The objective of this work was to evaluate the selection of immature bovine oocytes by brilliant cresyl blue dye (BCB) and expression of transcripts MATER and ZAR1. Cumulus-oocyte complexes (COCs) from slaughterhouse ovaries were exposed to BCB diluted in mDPBS and incubated for 60 min at 38.5 degrees C in humidified air. After exposure those COCs were distributed in two groups, according to their cytoplasm colour: BCB+ (coloured cytoplasm) or BCB- (colourless cytoplasm). The control group was submitted to in vitro maturation (IVM) immediately after morphological selection and holding control group COCs were exposed to mDPBS without BCB but in the same incubation conditions of BCB+ and BCB- group. The COCs of all groups were submitted to IVM, in vitro fertilization (IVF) and in vitro culture (IVC). Cleavage rate (72 h post-insemination) was similar between control (65.3%) and BCB+ (64.4%) groups, but greater than (p < 0.05) holding control (49.8%) and BCB- (51.3%) groups. Blastocyst rate (192 h post-insemination) was not different between BCB+ (18.5%) and control (16.3%) groups, but greater (p < 0.05) than BCB- (8.4%) group. No difference was found for blastocyst rate between holding control group (14.2%), control and BCB+ groups. The relative expression of MATER and ZAR1 genes was evaluated by real-time PCR in immature oocytes collected from the control, holding control, BCB+ and BCB- groups. Despite the relative expression of MATER in holding control, BCB+ and BCB- were down regulated in comparison to control group there was no statistical difference (p > 0.05) in the relative expression of MATER and ZAR1 transcripts among groups. The results indicate that the BCB dye detects immature oocyte populations with different developmental competence, although no improvement in in vitro embryo production using oocytes exposed or not to BCB was observed. Development competence of immature oocytes exposed to BCB does not seem to be associated with variations in the expression of MATER and ZAR1 transcripts.

The aim of this study was to evaluate the effect of culture media and stage of development in the osmotic ability of in vitro-fertilized bovine embryos and the expression of aquaporin 3 (Aqp3) and Na/K ATPase isoform 1 (ATPAse1) genes in embryos (i) with different ability to undergo rehydration and (ii) following vitrification. In experiment 1, in vitro fertilized presumptive zygotes were co-cultured in SOFaac or modified CR2aa medium and embryos at blastocyst and expanded blastocyst stages at day 7 post-insemination were exposed to NaCl hypertonic medium (900 mOsm) for 5 min following 120 min of culture in isotonic medium in order to evaluate dehydration and rehydration, respectively. No difference (P>0.05) on blastocyst rate was found between CR2aa and SOFaac medium but embryos co-cultured in SOFaac medium underwent greater (P<0.05) dehydration. Embryos at expanded blastocyst stage underwent greater dehydration but slower rehydration than embryos at blastocysts stage (P<0.05). In the experiment 2, the amount of Aqp3 and ATPase1 transcripts were quantified in blastocysts with high or low rehydration after exposure to hypertonic medium. No difference (P>0.05) on relative amount of transcripts was found in either genes. In the experiment 3, expanded blastocysts produced in a co-culture system were vitrified, warmed and then cultured for 72 h for analysis of embryo survival and amount of Aqp3 and ATPase1 transcripts. Lower (P<0.05) embryo survival rate was found for vitrified-warmed embryos (57.9%) than for their fresh counterparts (84.6%). There was no difference on expression of ATPase1 gene but lower (P<0.01) amount of Aqp3 transcripts was found in the vitrified-warmed embryos. In conclusion, embryo ability to undergo shrinkage and swelling is influenced by medium used in a co-culture system and by embryo stage. Rehydrating ability of embryos after exposure to NaCl hypertonic medium is not associated with variations on expression of Aqp3 and ATPase1 genes, but the vitrification can alter gene expression of in vitro-fertilized bovine embryos produced in a co-culture system.

The aim of this study was to evaluate the dose-response effect of insulin, plus follicle-simulating hormone (FSH) at a fixed concentration, in a serum-free defined culture medium (DCM) on the in vitro maturation of bovine cumulus-oocyte complexes (COCs). For oocyte nuclear maturation, the expression levels of GDF9, GLUT1, PRDX1 and HSP70.1 transcripts related to oocyte and embryo developmental competence were analysed. For in vitro maturation (IVM), cumulus-oocyte complexes from slaughterhouse ovaries were distributed into four groups based on insulin concentration added to serum-free DCM, which was composed of alpha minimum essential medium (α-MEM), as basal medium: (1) DCM control: 0 ng/ml; (2) DCM1: 1 ng/ml; (3) DCM10: 10 ng/ml; and (4) DCM100: 100 ng/ml. After IVM, the nuclear status of a sample of oocytes was analysed and the other oocytes were submitted for in vitro fertilization (IVF) and in vitro culture (IVC). Different concentrations of insulin did not affect significantly the nuclear maturation and cleavage rate (72 h post-insemination) across all groups. Blastocyst rate (192 h post-insemination) did not differ in DCM control (24.3%), DCM1 (27.0%) and DCM10 (26.3%) groups, but the DCM100 (36.1%) group showed a greater blastocyst rate (P 0.05) was observed at the different insulin concentrations. The results indicated that insulin added to DCM influenced levels of transcripts related to cellular stress (HSP70-1 and PRDX1) and oocyte competence (GDF9) in bovine oocytes and at higher concentrations enhanced blastocyst production.

Background: Pathologic pituitary hyperplasia (PH) in Pediatrics may be observed in several clinical settings. In PH secondary to primary hypothyroidism the increased TRH levels, due to a long-standing hypothyroidism state and loss of negative thyroxin feedback, causes thyrotroph and lactotroph cells hypertrophy, leading to an enlarged pituitary gland. Herein we report on two pediatric patients presenting this condition. Patient 1: A 9 year-old female was referred due to severe short stature (height Z-score: -5 SDS), face myxedema and a suprasellar mass on MRI. She had a past of headache, fatigue and learning disabilities.  Laboratory evaluation showed anaemia and dyslipidemia (total cholesterol 282 mg/ dL, LDL 222 mg/dL); severe hypothyroidism: TSH 1,300 IU/L (NR 0.27-4.2), fT4 0.14 ng/dL (NR 0.93-1.7), negative thyroid antibodies TgAb 0.9 mcg/L (NR <4); and hyperprolactinemia (PRL: 110 ng/mL; NR 4.79-23.3). Her bone age was delayed (2,6 yr).  Pituitary MRI revealed a 19×16×12-mm mass pushing the optic chiasm. Thyroid ultrasound revealed a diminished gland. After fourteen months on LT4 therapy, she presented catch up growth (height Z-score: -3,8 SDS); and improvement in school duties. Lab evaluation showed improvement on thyroid function (fT4 0.89 ng/dL, TSH 1.57 IU/L), PRL (18.25 ng/mL) and lipids (total cholesterol 151 mg/ dL, LDL 83 mg/dL). A follow up pituitary MRI evidenced a normal-sized pituitary gland inside a still enlarged sella. Patient 2: A 19 year-old female was referred due to severe short stature (height Z-score: -4.61 SDS), myxedema, madarosis, and delayed puberty (Tanner B2PH1). Neurological complaints were absent, except for a mild slowing of the speech. Bone age was delayed (10 yrs). Her labs showed a severe primary autoimune hypothyroidism: TSH 606.60 IU/L, fT4 < 0.023 ng/dL, anti-TPO 85.8 IU/mL (NR < 4); and a mildly elevated PRL 67.59 ng/mL. Thyroid ultrasound revealed a small gland volume with heterogeneous texture. MRI evidenced a pituitary macroadenoma measuring 11x9x8 mm. After eight months on LT4 therapy, her thyroid function (fT4 1.04ng/dL, TSH 1.48 IU/L) and PRL (11.73 ng/mL) were in the normal range. A follow up MRI revealed a normal-sized pituitary gland. Conclusion: Evaluation of the hypothalamic-pituitary axis must always be taken into account when investigating pituitary masses, since MRI alone is unable to reliably differentiate the underlying condition. In both reported cases, hormonal profile was unequivocal, unmasking the diagnosis of pituitary hyperplasia secondary to primary hypothyroidism. A specialized and integrated approach is essential to adequately define treatment and prevent unnecessary intervention.