Molecular Cloning of a Chitinase Gene from the Ovotestis of Kuroda’s Sea Hare Aplysia kurodai

In this study, we report that we successfully cloned and sequenced a chitinase gene from the ovotestis of Kuroda’s sea hare Aplysia kurodai. By using reverse transcription-polymerase chain reaction (RT-PCR) and a system for the 5’ and 3’ rapid amplification of cDNA ends, we obtained a 1352 bp chitinase gene (AkChi) from the ovotestis of A. kurodai. AkChi contains a 1263 bp open reading frame that encodes 421 amino acids. The domain structure predicted from the deduced amino acid sequence was an N-terminal signal peptide and a catalytic domain of glycoside hydrolase (GH) family 18 chitinase. A comparative analysis of the deduced amino acid sequences of AkChi with those of the acidic mammalian chitinase of the California sea hare Aplysia californica revealed the highest homology at 83%. The purified chitinase from the ovotestis was digested by trypsin, and 119 residues of digested peptides were consistent with the deduced amino acid sequence of AkChi. We used RT-PCR to evaluate the expression of AkChi in various tissues of A. kurodai, and we observed that AkChi was expressed only in the ovotestis. A phylogenetic tree analysis, performed using the amino acid sequences of AkChi and known GH family 18 chitinases, showed that AkChi was separated from the molluscan chitinases with a chitin binding domain. To our knowledge, this is the first study demonstrating the cDNA cloning of an ovotestis chitinase from a sea hare.

Gonadal Steroid Hormones Levels in a Bitch with True Bilateral Hermaphroditism

Intersexualism occurs in all species of maxnmals and the pathogenesis of abnormal development of the gonads is still unclear. Thus, the aim of this report is to describe the hormonal and histopathological findings from a case of bilateral true hermaphroditism in a mixed-breed bitch due to the few case reports published involving hormonal levels in dogs with intersexuality. Serum was obtained from the animal before surgery and was submitted to hormone measurements. Progesterone and testosterone were evaluated by radioimnunoassay and estrogen by ELISA （immune-enzymatic assay）. The values of progesterone, testosterone and estrogen concentrations were 2.26 ng/mL, 0.05 ng/mL and 9.7 pg/mL, respectively. The proportion of ovarian tissue found in the ovotestes after histopathological examination was higher in relation to seminiferous tubules, which may explain the low level of serum testosterone. Low concentration of serum testosterone may have contributed to the partial virilization of external genitalia, since the animal only had clitoral hypertrophy. Estrogen and progesterone levels found were compatible with the initial stage of estrus （preovulatory LH （luteinizing hormone） surge）, which shows that ovarian follicles found in the bilateral ovotestes contributed to the occurrence of the estrous cycle before surgery. Quantitation of serum hormones may also shed light on the cause of disturbances in sex differentiation in canine species. More studies are needed to elucidate the real cause of intersex in dogs.