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The article presents a study which evaluates triethylamine in the anaesthesia of Anopheles gambiae.

Granulosa cells (GC) and theca cells (TC) luteinize in culture because of the presence of serum in the medium and, as a consequence, the production of progesterone (P4) increases and that of 17β-estradiol (E2) decreases. The follicular phase of the bovine estrous cycle is characterized by the presence of E2, as well as aromatase activity, that is turned off in the atresia or in the luteal phase of the bovine estrous cycle. We propose to characterize 2 co-culture systems utilizing slices of follicular walls (explants) containing both GC and TC and to cultivate them in nondefined medium (NDM; TCM199+serum) or defined medium (DM; α MEM+polyvinyl alcohol). To prepare the explants, 80 follicles measuring 4 to 6 mm in diameter were selected. Concentrations of P4 and E2 were evaluated by RIA after 24, 48, and 72 h of culture and values were corrected by tissue weight (10 mg). Morphological analysis of the explants was stained with hematoxylin and eosin. The total mRNA from GC-TC explants was purified and the expression of the following genes was estimated by RT-PCR: bovine 3 beta-hydroxysteroid dehydrogenase (HSD3B1), cholesterol side-chain cleavage cytochrome P450 (CYP11A1), 17|3-hydroxylase (CYP17), aromatase cytochrome P450 (CYP19), steroidogenic acute regulatory protein (StAR), follicle-stimulating hormone receptor (FSHr), and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPD) as internal control. The results were analyzed by two-way ANOVA followedby Bonferroni least-significant tests. Microscopic analyses showed that cells cultured in NDM lost their polyhedral shape and acquired a fibroblast-like form. Defined medium maintained GC morphology with low cytoplasm:nucleus ratio and GC-GC contact, similar to in vivocells. In NDM, the concentration of P4 increased as opposed to that of E2, which decreased after 48 h of culture. In DM, the concentrations of P4 and E2 were maintained in the first 48 h of culture. The level of P4 in NDM was higher than in DM during all periods tested. Our data show that CYP11A1 expression remains absent in NDM for follicles explants that initially did not express CYP11A1, whereas in DM the expression of this gene appears after 24, 48, or 72 h of culture, evidence that indicates rescue from atresia. It is important to notice that gene expression of steroidogenic enzymes in DM were higher than in NDM. Very low levels of CYP11A1 and CYP19 were observed in the NDM group. On the other hand, in the DM group there was a progressive increase of all the steroidogenic enzymes and FSHr, which reached a peak at 48 h of culture. In conclusion, our results suggest that DM showed a stimulating effect on steroidogenic activity of GC-TC co-culture similar to that observed in vivofor functional growing follicle, whereas in NDM the explants seemed to mimic luteinizing follicles. Grants from FAP-DF, Finatec, CNPq, and Capes. [ABSTRACT FROM AUTHOR]

FSH at low concentrations affect embryo production. In vitroculture conditions also affect embryo production and embryonic expression of genes and alter oocyte competence to produce embryos. The search for better and less variable culture conditions simulating those in vivohas led to the development of several systems of oocyte in vitromaturation culture. To compare the efficiency of the systems of MIV we utilized 4 groups: (1) TCM-199 control; (2) α-minimal essential medium (MEM); 3) α-MEM + 1 ng of FSH; 4) α-MEM+ 10 ng of FSH. The medium of Group 1 is non-defined by the presence of fetal calf serum (10%). Groups 2, 3, and 4 are defined and polyvinyl alcohol (1%) was used as a macromolecule. Porcine FSH (1 IU mg-1) was used at 1 and 10 ng mL-1and at 100 ng in defined and non-defined medium, respectively. Bovine ovaries were collected at an abbatoir. Oocytes (n= 1718) with homogeneous cytoplasm and with more than 3 layers of granulosa cells were used. Mature oocytes from the 4 treatments (11 replicates of each treatment) were inseminated with frozen-thawed, motile sperm separated by Percoll, using Sperm TALP HEPES medium. Presumptive zygotes with up to 2 or 3 layers of cumulus cells were cultured in 50-mL drops of SOF medium, supplemented with 10% FCS and 1 mg mL-1BSA under mineral oil in a humid 5% CO2atmosphere at 38.5°C after. Cleavage rate was evaluated 72 h post-insemination (hpi), and blastocyst rate was evaluated 168-192 hpi. Cleavage and blastocyst rates were calculated on the basis of number of presumptive zygotes. The expression of the following genes (Bax, Bcl-2, and conexin 43) was evaluated in blastocysts by RT-PCR. One-way ANOVA was used to compare blastocyst number. There was no difference in the proportion of embryos with more than 8 blastomeres in all groups tested, indicating that the rate of development during the first 72 hpi was similar for oocytes matured in chemically defined medium and for oocytes matured in medium containing serum. Bax is a pro-apoptotic marker and Bcl-2 an antiapoptotic marker. Connexin 43 (Cx43) may be a marker of embryo competence. Glyceraldehyde 3-phosphate dehydrogenase was used as internal control. The Bax gene was not expressed in any group. The Bcl-2 and Cx43 genes were expressed, mainly in the α-MEM 10. Although no differences were observed in blastocyst rate among the groups (30% to 40%), the strong expression of Bcl-2 and of Cx43 on the group containing 10 ng mL-1of FSH may indicate that FSH could improve embryo quality under defined conditions. The authors thank FAP-DF, CNPq, FUNPE, FINATEC, CAPES, and Biovitro Tecnologia de Embrioes Ltda, for laboratory assistance and grants, and Frigorifico Ponte Alta, Brasília-DF, for supplying bovine ovaries. [ABSTRACT FROM AUTHOR]