Mammalian oocytes require pyruvate as an energy source for growth and resumption of meiosis. Because oocytes are not competent to carry out glycolysis, cumulus cells (CC) are responsible for metabolizing glucose into pyruvate and providing it to the oocyte through gap junctions. The understanding of the energetic metabolism of CC in culture conditions might provide basis for the improvement of COC in vitro maturation. The aim of this study was to determine the temporal patterns of mRNA expression of glycolytic enzymes [phosphofructokinase (PFKP), aldolase (ALDOA), triosephosphate isomerase (TPI), enolase (ENO1), pyruvate kinase (PKM2), and lactate dehydrogenase (LDHA)] in bovine CC during COC in vitro maturation with or without FSH. Immature COC (grades 1 and 2) were obtained from 2- to 8-mm follicles from abattoir ovaries (predominantly Bos indicus). Cumulus cells were separated from COC and frozen before (immature group) or after COC culture for 4, 8, 12, 16, and 20 hours with (10 ng/mL) or without FSH. Total RNA was extracted using RNeasy® (Qiagen, Valencia, CA, USA), and 100 ng of RNA was reverse transcribed using oligo dT primers and Omniscript® (Qiagen). Relative expression of target genes was assessed by real-time PCR using bovine-specific primers and Power SYBR green master mix in an ABI Prism® 7300. To select the most stable housekeeping gene for expression normalization, cyclophilin-A (CYC-A), GAPDH, and histone H2AFZ amplification profiles were compared using the geNorm applet for Microsoft Excel (Vandesompele J et al. 2002 Genome Biol. 3, 1-11); the most stable housekeeping gene was CYC-A. Relative expression values were calculated using the AACt method with efficiency correction (Pfaffl MW 2001 Nucleic Acids Res. 29, 2002-2007). Effects of time in culture and of FSH treatment were tested by ANOVA, and groups were compared by Tukey-Kramer Honestly Significant Difference test. Nonparametric analysis was used when data were not normally distributed. Abundance of mRNA of all glycolytic enzymes decreased during in vitro maturation with or without FSH. Expression of PFKP, ALDOA, TPI1, ENO1, and LDHA genes was decreased to around half of the initial value (time 0) by 4 to 8 h of culture (P < 0.05) and did not increase thereafter. A similar expression pattern was observed for PKM2, although mRNA abundance was reduced later in comparison with other enzymes; levels were decreased by 16 (without FSH) to 20 h (with FSH) of culture. The presence of FSH did not alter the overall temporal pattern of gene expression but decreased mRNA abundance for PFKP, ALDOA, and TPI1 at 20, 16 and 16 h of culture, respectively. In conclusion, gene expression of glycolytic enzymes decreased with time during COC in vitro maturation in cattle, and FSH did not have a major influence on this expression pattern. This study was supported by CAPES and FAPESP.

The selective serotonin reuptake inhibitor fluoxetine is one of the most commonly administered psychotropic medications; however, it has been recognized as toxic to aquatic life. In this study, we showed that stress responses and feeding motivation in Nile tilapia were affected by acute exposure to fluoxetine. To reach that conclusion, we exposed Nile tilapia to 0, 1 or 10 µg/L (environmentally/biologically relevant doses) of fluoxetine over a 24 h period and then exposed them to a handling stressor. We found that the 10 µg/L dose enhanced cortisol response to stress but caused an earlier decrease in the ventilation boost induced by that stressor. An immediate ventilation boost after stressful stimuli indicates sympathetic activation. Thus, this suggests that fluoxetine decreased sympathetic nervous system activity but augmented hypothalamus–pituitary–interrenal axis activity in the fish. Both feeding latency and ingestion were similar among the tested conditions; however, a multiple logistic regression model revealed that in the presence of a stressor or fluoxetine, the Nile tilapia tended to ingest less food but there was a higher probability of this decrease to be associated with fluoxetine. We concluded that acute exposure to environmentally/biologically relevant fluoxetine concentrations over 24 h acted as a modifying factor for Nile tilapia stress physiology and tended to interfere with feeding motivation. An acute stress response is an emergency reaction that contributes to the recovery of homeostasis. In the presence of fluoxetine, modifications of acute stress responses and the tendency to reduce food intake, which restricts the ability to replace the energy spent on stress responses, could compromise the resumption of homeostasis and an animal’s adjustment to different environmental contexts, such as those associated with aquaculture, in which anthropogenic stressors inevitably occur.

Recent findings suggest a role for estradiol in the regulation of early folliculogenesis. Estradiol production is greatest in the fetal ovary during early gestation in cattle, and both estradiol and progesterone inhibit primordial follicle activation (Yang MY and Fortune JE 2008 Biol. Reprod. 78 (Suppl 6), 1153-1161). Aromatase expression is detected in early stages of bovine pregnancy (Garverick HA et al. 2009 Anim. Reprod. Sci. in press). The mechanisms controlling steroidogenesis in the bovine fetal ovary remain to be fully elucidated. The objective of this study was to assess mRNA expression patterns of enzymes involved in steroid production [steroidogenic acute regulatory protein (STAR), side-chain cleavage P450 (CYP11), cytochrome P450 17 alpha-hydroxylase (CYP17A1), 3 beta-hydroxysteroid dehydrogenase (3fi-HSD), aromatase cytochrome P450 (CYP19), and 17 beta-hydroxysteroid dehydrogenase (17fi-HSD)] in bovine fetal ovaries during gestation. Bovine fetal ovaries were obtained in a local slaughterhouse, fetal age was estimated by the crown-rump length, and samples were grouped according with days of gestation as follows: 60 (n = 5), 75 (n = 8), 90 (n = 6), 120 (n = 7), 150 (n = 7), and 210 (n = 6). Expression of mRNA encoding steroidogenic enzymes was determined by semiquantitative real-time RT-PCR using bovine-specific primers and cyclophilin A as endogenous control. Reverse transcription was performed with SuperScriptIII® (Invitrogen, Carlsbad, CA, USA) and PCR with Power SYBR green master mix (Applied Biosystems, Foster City, CA, USA) in an ABI Prism® 7500 (Applied Biosystems). Gene expression values were determined by the Pfaffl equation and effect of day of gestation on gene expression was analyzed with Fisher’s protected test, except when data were not normally distributed and nonparametric analysis was performed. Expression of mRNA encoding all steroidogenic enzymes was detected throughout gestation. The mRNA abundance of CYP17A1 and CYP19 was highest at 60 days of gestation and decreased thereafter (P < 0.05). Expression of all other genes did not significantly vary with time of gestation. In conclusion, all major enzymes required for steroidogenesis were expressed in the bovine fetal ovary. Expression of CYP17A1 and CYP19 was suppressed after 60 days of gestation, suggesting that these enzymes may be involved in the mechanisms controlling estradiol production and follicle formation in the bovine fetal ovary. Supported by CAPES and FAPESP.