Perfluorooctanoic acid (PFOA) inhibits steroidogenesis and mitochondrial function in bovine granulosa cells in vitro.

Perfluorooctanoic acid (PFOA) is a persistent environmental contaminant. Due to the ubiquitous presence of PFOA in the environment, the impacts of PFOA exposure not only affect human reproductive health but may also affect livestock reproductive health. The focus of this study was to determine the effects of PFOA on the physiological functions of bovine granulosa cells in vitro. Primary bovine granulosa cells were exposed to 0, 4, and 40 µM PFOA for 48 and 96 h followed by analysis of granulosa cell function including cell viability, steroidogenesis, and mitochondrial activity. Results revealed that PFOA inhibited steroid hormone secretion and altered the expression of key enzymes required for steroidogenesis. Gene expression analysis revealed decreases in mRNA transcripts for CYP11A1, HSD3B, and CYP19A1 and an increase in STAR expression after PFOA exposure. Similarly, PFOA decreased levels of CYP11A1 and CYP19A1 protein. PFOA did not impact live cell number, alter the cell cycle, or induce apoptosis, although it reduced metabolic activity, indicative of mitochondrial dysfunction. We observed that PFOA treatment caused a loss of mitochondrial membrane potential and increases in PINK protein expression, suggestive of mitophagy and mitochondrial damage. Further analysis revealed that these changes were associated with increased levels of reactive oxygen species. Expression of autophagy related proteins phosphoULK1 and LAMP2 were increased after PFOA exposure, in addition to an increased abundance of lysosomes, characteristic of increased autophagy. Taken together, these findings suggest that PFOA can negatively impact granulosa cell steroidogenesis via mitochondrial dysfunction.

Effects of incubator oxygen and carbon dioxide concentrations on hatchability of fertile eggs, some blood parameters, and histopathological changes of broilers with different parental stock ages in high altitude.

The effects of incubator carbon dioxide (CO2) and oxygen (O2) concentrations with parental stock age (PSA) on embryonic deaths (ED), hatchability of fertile eggs (HFE), some blood parameters, and the tissue development of broilers were investigated. Four consecutive repetitions following the similar materials and methods were carried. From 3 different aged ROSS 308 broiler parental flocks 7,680 hatching eggs were obtained and classified as young (Y; 29 wk), middle (M; 37 wk) and old (O; 55 wk) as regards PSA, and randomly distributed. Four different incubator ventilation programs (IVP) as control (C; 0.67% CO2 and 20.33% O2), high CO2 (HC; 1.57% CO2 and 20.26% O2), high O2 (HO; 0.50% CO2 and 21.16% O2), and high CO2 + O2 (HCO; 1.17% CO2 21.03% O2) were applied with oxygen concentrator, and ED and HFE were investigated. Lung and heart tissues, hemoglobin value, packed cell volume, and red blood cell count, triiodothyronine, thyroxine, adrenocorticotropic hormone (ACTH) values of the chicks were analyzed. It was found that IVP affected ED and HFE. Higher rate of early ED (EED) was obtained from the HC than HCO, and higher middle+late stage+pipped but unhatched ED (MLPED) with a lower rate of HFE was observed in the C group than HO and HCO (P < 0.05). Association was found between PSA and IVP (P < 0.05), being more evident in EED for young PSA, in MLPED with HFE for Y and O PSA. From hematological values, no statistical difference in RBC, PCV, and Hb values were found among the treatment groups, ACTH concentration known as a response to stress was found to be higher than C in all groups, triiodothyronine concentration was higher in the HO group than C. In the histopathological examination, used IVPs were found to have negative effects on the lung and heart such as vacuolization, hemorrhage in all PSA groups except for C. Conclusively, PSA and IVP affected some hatching, blood and tissue development parameters of the broiler chicks.

Assessment of tebuconazole exposure on bovine testicular cells and epididymal spermatozoa.

This study is the first to investigate the effects of tebuconazole (TEB) on the physiological functions of bovine testicular cells and epididymal spermatozoa. Motility and plasma membrane integrity of spermatozoa exposed to TEB (0.001-100 µM) were evaluated at different incubation times (0-6 h), while TEB-induced spermiotoxicity was assessed after 24 h in cell cultures. Testicular cells, obtained from the parenchyma of bovine testes, were seeded at 1.0 × 104 and 1.5 × 106 cells/well in 96- and 12-well culture plates and incubated for 48 h in culture media containing TEB (0.001-100 µM) to evaluate cytotoxicity and hormone release, respectively. TEB did not affect the motility and plasma membrane integrity. However, significant spermiotoxicity occurred at higher TEB (1-100 µM) concentrations (P < 0.05) compared to control and lower doses. Although no dose caused cytotoxicity in testicular cells (P > 0.05), 1 and 100 µM TEB caused a significant increase in testosterone secretion (P < 0.05). As a result, high doses of TEB (1-100 µM) had slightly suppressive effects on spermatozoa; however, these doses had stimulatory effects on testosterone secretion by testicular cells. It appears that the disruption of hormonal homeostasis of testicular cells after TEB exposure may result in metabolic and especially reproductive adverse effects in bulls.

Effects of bisphenol A, diethylhexyl phthalate and pentabrominated diphenyl ether 99 on steroid synthesis in cultured bovine luteal cells.

Bisphenol A (BPA), diethylhexyl phthalate (DEHP) and pentabrominated diphenyl ether 99 (PBDE 99) are environmental toxicants belonging to the endocrine disrupting compounds (EDCs). They exert adverse effects on the various physiological systems, especially the reproductive system of humans and animals. The aim of this study was to investigate the effects of BPA, DEHP and PBDE 99 on progesterone (P4) synthesis in cultured bovine luteal cells. The bovine luteal cells isolated from the mid-luteal corpora lutea were exposed to different concentrations of BPA (1, 3, 10 and 30 µM), DEHP (1, 3, 10 and 30 µM) and PBDE 99 (0.1, 0.3, 1 and 3 µM) in a serum-free culture media for 48 and 96 hr. At 48 hr, the P4 level in the luteal cells decreased after treatment with all concentrations of BPA; 3, 10 and 30 µM of DEHP; and 3 µM of PBDE 99 compared to the control (p < .05). Treatment of cells with 3-30 µM of BPA, 1-30 µM of DEHP and 1-3 µM of PBDE 99 for 96 hr resulted in reduction in P4 synthesis (p < .05). However, lower concentrations of PBDE 99 (0.1 and 0.3 µM) increased P4 levels at 48 and 96 hr. Synthesis of P4 was lower at 96 hr compared to the 48 hr in the groups treated with BPA (30 µM), DEHP (1-30 µM), PBDE 99 (0.3-3 µM) and control group. Our results showed that BPA, DEHP and PBDE 99 are able to alter luteal steroidogenesis in bovine cells and can disrupt hormonal balance in the ovary. However, it is necessary to evaluate the exact mechanism underlying these effects in future studies.

Inhibitory effect of Bisphenol A on in vitro feline uterine contractions.

Bisphenol A (BPA) is an environmental pollutant used as a plasticizer in the manufacture of many plastic products, such as packaging, containers, and water and beverage bottles. There are deleterious effects of BPA on metabolic, endocrine, nervous, and reproductive systems. This is the first study in which there was investigation of the in vitro effect of BPA on the spontaneous contractions of the cat uterus. The tubal uterine segments (1 cm) collected from queens in estrus were suspended in an isolated organ bath. Following tissue stabilization, spontaneous contractions were recorded for 10 min to constitute the control group. The effects of the solvent (alcohol) and BPA at different concentrations (1, 10, and 100 µM) on uterine contractions were subsequently evaluated at 10 min intervals in terms of frequency and mean amplitude variables. The ethanol vehicle did not alter the uterine contractions compared to the control group. All concentrations of BPA used in the study resulted in a reduction (P < 0.05) in amplitude of uterine contractions in a dose-dependent manner, while only the largest dose of BPA decreased the frequency of contractions (P < 0.05). In reproductive physiology, regular uterine contractions facilitate successful fertilization, migration, implantation, and maintenance of pregnancy, as well as fetus expulsion. The results of this study indicate BPA has an inhibitory effect on spontaneous contractions of the cat uterus. It is proposed that this suppressive effect of BPA on uterine contractions might lead to queen infertility.

Effects of mancozeb, metalaxyl and tebuconazole on steroid production by bovine luteal cells in vitro.

Mancozeb, metalaxyl and tebucanazole are widely used pesticides in agriculture and industry to treat plant pathogenic fungi. Livestock may be exposed to such substances by consuming contaminated plants. The present study was designed to evaluate the effects of these three fungicides on bovine luteal cell steroidogenesis. Luteal slices from mid-cycle corpus luteum were dissociated into single cell suspension in aerated (O2) culture media (DMEM/F12) by enzymatic digestion. The cells were incubated in newborn calf serum (10%) for 18 h and then with serum-free media containing mancozeb (0.01 µM, 0.1 µM, 1 µM), tebuconazole (1 µM, 10 µM, 100 µM) or metalaxyl (100 µM, 500 µM, 2500 µM) for additional 96 h. The medium was replaced on day 1 and 3; and the retrieved medium was stored at -20 °C until progesterone assay. Treatment of cells with three different fungicides induced dose dependent variable decrease in steroid synthesis during incubation periods. Incubation of cells with 1 µM mancozeb exhibited a 33% decline in steroid synthesis on day 3 and 48% decline on day 5 compared with controls. Treatment of cells with 100 µM tebuconazole and 500 µM metalaxyl resulted in a 65% and 31% decrease, respectively, in progesterone accumulation on day 5 of incubation. Fungicide induced suppressive effects on luteal steroidogenesis were as metalaxyl < tebuconazole < mancozeb. Results of the present study suggest that designated concentrations of all three fungicides studied might have varying degrees of adverse effects on luteal steroidogenesis.

Effect of resveratrol on hematological and biochemical alterations in rats exposed to fluoride.

We investigated the protective effects of resveratrol on hematological and biochemical changes induced by fluoride in rats. A total of 28 rats were divided into 4 groups: control, resveratrol, fluoride, and fluoride/resveratrol (n = 7 each), for a total of 21 days of treatment. Blood samples were taken and hematological and biochemical parameters were measured. Compared to the control group, the fluoride-treated group showed significant differences in several hematological parameters, including decreases in WBC, RBC, and PLT counts and neutrophil ratio. The group that received resveratrol alone showed a decrease in WBC count compared to the control group. Furthermore, in comparison to the control group, the fluoride group showed significantly increased ALT enzyme activity and decreased inorganic phosphorus level. The hematological and biochemical parameters in the fluoride + resveratrol treated group were similar to control group. In the fluoride + resveratrol group, resveratrol restored the changes observed following fluoride treatment, including decreased counts of WBC, RBC, and PLT, decreased neutrophil ratio and inorganic phosphorus levels, and elevated ALT enzyme activity. The present study showed that fluoride caused adverse effects in rats and that resveratrol reduced hematological and biochemical alterations produced by fluoride exposure.