Differentiating between the effects of heat stress and lipopolysaccharide on the porcine ovarian heat shock protein response1.

Heat stress (HS) negatively affects both human and farm-animal health and undermines efficiency in a variety of economically important agricultural variables, including reproduction. HS impairs the intestinal barrier, allowing for translocation of the resident microflora and endotoxins, such as lipopolysaccharide (LPS), from the gastrointestinal lumen into systemic circulation. While much is known about the cellular function of heat shock proteins (HSPs) in most tissues, the in vivo ovarian HSP response to stressful stimuli remains ill-defined. The purpose of this study was to compare the effects of HS or LPS on ovarian HSP expression in pigs. We hypothesized that ovarian HSPs are responsive to both HS and LPS. Altrenogest (15 mg/d) was administered per os for estrus synchronization (14 d) prior to treatment and three animal paradigms were used: (i) gilts were exposed to cyclical HS (31 ± 1.4 °C) or thermoneutral (TN; 20 ± 0.5 °C) conditions immediately following altrenogest withdrawal for 5 d during follicular development; (ii) gilts were subjected to repeated (4×/d) saline (CON) or LPS (0.1 µg/kg BW) i.v. infusion immediately following altrenogest withdrawal for 5 d; and (iii) gilts were subjected to TN (20 ± 1 °C) or cyclical HS (31 to 35 °C) conditions 2 d post estrus (dpe) until 12 dpe during the luteal phase. While no differences were detected for transcript abundances of the assessed ovarian HSP, the protein abundance of specific HSP was influenced by stressors during the follicular and luteal phases. HS during the follicular phase tended (P < 0.1) to increase ovarian protein abundance of HSP90AA1 and HSPA1A, and increased (P ≤ 0.05) HSF1, HSPD1, and HSPB1 compared with TN controls, while HS decreased HSP90AB1 (P = 0.01). Exposure to LPS increased (P < 0.05) HSP90AA1 and HSPA1A and tended (P < 0.1) to increase HSF1 and HSPB1 compared with CON gilts, while HSP90AB1 and HSPD1 were not affected by LPS. HS during the luteal phase increased (P < 0.05) abundance of HSPB1 in corpora lutea (CL), decreased (P < 0.05) CL HSP90AB1, but did not impact HSF1, HSPD1, HSP90AA1, or HSPA1A abundance. Thus, these data support that HS and LPS similarly regulate expression of specific ovarian HSP, which suggest that HS effects on the ovary are in part mediated by LPS.

Impact of heat stress during the follicular phase on porcine ovarian steroidogenic and phosphatidylinositol-3 signaling.

Environmental conditions that impede heat dissipation and increase body temperature cause heat stress (HS). The study objective was to evaluate impacts of HS on the follicular phase of the estrous cycle. Postpubertal gilts (126.0 ± 21.6 kg) were orally administered altrenogest to synchronize estrus, and subjected to either 5 d of thermal-neutral (TN; 20.3 ± 0.5 °C; n = 6) or cyclical HS (25.4 - 31.9 °C; n = 6) conditions during the follicular phase preceding behavioral estrus. On d 5, blood samples were obtained, gilts were euthanized, and ovaries collected. Fluid from dominant follicles was aspirated and ovarian protein homogenates prepared for protein abundance analysis. HS decreased feed intake (22%; P = 0.03) and while plasma insulin levels did not differ, the insulin:feed intake ratio was increased 3-fold by HS (P = 0.02). Insulin receptor protein abundance was increased (29%; P < 0.01), but insulin receptor substrate 1, total and phosphorylated protein kinase B, superoxide dismutase 1, and acyloxyacyl hydrolase protein abundance were unaffected by HS (P > 0.05). Plasma and follicular fluid 17ß-estradiol, progesterone, and lipopolysaccharide-binding protein concentrations as well as abundance of steroid acute regulatory protein, cytochrome P450 19A1, and multidrug resistance-associated protein 1 were not affected by HS (P > 0.05). HS increased estrogen sulfotransferase protein abundance (44%; P = 0.02), toll-like receptor 4 (36%; P = 0.05), and phosphorylated REL-associated protein (31%; P = 0.02). Regardless of treatment, toll-like receptor 4 protein was localized to mural granulosa cells in the porcine ovary. In conclusion, HS altered ovarian signaling in postpubertal gilts during their follicular phase in ways that likely contributes to seasonal infertility.

Phosphoramide mustard induces autophagy markers and mTOR inhibition prevents follicle loss due to phosphoramide mustard exposure.

Phosphoramide mustard (PM) is an ovotoxic metabolite of cyclophosphamide. Postnatal day 4 Fisher 344 rat ovaries were exposed to vehicle control (1% DMSO) or PM (60µM)±LY294002 or rapamycin for 2 or 4 d. Transmission election microscopy revealed abnormally large golgi apparatus and electron dense mitochondria in PM-exposed ovaries prior to and at the time of follicle depletion. PM exposure increased (P<0.05) mRNA abundance of Bbc3, Cdkn1a, Ctfr, Edn1, Gstp1, Nqo1, Tlr4, Tnfrsfla, Txnrd1 and decreased (P<0.05) Casp1 and Il1b after 4d. PM exposure increased (P<0.1) BECN1 and LAMP, decreased (P<0.1) ABCB1 and did not alter ABCC1 protein. LY294002 did not impact PM-induced ovotoxicity, but decreased ABCC1 and ABCB1 protein. Rapamycin prevented PM-induced follicle loss. These data suggest that the mammalian target of rapamycin, mTOR, may be a gatekeeper of PM-induced follicle loss.