RESUMO
Sexual dimorphism of the rodent brain is manifested by the epigenetic action of gonadal steroids. Our previous research identified the granulin (grn) precursor gene as a sex steroid-inducible gene, which was shown to be expressed more abundantly in male than female neonates at the mediobasal hypothalamic area. Grn is a 6-kDa polypeptide promoting or inhibiting the growth of epithelial cells and hematocytes in vitro. In this study, effects on male sexual behavior of male were pursued under conditions in which grn gene expression was suppressed during the critical period. To this end, an antisense oligodeoxynucleotide (ODN) of the grn precursor gene was designed, incorporated into inactivated Sendai virus (HVJ)-liposome complexes, and infused into the third ventricle of 2-day-old male rats. Two different control treatments were used: the first consisted of a control sequence ODN that had little homology to known mRNAs; the second of vehicle (HVJ-liposome) alone. After maturation, animals treated with antisense ODN of grn displayed significantly lower scores than control males on various parameters assessing sexual behavior; i.e., mount, intromission, and ejaculation. The antisense ODN, however, did not affect body growth or serum concentrations of testosterone and luteinizing hormone. Further, there was no significant difference in the volume of the sexual dimorphic nucleus of the preoptic area between antisense ODN-treated and control animals. It was shown that inadequate expression of the grn gene in the brain of male neonatal rats during the critical period suppressed the induction of some type of male sexual behavior, suggesting the grn was involved in the process of masculinization of the rat brain.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Precursores de Proteínas/antagonistas & inibidores , Precursores de Proteínas/genética , Comportamento Sexual Animal/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Células Cultivadas , Portadores de Fármacos , Humanos , Hipotálamo/química , Hipotálamo/metabolismo , Injeções Intraventriculares , Lipossomos , Hormônio Luteinizante/sangue , Masculino , Área Pré-Óptica/anatomia & histologia , Área Pré-Óptica/efeitos dos fármacos , Progranulinas , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Ratos Wistar , Respirovirus/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Comportamento Sexual Animal/fisiologia , Testosterona/sangue , Terceiro VentrículoRESUMO
We developed a two-step extraction method for measuring fecal steroid concentrations. In the first step, distilled water was used to extract steroids from fecal samples. In the second step, a mixture of organic solvents (hexane and ether) was used to re-extract water extracts that had been transferred to a glass tube. A portion of the upper layer of the organic solvents was transferred to separate assay-tubes for measurement of estradiol (E2) or progesterone (P), and the organic solvents were evaporated in vacuo. After phosphate-buffered saline was added to each tube, commercially supplied radioimmunoassay (RIA) kits were used to determine the steroids. We demonstrated the advantages and reliability of this method by using it to assay the steroid hormone concentrations in fecal samples and serum samples collected on the same day from female cynomolgus monkeys who showed normal menstrual cycles and from monkeys who had induced hyperfunction of ovarian steroidgenesis. Different fecal samples from each monkey were used to determine the recovery rate of each steroid in water extraction from the fecal samples and the reproductivity of hormone concentrations in the fecal samples. The results demonstrate that this two-step method is simple and effective for measuring fecal steroids for monitoring the reproductive status of cynomolgus monkeys, without having to collect serum samples.
