Shanthi Ganesan

Increased prevalence of torque teno viruses in porcine respiratory disease complex affected pigs.

The role of swine torque teno sus viruses (TTSuVs) as co-factors in disease syndromes involving porcine circovirus strain 2 (PCV2) and porcine reproductive and respiratory disease syndrome virus (PRRSV) has been a debatable subject. In this study, the prevalence of TTSuVs in Iowa, the leading pork producing state in the U.S., was estimated by a duplex PCR. The PCR is capable of simultaneously detecting both teno sus viruses 1 and 2 (TTSuV1 and 2). Based on an analysis of 300 random samples representing six major geographical regions of the state, the overall prevalence rates for TTSuV1 and 2 were 47.34% and 24.67% respectively while the combined prevalence rate was 52.33%. The epidemiological association of TTSuV1 and 2 with the common etiological agents of the porcine respiratory disease complex (PRDC) namely porcine PRRSV, PCV2, Mycoplasma hyopneumoniae and swine influenza virus (SIV) was estimated in lung tissue derived from 45 pigs showing clinical signs of PRDC. Notably, 86.67% of the PRDC-suspect samples were positive for TTSuV1 in comparison to the baseline population prevalence rate of 47.34%. However, the prevalence of TTSuV2 (26.67%) was not significantly different. TTSuV1 was detected in 80.00%, 81.81%, 75.00% and 77.78% of the PRRSV, SIV, M. hyopneumoniae and PCV2 positive PRDC-suspect samples respectively. Our results indicate that TTSuV1 is strongly associated with clinical PRDC and support the hypothesis that TTSuVs might function as co-factors in PRDC. Further studies to define their possible role in the pathogenesis of swine respiratory diseases are warranted.

Progressive Obesity Alters Ovarian Folliculogenesis with Impacts on Pro-Inflammatory and Steroidogenic Signaling in Female Mice

ABSTRACT Diet-induced obesity induces immune cell infiltration and inflammation in peri-ovarian adipose tissue and mRNA expression of inflammatory markers in ovarian tissue. Whether these changes are associated with obesity-related ovarian dysfunction remains unknown. In the present study, qRT-PCR and Western blotting techniques were used to compare mRNA and protein abundance of ovarian immune cell and inflammation markers, along with NF-kappaB and steroidogenic pathway members in normal wild-type non-agouti (a/a; lean) and lethal yellow mice (KK.CG-Ay/J; obese) at 6, 12, 18, or 24 wk of age. Our data revealed that, beginning at 12 wk of age, NF-kappaB inflammatory signaling members were elevated (P < 0.05) in obese females. Interestingly obesity had opposing and temporal effects on the steroidogenic enzyme pathway. Obesity decreased (P < 0.05) STAR protein at 12, 18, and 24 wk of age. CYP11A1 and CYP19A1 proteins were increased (P < 0.05) at 12 wk but were decreased (P < 0.05) at 18 and 24 wk. Interestingly, CYP19A1 was increased in lethal yellow mouse ovaries at 6 wk of age, potentially indicating early puberty onset. These data demonstrate that obesity alters expression of ovarian inflammatory and steroidogenic pathway genes in ways which could adversely affect ovarian function.

Impact of Obesity on Ovotoxicity Induced by 7,12-Dimethylbenz[a]anthracene in Mice

ABSTRACT Insulin, elevated during obesity, regulates xenobiotic biotransformation enzymes, potentially through phosphatidylinositol 3-kinase (PI3K) signaling, in extraovarian tissues. PI3K regulates oocyte viability, follicular activation, and ovarian chemical biotransformation. 7,12-Dimethylbenz[a]anthracene (DMBA), a carcinogen and ovotoxicant, destroys all stages of follicles, leading to premature ovarian failure. Obesity has been reported to promote DMBA-induced tumors, but it remains unknown whether obesity affects ovarian xenobiotic metabolism. Therefore, we investigated ovarian expression of xenobiotic metabolism genes—microsomal epoxide hydrolase (Ephx1), glutathione S-transferase (GST) class Pi (Gstp1) and class mu 1 (Gstm1), and PI3K-signaling members (protein kinase B [AKT] alpha [Akt1], beta [Akt2], and the forkhead transcription factor subfamily 3 [Foxo3])—in lean and obese female mice after DMBA exposure (1 mg/kg; intraperitoneal injection for 14 days). Relative to lean, obese mice had decreased (P < 0.05) healthy primordial and primary follicle numbers but increased (P < 0.05) secondary and preovulatory follicles numbers. Obesity increased (P < 0.05) Akt1, Akt2, Gstm1, and Ephx1 mRNA and pAKTSer473/Thr308, GSTM1, GSTP1, and EPHX1 protein levels. DMBA decreased (P < 0.05) ovarian weight in lean and obese mice, however, obese DMBA-treated females had a greater reduction (P < 0.05) in ovarian weight. In both lean and obese mice, DMBA decreased (P < 0.05) all stages of healthy follicle numbers, increased Gstp1 and Ephx1 mRNA as well as GSTM1, GSTP1, and EPHX1 protein levels, and decreased Akt1 and Akt2 mRNA as well as pAKTSer473 or pAKTThr308, FOXO3, and pFOXO3Ser253 protein expression. There was an additive effect between obesity and DMBA exposure for increased Gstm1 and Ephx1 mRNA as well as GSTM1 and EPHX1 protein expression.

7,12-Dimethylbenz[a]anthracene exposure induces the DNA repair response in neonatal rat ovaries

7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles at all stages of development. This study investigated DMBA-induced DNA double strand break (DSB) formation with subsequent activation of the ovarian DNA repair response in models of pre-antral or pre-ovulatory follicle loss. Postnatal day (PND) 4 Fisher 344 (F344) rat ovaries were cultured for 4 days followed by single exposures of vehicle control (1% DMSO) or DMBA (12.5 nM or 75 nM) and maintained in culture for 4 or 8 days. Alternately, PND4 F344 rat ovaries were exposed to 1 μM DMBA at the start of culture for 2 days. Total RNA or protein was isolated, followed by qPCR or Western blotting to quantify mRNA or protein level, respectively. γH2AX and phosphorylated ATM were localized and quantified using immunofluorescence staining. DMBA exposure increased caspase 3 and γH2AX protein. Additionally, DMBA (12.5 nM and 1 μM) increased levels of mRNA encoding Atm, Xrcc6, Brca1 and Rad51. In contrast, Parp1 mRNA was decreased on d4 and increased on d8 of DMBA exposure, while PARP1 protein increased after 8 days of DMBA exposure. Total ATM increased in a concentration-dependent temporal pattern (75 nM d4; 12.5 nM d8), while pATM was localized in large primary and secondary follicles and increased after 8 days of 75 nM DMBA exposure compared to both control and 12.5 nM DMBA. These findings support that, despite some concentration effects, DMBA induces ovarian DNA damage and that DNA repair mechanisms are induced as a potential mechanism to prevent follicle loss.

Bisphenol A-Induced Ovotoxicity Involves DNA Damage Induction to Which the Ovary Mounts a Protective Response Indicated by Increased Expression of Proteins Involved in DNA Repair and Xenobiotic Biotransformation.

Bisphenol A (BPA) is an endocrine disrupting chemical with ubiquitous human exposure. BPA causes primordial follicle loss and DNA damage in germ cells, thus we hypothesized that BPA induces ovarian DNA damage, thereby precipitating follicle loss. We also anticipated that the ovary activates DNA repair and xenobiotic biotransformation to minimize oocyte damage and/or, activate cell death signaling to deplete follicles. Postnatal day 4 F344 rat ovaries were cultured in medium containing vehicle control (1% dimethylsulfoxide [DMSO]) ± BPA (440 µM) for 2-8 days. BPA reduced (P < 0.05) small primary, large primary and secondary follicle numbers after 2 days, followed by a reduction (P < .05) in primordial follicle numbers after 4 days. Phosphorylated H2AX (γH2AX) and Ataxia-telangiectasia mutated (ATM), markers of DNA double-strand breaks, were increased (P < .05) in abundance prior to observed follicle loss. DNA repair genes (Atm, Prkdc, Xrcc6, Brca1, Mre11a, Rad50, and Smc1a) were increased (P < .05) after 1 day of BPA exposure. mRNA encoding Meh, Gstm, c-kit, Kitlg, and Akt were increased (P < .05), as was MEH, AKT, pAKT, Jun N-terminal kinase, and P53 protein abundance, while GST isoforms pi and Nuclear factor erythroid-related factor 2 proteins were decreased (P < .05) by BPA exposure. These data demonstrate the dynamic ovarian response to BPA exposure, which indicates that BPA, via biotransformation, may be converted to a DNA alkylating agent, causing ovarian DNA damage, to which the ovary mounts a protective response and further our knowledge on the biological impacts of BPA on the female germline.

Twelve hours of heat stress induces inflammatory signaling in porcine skeletal muscle

Heat stress causes morbidity and mortality in humans and animals and threatens food security by limiting livestock productivity. Inflammatory signaling may contribute to heat stress-mediated skeletal muscle dysfunction. Previously, we discovered increased circulating endotoxin and intramuscular oxidative stress and TNF-α protein abundance, but not inflammatory signaling following 24 and 72 h of heat stress. Thus the purpose of this investigation was to clarify the role of inflammatory signaling in heat-stressed skeletal muscle. Crossbred gilts (n = 8/group) were assigned to either thermal neutral (24°C), heat stress (37°C), or pair-fed thermal neutral (24°C) conditions for 12 h. Following treatment, animals were euthanized, and the semitendinosus red (STR) and white (STW) were recovered. Heat stress did not alter inflammatory signaling in STW. In STR, relative heat shock protein abundance was similar between groups, as was nuclear content of heat shock factor 1. In whole homogenate, relative abundance of the NF-κB activator inhibitory κB kinase-α was increased by heat stress, although abundance of NF-κB was similar between groups. Relative abundance of phosphorylated NF-κB was increased by heat stress in nuclear fractions. Activator protein-1 (AP-1) signaling was similar between groups. While there were few differences in transcript expression between thermal neutral and heat stress, 80 and 56% of measured transcripts driven by NF-κB or AP-1, respectively, were increased by heat stress compared with pair-fed thermal neutral. Heat stress also caused a reduction in IL-6 transcript and relative protein abundance. These data demonstrate that short-term heat stress causes inflammatory signaling through NF-κB in oxidative, but not glycolytic, skeletal muscle.

Impact of 7,12-dimethylbenz[a]anthracene exposure on connexin gap junction proteins in cultured rat ovaries

7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles in a concentration-dependent manner. The impact of DMBA on connexin (CX) proteins that mediate communication between follicular cell types along with pro-apoptotic factors p53 and Bax were investigated. Postnatal day (PND) 4 Fisher 344 rat ovaries were cultured for 4days in vehicle medium (1% DMSO) followed by a single exposure to vehicle control (1% DMSO) or DMBA (12.5nM or 75nM) and cultured for 4 or 8days. RT-PCR was performed to quantify Cx37, Cx43, p53 and Bax mRNA level. Western blotting and immunofluorescence staining were performed to determine CX37 or CX43 level and/or localization. Cx37 mRNA and protein increased (P<0.05) at 4days of 12.5 nM DMBA exposure. Relative to vehicle control-treated ovaries, mRNA encoding Cx43 decreased (P<0.05) but CX43 protein increased (P<0.05) at 4days by both DMBA exposures. mRNA expression of pro-apoptotic p53 was decreased (P<0.05) but no changes in Bax expression were observed after 4days of DMBA exposures. In contrast, after 8days, DMBA decreased Cx37 and Cx43 mRNA and protein but increased both p53 and Bax mRNA levels. CX43 protein was located between granulosa cells, while CX37 was located at the oocyte cell surface of all follicle stages. These findings support that DMBA exposure impacts ovarian Cx37 and Cx43 mRNA and protein prior to both observed changes in pro-apoptotic p53 and Bax and follicle loss. It is possible that such interference in follicular cell communication is detrimental to follicle viability, and may play a role in DMBA-induced follicular atresia.

Short‐term heat stress alters redox balance in porcine skeletal muscle

Heat stress contributes to higher morbidity and mortality in humans and animals and is an agricultural economic challenge because it reduces livestock productivity. Redox balance and associated mitochondrial responses appear to play a central role in heat stress‐induced skeletal muscle pathology. We have previously reported increased oxidative stress and mitochondrial content in oxidative muscle following 12 h of heat stress. The purposes of this investigation were to characterize heat stress‐induced oxidative stress and changes in mitochondrial content and biogenic signaling in oxidative skeletal muscle. Crossbred gilts were randomly assigned to either thermal neutral (21°C; n = 8, control group) or heat stress (37°C) conditions for 2 h (n = 8), 4 h (n = 8), or 6 h (n = 8). At the end, their respective environmental exposure, the red portion of the semitendinosus muscle (STR) was harvested. Heat stress increased concentration of malondialdehyde (MDA) following 2 and 4 h compared to thermal neutral and 6 h, which was similar to thermal neutral, and decreased linearly with time. Protein carbonyl content was not influenced by environment. Catalase activity was increased following 4 h of heat stress and superoxide dismutase activity was decreased following 6 h of heat stress compared to thermal neutral conditions. Heat stress‐mediated changes in antioxidant activity were independent of altered protein abundance or transcript expression. Mitochondrial content and mitochondrial biogenic signaling were similar between groups. These data demonstrate that heat stress caused a transient increase in oxidative stress that was countered by a compensatory change in catalase activity. These findings contribute to our growing understanding of the chronology of heat stress‐induced intracellular dysfunctions in skeletal muscle.

Short-term heat stress causes altered intracellular signaling in oxidative skeletal muscle

Heat stress (HS) causes morbidities and mortalities, in part by inducing organ-specific injury and dysfunction. Further, HS markedly reduces farm animal productivity, and this is especially true for lean tissue accretion. The purpose of this investigation was to determine the extent to which short-term HS caused muscle dysfunction in skeletal muscle. We have previously found increased free radical injury in skeletal muscle following 24 h of HS. Thus, we hypothesized that HS would lead to apoptosis, autophagy, and decreased mitochondrial content in skeletal muscle. To test this hypothesis, crossbred gilts were divided into 3 groups ( = 8/group): thermal neutral (TN: 21°C), HS (37°C), and pair-fed thermal neutral (PFTN: feed intake matched with heat-stressed animals). Following 12 h of treatment, animals were euthanized and red (STR) and white (STW) portions of the semitendinosus were recovered. Heat stress did not alter intracellular signaling in STW. In STR, the oxidative stress marker malondialdehyde protein and concentration were increased in HS ( = 0.007) compared to TN and PFTN, which was matched by an inadequate antioxidant response, including an increase in superoxide dismutase (SOD) I ( = 0.03) and II relative protein abundance ( = 0.008) and total SOD activity ( = 0.02) but a reduction ( = 0.006) in catalase activity in HS compared to TN. Further, B-cell lymphoma 2-associated X protein ( = 0.02) and apoptotic protease activating factor 1 ( = 0.01) proteins were increased by HS compared to TN and PFTN. However, caspase 3 activity was similar between groups, indicating a lack of apoptotic execution. Despite increased initiation, autophagy appeared to be inhibited by HS as the microtubule-associated protein A/B light chain 3 II/I ratio and mitofusin-2 proteins were decreased ( < 0.03) and sequestosome 1(p62) protein abundance was increased ( = 0.001) in HS compared to TN and PFTN. Markers of mitochondrial content cytochrome c, cytochrome c oxidase IV, voltage-dependent anion channel, pyruvate dehydrogenase, and prohibitins 1 were increased ( < 0.05) in HS compared to TN, whereas mitochondrial biogenesis and mitophagy markers were similar between groups. These data demonstrate that HS caused aberrant intracellular signaling, which may contribute to HS-mediated muscle dysfunction.

The ovarian DNA damage repair response is induced prior to phosphoramide mustard-induced follicle depletion, and ataxia telangiectasia mutated inhibition prevents PM-induced follicle depletion.

Phosphoramide mustard (PM) is an ovotoxic metabolite of cyclophosphamide and destroys primordial and primary follicles potentially by DNA damage induction. The temporal pattern by which PM induces DNA damage and initiation of the ovarian response to DNA damage has not yet been well characterized. This study investigated DNA damage initiation, the DNA repair response, as well as induction of follicular demise using a neonatal rat ovarian culture system. Additionally, to delineate specific mechanisms involved in the ovarian response to PM exposure, utility was made of PKC delta (PKCδ) deficient mice as well as an ATM inhibitor (KU 55933; AI). Fisher 344 PND4 rat ovaries were cultured for 12, 24, 48 or 96h in medium containing DMSO ±60μM PM or KU 55933 (48h; 10nM). PM-induced activation of DNA damage repair genes was observed as early as 12h post-exposure. ATM, PARP1, E2F7, P73 and CASP3 abundance were increased but RAD51 and BCL2 protein decreased after 96h of PM exposure. PKCδ deficiency reduced numbers of all follicular stages, but did not have an additive impact on PM-induced ovotoxicity. ATM inhibition protected all follicle stages from PM-induced depletion. In conclusion, the ovarian DNA damage repair response is active post-PM exposure, supporting that DNA damage contributes to PM-induced ovotoxicity.