Low-dose lipopolysaccharide exposure during oocyte maturation disrupts early bovine embryonic development.

Gram-negative bacteria release of lipopolysaccharide (LPS) endotoxin elicits robust immune responses capable of disrupting normal ovarian function contributing to female infertility. However, effects of subclinical or non-detectable infections on oocyte competence and subsequent embryo development remain to be fully elucidated. The aim of this study was to investigate the effects of exposing bovine oocytes to low LPS doses on oocyte and embryo competence. Bovine oocytes were collected from slaughterhouse-derived ovaries and matured with vehicle-control or increasing doses of LPS (0.01, 0.1, and 1 µg/mL) for 21 h. Oocytes (n = 252) were evaluated for nuclear maturation. A set of embryos from LPS-matured oocytes (n = 300) were cultured for 8 d to evaluate day 3 cleavage rates and day 8 blastocyst rates along with blastocyst cell counts. A subset of oocytes (n = 153) was fertilized and cultured for time-lapse image capture and analysis of embryo development. Results demonstrate no significant treatment differences among treatment groups in percent of oocytes at germinal vesicle (GV; P = 0.90), germinal vesicle breakdown (GVBD; P = 0.13), meiosis I (MI; P = 0.26), or metaphase II (MII; P = 0.44). Likewise, treatment differences were not observed in cleavage rates (P = 0.97), or blastocyst rates (P = 0.88) evaluated via traditional microscopy. Treatment with LPS did not affect total blastocyst cell count (P = 0.68), as indicated by trophectoderm (P = 0.83), and inner cell mass (P = 0.21) cell counts. Time-lapse embryo evaluation demonstrated no differences among control or LPS matured oocytes in number of zygotes that did not cleave after fertilization (P = 0.84), or those that cleaved but arrested at the 2-cell stage (P = 0.50), 4-cell (P = 0.76), prior to morula (P = 0.76). However, embryos derived from oocytes challenged with 0.1 µg/mL LPS tended to have reduced development to the morula stage compared with vehicle-treated controls (P = 0.06). Additionally, the percentage of blastocysts derived from oocytes matured in 0.01 µg/mL LPS tended to decrease compared to vehicle-treated controls (11.38 and 25.45 %, respectively; P = 0.09). Similarly, the proportion of oocytes that developed to the blastocyst stage was greater in vehicle-treated controls (25.45 %) compared with embryos derived from oocytes matured in 0.1 and 1 µg/mL (5.92 and 6.55 %, respectively; P = 0.03) LPS. These data suggest LPS-matured oocytes that subsequently underwent in vitro fertilization, experienced decreased competence to develop to the blastocyst stage.

Protein kinase B is required for follicle-stimulating hormone mediated beta-catenin accumulation and estradiol production in granulosa cells of cattle.

Follicle-stimulating hormone regulation of ovarian estradiol (E2) production requires involvement of beta-catenin (CTNNB1), a transcriptional co-factor. In cultured granulosa cells (GC) of cattle, FSH treatment increased protein abundance of CTNNB1 as well as protein kinase B (AKT), a molecule known to regulate components of the CTNNB1 degradation complex. However, whether FSH induction of CTNNB1 is through direct modulation of AKT remains to be determined. To investigate specific contributions of AKT to CTNNB1 accumulation, GC were treated with insulin-like growth factor-I (IGF-I), a well-established AKT activator, in the presence or absence of FSH. Granulosa cells treated with FSH, IGF-I, and IGF-I plus FSH had increased CTNNB1 accumulation compared with controls (P ≤ 0.02; n=6). E2 medium concentrations were greater (P=0.09; n=4) in FSH treated cells compared to controls (166 and 100 ± 28 pg/mL, respectively). Treatment with IGF-I and IGF-I plus FSH increased (P<0.01) E2 to comparable concentrations. Subsequently, GC treated with lithium chloride (LiCl), a pharmacological activator of AKT, provided a response consistent with IGF-I treated cells, as LiCl, FSH, and FSH plus LiCl increased CTNNB1 accumulation compared with non-treated controls (P ≤ 0.03; n=3). In contrast, inhibition of AKT signaling with LY294002 suppressed the ability of FSH and IGF-I to regulate CTNNB1. Additionally, LY294002 treatment reduced FSH and IGF-I mediated E2 medium concentrations (P ≤ 0.004). These results demonstrate that activation of AKT is required for gonadotropin regulation of CTNNB1 accumulation and subsequent ovarian E2 production.

The role of WNT signaling in adult ovarian folliculogenesis.

Wingless-type mouse mammary tumor virus integration site (WNT) signaling molecules are locally secreted glycoproteins that play important role in regulation of ovarian follicle maturation and steroid production. Components of the WNT signaling pathway have been demonstrated to impact reproductive functions, including embryonic development of the sex organs and regulation of follicle maturation controlling steroidogenesis in the postnatal ovary. Emerging evidence underscores the complexity of WNT signaling molecules in regulation of dynamic changes that occur in the ovary during the reproductive cycle. While disruption in the WNT signaling cascade has been recognized to have deleterious consequences to normal sexual development, more recent studies are beginning to highlight the importance of these molecules in adult ovarian function related to follicle development, corpus luteum formation, steroid production and fertility. Hormonal regulation of WNT genes and expression of members of the WNT signaling network, including WNT ligands, frizzled receptors, and downstream signaling components that are expressed in the postnatal ovary at distinct stages of the estrous cycle suggest a crucial role in normal ovarian function. Similarly, FSH stimulation of T-cell factor-dependent gene expression requires input from ß-catenin, a lynchpin molecule in canonical WNT signaling, further indicating ß-catenin participation in regulation of follicle maturation. This review will focus on the multiple functions of WNT signaling in folliculogenesis in the adult ovary.

Cattle with increased severity of bovine respiratory disease complex exhibit decreased capacity to protect against histone cytotoxicity.

Bovine respiratory disease complex (BRDC) is the leading cause of morbidity and mortality in feedlot cattle. Significant inflammation and lesions are often observed in lungs of infected cattle. During acute inflammatory responses, histones contribute to mortality in rodents and humans and serum proteins can protect against histone-induced cytotoxicity. We hypothesized that cattle experiencing chronic or fatal cases of BRDC have reduced ability to protect against cytotoxic effects of histones. Serum samples were collected from 66 bull calves at the time of normal feedlot processing procedures. Animals were retrospectively assigned to groups consisting of calves never treated for BRDC (control [CONT]; n = 10), calves treated with antimicrobials once for BRDC (1T; n = 16), calves treated twice for BRDC (2T; n = 13), calves treated 3 times for BRDC (3T; n = 14), or calves treated 4 times for BRDC (4T; n = 13). Samples were also collected each time animals received antimicrobial treatment; animals within a group were further sorted by calves that recovered and calves that died to test histone cytotoxicity. Bovine kidney cells were cultured in duplicate in 96-well plates and exposed to 0 or 50 µg/mL of total histones for 18 h with 1% serum from each animal. Cell viability was assessed by the addition of resazurin for 6 h followed by fluorescent quantification. Fluorescent values from serum alone were subtracted from values obtained for histone treatment for each animal. Serum from CONT, 1T, and 2T at initial processing all exhibited a similar (P > 0.10) response to histone treatment with fluorescent values of -312 ± 557, -1,059 ± 441, and -975 ± 489, respectively. However, 3T and 4T demonstrated an impaired capacity (P < 0.05) to protect against histones (-2,778 ± 471 and -3,026 ± 489) at initial processing when compared to the other groups. When sorted by mortality within group, calves that were treated twice and recovered (-847 ± 331) demonstrated a greater (P < 0.05) protective capacity than calves that were treated twice and died (-2,264 ± 412), indicating that calves that contract BRDC and ultimately die might have reduced protective capacity against histone cytotoxicity. Results suggest that calves that require multiple treatments for BRDC have reduced ability to protect against cytotoxicity of histones. Understanding the primary mechanism responsible for protecting against histone cytotoxicity could lead to improved identification of animals susceptible to severe cases of BRDC, improved focus and use of available resources, or better treatments for severe cases of BRDC

Effect of anabolic implants on adrenal cortisol synthesis in feedlot beef cattle implanted early or late in the finishing phase.

Implantation of anabolic steroids to increase growth rate in beef cattle impacts adrenal glucocorticoid production. The mechanism by which combination androgen and estrogen implants reduce cortisol biosynthesis in heifers is not clear. The objective of this study was to identify whether pituitary or adrenal gene expression and liver enzyme activity may contribute to altered serum cortisol concentrations in heifers receiving a combination implant. On d 0 of a 122-d finishing phase, 187 predominantly Angus heifers (361 kg) approximately 14 months old were randomly assigned to one of three implant groups: (1) non-implanted control, (2) implanted at the beginning of the finishing phase (d 0; early implant) with a combination implant (200mg TBA+20mg E2; Revalor 200®), and (3) implanted during the late stage of the finishing phase (d 56; late implant) with Revalor 200®. At d 56, body weight (BW) was greater (P<0.0001) for the early implanted heifers (456 ± 1.9 kg) compared to 437 and 435 (± 1.8) kg for control and late implanted heifers, respectively. Final BW (d 122) was similar between both implanted groups and heavier than non-implanted controls (P<0.0001). Serum cortisol was similar among groups at d 0 (P=0.86) however, by d 28 heifers receiving the combination implant had reduced (P<0.05) serum cortisol concentrations (31.2 ng/mL) compared to controls (49.4 ng/mL) and late (48.2 ng/mL) groups. On d 84 cortisol was similar (P=0.75) among implanted heifers and was less (P<0.01) than non-implanted heifers. Expression of pituitary and adrenal genes involved in glucocorticoid synthesis was evaluated at d 28/29 or 84/85; however, despite decreased serum cortisol in implanted heifers, no change in mRNA expression was demonstrated. Liver CYP3A enzyme activity at d 28/29 was decreased 59% in early implanted heifers compared to control heifers (P=0.01). Additionally, at d 84/85 AKR1C activity was greatest (P=0.01) in control heifers compared to both implanted groups. Data suggest that components of hypothalamic-pituitary-adrenal axis are influenced by exposure to exogenous hormones and this should be recognized when considering cortisol levels as a marker for stress response.

Follicle-stimulating hormone regulation of estradiol production: possible involvement of WNT2 and ß-catenin in bovine granulosa cells.

Follicle-stimulating hormone regulation of estrogen biosynthesis in the adult rodent ovary requires ß-catenin (CTNNB1), but whether CTNNB1 is involved in FSH-induced estrogen production in cattle is unknown. To elucidate the effect of FSH in regulating specific wingless-type mouse mammary tumor virus integration site (WNT)/CTNNB1 pathway components in bovine folliculogenesis and steroidogenesis, granulosa cells and follicular fluid were collected from large antral follicles (8 to 22 mm) from ovaries containing stage-III corpora lutea (d 11 to 17 of an estrous cycle). Follicles were categorized as high estradiol (n = 3; ≥ 25 ng/mL) or low estradiol (n = 3; ≤ 14 ng/mL) based on intra-follicular estradiol concentrations. Protein fractions were collected from granulosa cells and CTNNB1 abundance was analyzed by Western blot. Follicles with increased estradiol concentrations had 6-fold greater (P < 0.001) abundances of CTNNB1 compared with those classified as low-estradiol follicles, indicating that the hormonal milieu responsible for increased estradiol content could result in CTNNB1 accumulation. To ascertain specific contributions of FSH to increases in CTNNB1 protein abundances, granulosa cells were isolated from small ovarian follicles (1 to 5 mm) and cultured in the presence or absence of 100 ng/mL FSH for 24 or 48 h. Real-time PCR quantification of aromatase (CYP19A1) and select WNT family members were evaluated in response to FSH treatment. Successful stimulation of granulosa cells with FSH was confirmed by induction of CYP19A1 mRNA and parallel temporal increases of medium estradiol concentrations. Additionally, protein kinase b (AKT), a known FSH target, increased 1.7-fold (P = 0.07). Of the WNT family members analyzed, only WNT2 mRNA was induced after 24 h of FSH treatment compared with controls (0.12-fold and 3.7-fold for control and FSH-treated, respectively; P < 0.05), and WNT2 expression tended (P = 0.11) to remain increased at 48 h in FSH-treated cells compared with controls (1.0- and 3.14-fold, respectively). Furthermore, FSH-treated granulosa cells had greater abundances of total CTNNB1 (P = 0.04) protein. These data demonstrate for the first time that FSH regulates CTNNB1 protein and WNT2 mRNA expressions in bovine granulosa cells, suggesting a potential role of canonical WNT signaling in ovarian steroidogenesis and follicular growth of cattle. Future studies are necessary to determine if FSH directly regulates CTNNB1 through modulation of AKT or indirectly by up regulating WNT2, which subsequently activates the canonical WNT pathway.