Effects of Heat Stress on Ovarian Physiology in Growing Pigs

Ovaries were obtained from growing pigs that had been heat-stressed and were evaluated for alterations in a signaling pathway known to play a critical role in ovarian physiology. Our results indicate that hyperthermia alters this pathway in a short space of time (after 7 days). Identifying how and why heat stress alters ovarian physiology are important in developing therapeutic approaches to prevent the reduction in reproductive performance associated with warm summer months

Dual protective role for Glutathione S-transferase class pi against VCD-induced ovotoxicity in the rat ovary

The occupational chemical 4-vinylcyclohexene diepoxide (VCD) selectively destroys ovarian small pre-antral follicles in rats and mice via apoptosis. Detoxification of VCD can occur through glutathione conjugation, catalyzed by glutathione S-transferase (GST) enzymes. Further, GST class pi (GSTp) can negatively regulate JNK activity through protein:protein interactions in extraovarian tissues. Dissociation of this protein complex in the face of chemical exposure releases the inhibition of pro-apoptotic JNK. Increased JNK activity during VCD-induced ovotoxicity has been shown in isolated ovarian small pre-antral follicles following in vivo dosing of rats (80mg/ Kg/d; 15d, i.p). The present study investigated the pattern of ovarian GSTp expression during VCD exposure. Additionally, the effect of VCD on an ovarian GSTp:JNK protein complex was investigated. PND4 F344 rat ovaries were incubated in control medium ± VCD (30 μM) for 2-8d. VCD increased ovarian GSTp mRNA (P \u3c0.05) relative to control on d4-d8; whereas GSTp protein was increased (P \u3c 0.05) on d6-d8. A GSTp:JNK protein complex was detected by immunoprecipitation and Western blotting in ovarian tissues. Relative to control, the amount of GSTp-bound JNK was increased (P = 0.09), while unbound JNK was decreased (P \u3c 0.05) on d6 of VCD exposure. The VCD-induced decrease in unbound JNK was preceded by a decrease in phosphorylated c-Jun which occurred on d4. These findings are in support of a possible dual protective role for GSTp in the rat ovary, consisting of metabolism of VCD and inhibition of JNKinitiated apoptosis

Developmental Origins of Ovarian Disorder: Impact of Maternal Lean Gestational Diabetes on the Offspring Ovarian Proteome in Mice

Gestational diabetes mellitus (GDM) is an obstetric disorder affecting approximately 10% of pregnancies. The 4HFHS (High Fat High Sucrose) mouse model emulates GDM in lean women. Dams are fed a HFHS diet one week prior to mating and throughout gestation resulting in inadequate insulin response to glucose in mid-late pregnancy. The offspring of HFHS dams have increased adiposity, thus, we hypothesized that maternal metabolic alterations during lean GDM would compromise ovarian function in offspring both basally and in response to a control or HFHS diet in adulthood. Briefly, DLPL were lean dams and control diet pups; DLPH were lean dams and HFHS pups; DHPL were HFHS dams and control diet pups and DHPH were HFHS dams and HFHS pups. A HFHS challenge in the absence of maternal GDM (DLPL vs. DLPH) increased 3 and decreased 30 ovarian proteins. Maternal GDM in the absence of a dietary stress (DLPL vs. DHPL) increased abundance of 4 proteins and decreased abundance of 85 proteins in the offspring ovary. Finally, 87 proteins increased, and 4 proteins decreased in offspring ovaries due to dietary challenge and exposure to maternal GDM in utero (DLPL vs. DHPH). Canopy FGF signaling regulator 2 (CNPY2), Deleted in azoospermia-associated protein 1 (DAZAP1), Septin 7 (SEPT7), and Serine/arginine rich splicing factor 2 (SRSF2) were altered across multiple offspring groups. Together, these findings suggest a possible impact on fertility and oocyte quality in relation to GDM exposure in utero as well as in response to a western diet in later life

FOXO3 Expression and Function in the Pig Oocyte and Embryo

Forkhead box O3 (FOXO3), a member of the FOXO subfamily of Forkhead transcription factors, has been shown to play critical roles in apoptosis, oxidative stress, cell cycle and DNA repair. The objective of this study was to characterize FOXO3 expression and its function during oocyte maturation and early embryo development in the pig.We found: (1) FOXO3 is dynamically expressed at both mRNA and protein level in the maturing oocyte and early in vitro fertilized embryos. (2) FOXO3 protein is localized in the cytoplasm of pig maturing oocytes. (3) Co-culture with Doxorubicin (DOX, 2 µM) significantly altered the total FOXO3 protein level during in vitro maturation. (4) in vitromaturation with DOX (2µM) numerically increased maturation rate, but significantly decreased embryo development to the blastocyst stage after oocyte parthenogenetic activation. Our work provides useful data for functional study of FOXO3 protein in female gametogenesis and the potential impact on developing pig embryos, which could benefit animal reproductive health and provide foundational knowledge for improving swine reproductive efficiency

Intestinal integrity, endotoxin transport and detoxification in pigs divergently selected for residual feed intake

Microbes and microbial components potentially impact the performance of pigs through immune stimulation and altered metabolism. These immune modulating factors can include endotoxin from gram negative bacterial outer membrane component, commonly referred to as lipopolysaccharide (LPS). In this study, our objective was to examine the relationship between intestinal barrier integrity, endotoxin and inflammation with feed efficiency (FE), using pig lines divergently selected for residual feed intake (RFI) as a model. Twelve gilts (62 ± 3 kg BW) from the low RFI (LRFI, more efficient) and 12 from the high RFI (HRFI, less efficient) were used. Individual performance data was recorded for 5 wk. At the end of the experimental period, ADFI of LRFI pigs was less (P \u3c 0.001), ADG not different between the 2 lines (P = 0.72) but the G:F of LRFI pigs was greater than for HRFI pigs (P = 0.019). Serum endotoxin concentration (P \u3c 0.01) and the acute phase protein haptoglobin (P \u3c 0.05) were greater in HRFI pigs. Transepithelial resistance of the ileum, transport of fluorescein isothiocyanate labeled-Dextran and-LPS in ileum and colon, as well as tight junction protein mRNA expression in ileum, did not differ between the lines, indicating the 2 lines did not differ in transport characteristics at the intestinal level. Ileum inflammatory markers, myeloperoxidase (P \u3c 0.05) and IL-8 (P \u3c 0.10), were found to be greater in HRFI pigs. Alkaline phosphatase (ALP) activity was significantly increased in the LRFI pigs in ileum and liver tissues and negatively correlated with blood endotoxin (P \u3c 0.05). Lysozyme activity in the liver was not different between the lines; however, the LRFI pigs had a twofold greater lysozyme activity in ileum (P \u3c 0.05). Despite the difference in their activity, ALP or lysozyme mRNA expression was not different between the lines in either tissue. Decreased endotoxin and inflammatory markers and the enhanced activities of antimicrobial enzymes in the LRFI line may not fully explain the difference in the FE between the lines, but they have the potential to prevent the growth potential in HRFI pigs. Further studies are needed to identify the other mechanisms that may contribute to the greater endotoxin and acute phase proteins in the HRFI pigs and the greater FE in the LRFI pigs

Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P \u3c 0.05) after 48 h of exposure to 3 or 6 μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response

Physiological mechanisms through which heat stress compromises reproduction in pigs

Seasonal variations in environmental temperatures impose added stress on domestic species bred for economically important production traits. These heat‐mediated stressors vary on a seasonal, daily, or spatial scale, and negatively impact behavior and reduce feed intake and growth rate, which inevitably lead to reduced herd productivity. The seasonal infertility observed in domestic swine is primarily characterized by depressed reproductive performance, which manifests as delayed puberty onset, reduced farrowing rates, and extended weaning‐to‐estrus intervals. Understanding the effects of heat stress at the organismal, cellular, and molecular level is a prerequisite to identifying mitigation strategies that should reduce the economic burden of compromised reproduction. In this review, we discuss the effect of heat stress on an animal\u27s ability to maintain homeostasis in multiple systems via several hypothalamic‐pituitary‐end organ axes. Additionally, we discuss our understanding of epigenetic programming and how hyperthermia experienced in utero influences industry‐relevant postnatal phenotypes. Further, we highlight the recent recognized mechanisms by which distant tissues and organs may molecularly communicate via extracellular vesicles, a potentially novel mechanism contributing to the heat‐stress response

Mechanisms of Ovarian Atresia Induced by Xenobiotic Exposures

The focus of this chapter is the mechanisms of apoptosis that occur during “normal” (physiologically-induced) or “abnormal” (chemical- or pathology-induced) attrition of ovarian oocytes. One of the primary functions of the ovary is development and maturation of oocytes, which occur within a follicular structure. During fetal development of the ovary, primordial germ cells (oogonia) are formed and become oocytes when they stop dividing, and are arrested at the diplotene stage (prophase) of the first meiotic division. At this stage the oocyte is surrounded by a single layer of flattened somatic cells (pre-granulosa cells) and a basement membrane to form primordial follicles (Hirshfield, 1991), the most immature follicular stage of development. As a result, the lifetime supply of oocytes is set at the time of birth, and is irreplaceable. Association of the granulosa cells with the oocyte is critical for maintenance of oocyte viability and follicle development (Buccione et al., 1990). Development and progression of a recruited follicle also requires the appropriate expression of numerous factors, including critical members of the transforming growth factor β superfamily, such as growth differentiation factor 9 (GDF9) and bone morphogenic protein (BMP15) (Paulini and Melo 2011). Characterization of GDF9 function has a demonstrated a required role in promoting somatic cells of the follicle to undergo mitosis and initiates paracrine signaling between the oocyte and the follicular cells surrounding it (Carabatsos et al. 1998; Hreinsson et al. 2002; Nilsson and Skinner 2002). The impaired fertility in GDF9 mice appears to primarily be a result of loss of function in somatic cells since oocytes of GDF knockout mice remain capable of undergoing in vitro maturation and progressing to metaphase II of meiosis (Carabatsos et al. 1998)