4.03, 3.71, 0.43-fold, respectively, whereas histone variant H1.2 and linker histone, H15 increased by 2.72- and 3.07-fold, respectively (P < 0.05). Thus, DMBA exposure alters histones and histone variants, and responsivity is more pronounced during obesity, potentially altering ovarian transcriptional regulation.

Maternal impacts of pre-conceptional glyphosate exposure.

Maternal glyphosate (GLY) impacts remain unclear despite associations between urinary GLY and birth outcomes. Whether maternal pre-conceptional GLY exposure would have phenotypic and molecular impacts in the dam and offspring was tested. Female C57BL6 mice (6 wk) were exposed to saline (CT; n = 20) or GLY (2 mg/kg; n = 20) per os five d per week for 20 wk. Females were housed with males and on gestation day (GD) 14, divided into: CT non-pregnant (CNP), CT pregnant (CP), GLY non-pregnant (GNP), GLY pregnant (GP). Another cohort (CT; n = 10 or GLY; n = 10) completed three pregnancy rounds and pregnancy index (PI), number of pups per litter and pups surviving to postnatal day (PND) 5 calculated. The PI in GLY mice was higher in breeding rounds 1 and 2, but lower in round 3. Pregnancy increased (P ≤ 0.1) GD14 liver and ovary weight. Spleen weight was increased (P < 0.05) in GP relative to GNP mice. No offspring phenotypic impacts were observed. Approximately six months after cessation of exposure, secondary follicle number was reduced (P < 0.05) by pre-conceptional GLY exposure. The ovarian proteome analyzed by LC-MS/MS was altered (P < 0.05) by pregnancy (49 increased, 43 decreased) and GLY exposure (non-pregnant: 75 increased, 22 decreased, pregnant: 27 increased, 29 decreased; aged dams: 60 increased, 98 decreased) with several histone proteins being altered. These findings support ovarian transient and persistent impacts of GLY exposure and identify pathways as potential modes of action.

Hepatic and ovarian effects of perfluorooctanoic acid exposure differ in lean and obese adult female mice.

Obesity and overweight cause poor oocyte quality, miscarriage, infertility, polycystic ovarian syndrome, and offspring birth defects and affects 40% and 20% of US women and girls, respectively. Perfluorooctanoic acid (PFOA), a per- and poly-fluoroalkyl substance (PFAS), is environmentally persistent and has negative female reproductive effects including endocrine disruption, oxidative stress, altered menstrual cyclicity, and decreased fertility in humans and animal models. PFAS exposure is associated with non-alcoholic fatty liver disease which affects ∼24-26% of the US population. This study investigated the hypothesis that PFOA exposure impacts hepatic and ovarian chemical biotransformation and alters the serum metabolome. At 7 weeks of age, female lean, wild type (KK.Cg-a/a) or obese (KK.Cg-Ay/J) mice received saline (C) or PFOA (2.5 mg/Kg) per os for 15 d. Hepatic weight was increased by PFOA exposure in both lean and obese mice (P < 0.05) and obesity also increased liver weight (P < 0.05) compared to lean mice. The serum metabolome was also altered (P < 0.05) by PFOA exposure and differed between lean and obese mice. Exposure to PFOA altered (P < 0.05) the abundance of ovarian proteins with roles in xenobiotic biotransformation (lean - 6; obese - 17), metabolism of fatty acids (lean - 3; obese - 9), cholesterol (lean - 8; obese - 11), amino acids (lean - 18; obese - 19), glucose (lean - 7; obese - 10), apoptosis (lean - 18; obese - 13), and oxidative stress (lean - 3; obese - 2). Use of qRT-PCR determined that exposure to PFOA increased (P < 0.05) hepatic Ces1 and Chst1 in lean but Ephx1 and Gstm3 in obese mice. Also, obesity basally increased (P < 0.05) Nat2, Gpi and Hsd17b2 mRNA levels. These data identify molecular changes resultant from PFOA exposure that may cause liver injury and ovotoxicity in females. In addition, differences in toxicity induced by PFOA exposure occurs in lean and obese mice.

Maternal pre-conceptional glyphosate exposure impacts the offspring hepatic and ovarian proteome.

Glyphosate (GLY) is an herbicide used for rural and urban weed control. Urinary GLY in women is associated with shortened gestational length yet effects of GLY on offspring due to maternal exposure are unclear. This study tested the hypothesis that maternal chronic pre-conceptional GLY exposure would cause phenotypic and molecular changes in F1 offspring. Female C57BL/6 mice (7-week-old; n = 40) received saline vehicle control (CT; n = 20) or GLY (2 mg/kg; n = 20) daily per os for 10 weeks. At dosing completion, females were housed with unexposed males and divided into Cohort 1 who were euthanized at gestation day 14 (n = 10 per treatment) and Cohort 2 who completed gestation (n = 10 per treatment). F1 female ovarian and liver samples underwent LC-MS/MS and bioinformatic analysis. Maternal exposure did not affect litter (P > .05) sex ratio, or embryonic or neonatal gross phenotypes. In Cohort 2 offspring, no treatment effect on (P > .05) offspring anogenital distance, puberty onset, or ovarian follicular composition was noted. Body weight was increased (P < .05) in male GLY-exposed compared with CT dam offspring. F1 females from GLY-exposed dams had altered (P < .05) abundance of 54 ovarian and 110 hepatic proteins. Pathways altered in the ovary (false discovery rate [FDR] ≤ 0.07) included thermogenesis and phosphatidylinositol-3 kinase-AKT signaling and in liver (FDR ≤ 0.08) included metabolic, glutathione metabolism, oxidative phosphorylation, non-alcoholic fatty liver disease, and thermogenesis. Thus, pre-conceptional GLY exposure affected offspring phenotypic and molecular profiles potentially impacting reproductive health.

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.

Obesity partially potentiates dimethylbenz[a]anthracene-exposed ovotoxicity by altering the DNA damage repair response in mice†.

Obesity adversely affects reproduction, impairing oocyte quality, fecundity, conception, and implantation. The ovotoxicant, dimethylbenz[a]anthracene, is biotransformed into a genotoxic metabolite to which the ovary responds by activating the ataxia telangiectasia mutated DNA repair pathway. Basal ovarian DNA damage coupled with a blunted response to genotoxicant exposure occurs in obese females, leading to the hypothesis that obesity potentiates ovotoxicity through ineffective DNA damage repair. Female KK.Cg-a/a (lean) and KK.Cg-Ay/J (obese) mice received corn oil or dimethylbenz[a]anthracene (1 mg/kg) at 9 weeks of age for 7 days via intraperitoneal injection (n = 10/treatment). Obesity increased liver weight (P < 0.001) and reduced (P < 0.05) primary, preantral, and corpora lutea number. In lean mice, dimethylbenz[a]anthracene exposure tended (P < 0.1) to increase proestrus duration and reduced (P = 0.07) primordial follicle number. Dimethylbenz[a]anthracene exposure decreased (P < 0.05) uterine weight and increased (P < 0.05) primary follicle number in obese mice. Total ovarian abundance of BRCA1, γH2AX, H3K4me, H4K5ac, H4K12ac, and H4K16ac (P > 0.05) was unchanged by obesity or dimethylbenz[a]anthracene exposure. Immunofluorescence staining demonstrated decreased (P < 0.05) abundance of γH2AX foci in antral follicles of obese mice. In primary follicle oocytes, BRCA1 protein was reduced (P < 0.05) by dimethylbenz[a]anthracene exposure in lean mice. Obesity also decreased (P < 0.05) BRCA1 protein in primary follicle oocytes. These findings support both a follicle stage-specific ovarian response to dimethylbenz[a]anthracene exposure and an impact of obesity on this ovarian response.

A systematic evidence map for the evaluation of noncancer health effects and exposures to polychlorinated biphenyl mixtures.

Assessing health outcomes associated with exposure to polychlorinated biphenyls (PCBs) is important given their persistent and ubiquitous nature. PCBs are classified as a Group 1 carcinogen, but the full range of potential noncancer health effects from exposure to PCBs has not been systematically summarized and evaluated. We used systematic review methods to identify and screen the literature using combined manual review and machine learning approaches. A protocol was developed that describes the literature search strategy and Populations, Exposures, Comparators, and Outcomes (PECO) criteria used to facilitate subsequent screening and categorization of literature into a systematic evidence map of PCB exposure and noncancer health endpoints across 15 organs/systems. A comprehensive literature search yielded 62,599 records. After electronic prioritization steps, 17,037 studies were manually screened at the title and abstract level. An additional 900 studies identified by experts or supplemental searches were also included. After full-text screening of 3889 references, 1586 studies met the PECO criteria. Relevant study details such as the endpoints assessed, exposure duration, and species were extracted into literature summary tables. This review compiles and organizes the human and mammalian studies from these tables into an evidence map for noncancer health endpoints and PCB mixture exposure to identify areas of robust research as well as areas of uncertainty that would benefit from future investigation. Summary data are available online as interactive visuals with downloadable metadata. Sufficient research is available to inform PCB hazard assessments for most organs/systems, but the amount of data to inform associations with specific endpoints differs. Furthermore, despite many years of research, sparse data exist for inhalation and dermal exposures, which are highly relevant human exposure routes. This evidence map provides a foundation for future systematic reviews and noncancer hazard assessments of PCB mixtures and for strategic planning of research to inform areas of greater uncertainty.

PFOA-Induced Ovotoxicity Differs Between Lean and Obese Mice With Impacts on Ovarian Reproductive and DNA Damage Sensing and Repair Proteins.

Perfluorooctanoic acid (PFOA) is an environmentally persistent perfluoroalkyl substance that is widely used in consumer products. Exposure to PFOA is associated with reproductive and developmental effects including endocrine disruption, delayed puberty in girls, and decreased fetal growth. In the United States, obesity affects 40% of women and 20% of girls, with higher rates in minority females. Obesity causes infertility, poor oocyte quality, miscarriage, and offspring defects. This study proposed that PFOA exposure would impact estrous cyclicity, ovarian steroid hormones, and the ovarian proteome and further hypothesized that obesity would impact PFOA-induced ovotoxicity. Female wild type (KK.Cg-a/a; lean) or KK.Cg-Ay/J mice (obese) received saline (CT) or PFOA (2.5 mg/kg) per os for 15 days beginning at 7 weeks of age. There were no effects on food intake, body weight, estrous cyclicity, serum progesterone, and heart, spleen, kidney, or uterus weight (p > .05). Ovary weight was decreased (p < .05) by PFOA exposure relative to vehicle control-treated mice in lean but not obese mice. Liquid chromatography-tandem mass spectrometry was performed on isolated ovarian protein and PFOA exposure altered the ovarian abundance of proteins involved in DNA damage sensing and repair pathways and reproduction pathways (p < .05) differentially in lean and obese mice. The data suggest that PFOA exposure alters ovary weight and differentially targets ovarian proteins in lean and obese females in ways that might reduce female fecundity.

Pre-Conceptional Exposure to Glyphosate Affects the Maternal Hepatic and Ovarian Proteome.

Exposure to glyphosate (GLY), a commonly used herbicide, is supported by urinary detection and associated with shortened gestation in women. This study tested the hypothesis that chronic low-dose pre-conceptional GLY exposure would affect maternal ovarian function mid- and post-gestation. Mice (C57BL/6; n = 40) were exposed per os to saline vehicle control (CT; n = 20) or GLY (2 mg/kg; n = 20) daily for 10 weeks starting at 7 weeks of age. Post-exposure, females were impregnated and euthanized at gestation day 14 (GD14) or post-weaning (PW). Pregnancy success was reduced from 75% to 55% by GLY exposure. No treatment effect (p > .05) on body weight, maternal serum 17ß-estradiol, or litter size was noted. Ovarian weight was unaffected or reduced (p < .05) by GLY in GD14 and PW dams, respectively. Exposure to GLY decreased (p < .05) PW ovarian secondary follicle number with no other follicle composition impacts. Protein abundance analysis by LC-MS/MS identified that GLY altered (p < .05) 26 ovarian and 41 hepatic proteins in GD14 dams and 39 hepatic proteins in PW dams. In GD14 dams, GLY increased ovarian protein abundance of SEC16A (p < .05; 29-fold) and hepatic RPS27L and GM4952 (p < .05; ∼4-fold). In both GD14 and PW dams, GLY exposure increased (p < .05) hepatic RPS4 and decreased (p < .05) ECHDC3. Pathway analysis using DAVID identified 10 GLY hepatic pathway targets with FDR ≤ 0.07 in GD14 dams.

MicroRNA21 inhibition affects porcine oocyte maturation and alters protein expression critical for metabolic pathway function.

MicroRNA21 (MIR21) abundance in porcine oocytes and cumulus cells increases during in vitro maturation. The mechanism by which MIR21 regulates oocyte maturation and the effect on the developmental competence of subsequent embryos remains unclear. The objective of this study was to assess the function of MIR21 during porcine oocyte maturation and its effect on embryonic development. Treatment with peptide nucleic acid MIR21 inhibitor (MIR21-PNA), designed to specifically bind to and prevent MIR21 activity during in vitro oocyte maturation, decreased cumulus cell expansion, and the oocyte ability to achieve metaphase II maturation stage when compared to control groups. Following parthenogenetic activation, the cleavage rate at 48 h in the MIR21-PNA group was decreased (p ≤ 0.03) relative to the control groups. Additionally, liquid chromatography-mass spectrometry (LC-MS/MS) of oocyte and cumulus cell total protein following MIR21-PNA treatment during in vitro maturation identified changes in signaling pathways with primary involvement of glucose metabolism (GM) pathways. Furthermore, there was no difference (p = 0.21) in oocyte maturation of control and MIR21-PNA treated oocytes when cultured in pyruvate lacking medium. Finally, MIR21-PNA treatment decreased (p = 0.04) glutathione and increased (p = 0.07) reactive oxygen species production in the oocyte. These data suggest that MIR21 influences porcine oocyte maturation by regulating GM pathways in the cumulus-oocyte complex.

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.

Porcine endometrial heat shock proteins are differentially influenced by pregnancy status, heat stress, and altrenogest supplementation during the peri-implantation period.

Heat stress (HS) deleteriously affects multiple components of porcine reproduction and is causal to seasonal infertility. Environment-induced hyperthermia causes a HS response (HSR) typically characterized by increased abundance of intracellular heat shock proteins (HSP). Gilts exposed to HS during the peri-implantation period have compromised embryo survival, however if (or how) HS disrupts the porcine endometrium is not understood. Study objectives were to evaluate the endometrial HSP abundance in response to HS during this period and assess the effect of oral progestin (altrenogest; ALT) supplementation. Postpubertal gilts (n = 42) were artificially inseminated during behavioral estrus (n = 28) or were kept cyclic (n = 14), and randomly assigned to thermal neutral (TN; 21 ± 1 °C) or diurnal HS (35 ± 1 °C for 12 h/31.6 ± 1 °C for 12 h) conditions from day 3 to 12 postestrus (dpe). Seven of the inseminated gilts from each thermal treatment group received ALT (15 mg/d) during this period. Using quantitative PCR, transcript abundance of HSP family A (Hsp70) member 1A (HSPA1A, P = 0.001) and member 6 (HSPA6, P < 0.001), and HSP family B (small) member 8 (HSB8, P = 0.001) were increased while HSP family D (Hsp60) member 1 (HSPD1, P = 0.01) was decreased in the endometrium of pregnant gilts compared to the cyclic gilts. Protein abundance of HSPA1A decreased (P = 0.03) in pregnant gilt endometrium due to HS, while HSP family B (small) member 1 (HSPB1) increased (P = 0.01) due to HS. Oral ALT supplementation during HS reduced the transcript abundance of HSP90α family class B member 1 (HSP90AB1, P = 0.04); but HS increased HSP90AB1 (P = 0.001), HSPA1A (P = 0.02), and HSPA6 (P = 0.04) transcript abundance irrespective of ALT. ALT supplementation decreased HSP90α family class A member 1 (HSP90AA1, P = 0.001) protein abundance, irrespective of thermal environment, whereas ALT only decreased HSPA6 (P = 0.02) protein abundance in TN gilts. These results indicate a notable shift of HSP in the porcine endometrium during the peri-implantation period in response to pregnancy status and heat stress.

Heat stress (HS) deleteriously affects multiple components of porcine reproduction and causes seasonal infertility. Environment-induced hyperthermia causes a HS response (HSR) typically characterized by increased abundance of intracellular heat shock proteins (HSP). Gilts exposed to HS during the peri-implantation period have compromised embryo survival, however if (or how) HS disrupts the porcine endometrium is not understood. Study objectives were to evaluate the endometrial HSP abundance in response to HS during this period and assess the effect of oral progestin (altrenogest; ALT) supplementation. We evaluated the abundance of HSP90, HSP70, HSP60 and HSPB in the porcine endometrium during the peri-implantation period. We demonstrate how a physiological event such as pregnancy and an environmental stressor such as HS, individually and in combination, alter the endometrial abundance of these HSP. Moreover, supplementation of pregnant gilts subjected to HS with ALT also altered the abundance of these HSP in the porcine endometrium.

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.