ROLE OF STEROID HORMONES IN ECHINOID REPRODUCTIVE BIOLOGY

Echinoid reproductive cycle has been extensively studied in several species but the mechanisms regulating gametogenesis processes are still scarcely understood. Apart from environmental factors, different research have suggested a steroid role in gonad maturation and growth. Particularly, in echinoderms steroid involvement in reproduction has been suggested by both studies on seasonal changes of steroid levels during the gonadal cycle and experiments of hormone administration. Nevertheless, the steroid function in echinoid reproductive processes has not been clearly identified, probably due to the low number of studies and the big variability of results reported. Thus, the main aim of this research project was to shed light on echinoid endocrinology and, in particular, to clarify the involvement of sex-steroid hormones in sea urchin reproductive biology. This was achieved employing both in vivo and in vitro approaches. First of all, considering the lack of studies on the development of effective cell cultures from echinoderm gonads, primary cell cultures from ovaries of the edible sea urchin Paracentrotus lividus were developed. Ovary cell phenotypes, present in culture, were identified and characterized by different microscopic techniques. Although cell cultures could be produced from ovaries at all stages of maturation, the cells appeared healthier and viable, displaying a higher survival rate, when ovaries at early stages of gametogenesis were used. In terms of culture medium, ovarian cells were successfully cultured in modified Leibovitz-15 medium, whereas poor results were obtained in Minimum Essential Medium Eagle and Medium 199. Different substrates were tested but ovarian cells completely adhered only on poly-L-lysine. To improve in vitro conditions and stimulate cell proliferation different serum-supplements were tested. Fetal Calf Serum and an originally developed Pluteus Extract resulted to be detrimental to cell survival, apparently accelerating processes of cell death. In contrast, cells cultured with sea urchin Egg Extract appeared larger and healthier, displaying an increased longevity that allowed to maintain them for up to 1 month. Overall this study provides new experimental bases and procedures for producing successfully long-term primary cell cultures from sea urchin ovaries, providing a simple and versatile experimental tool for research in echinoderm reproductive biology. Subsequently, in vivo and in vitro experiments, specifically addressed to determine possible 17β-estradiol (E2) and testosterone (T) involvement in echinoid reproduction, were performed. An in vivo long-term experiment of steroid dietary administration was performed in adult specimens of P. lividus. The experimental plan was specifically designed in order to reduce individual variability and synchronize the experimental animals at the same starting maturative condition. We analysed and compared different reproductive parameters (Gonad Index, Maturative Index and maturative stages distribution) in 4 experimental groups: control group (CTL), E2 and T groups fed with pellets containing respectively 17β-estradiol and testosterone, and E2-4 weeks group fed with control pellets for the first 4 weeks and then treated with 17β-estradiol. This latter was chosen in order to verify the existence of a specific E2-sensitive gametogenic stage, as proposed in different asteroid species. Possible steroid effects on P. lividus female reproduction was also investigated with an in vitro approach. Cells, isolated by ovaries in the same maturative conditions considered in the in vivo experiments, were cultured in presence of E2 and T physiological concentrations for 2 weeks. Effects on ovarian cell morphology and behaviour were investigated. In addition, steroid regulation of the Major Yolk Protein (MYP) expression was analyzed 24 and 48 hours after E2 and T exposure. According to our results, E2 and T do not markedly influence echinoid gonad maturation and, particularly, they do not promote gamete maturation. Hormonal dietary administration did not induce striking variations in the considered reproductive parameters and no effect was observed also when males and females were analyzed separately. In addition, no specific maturative stage sensitive to E2 was found, suggesting the existence of different hormonal mechanisms in asteroids and echinoids. Similar considerations could be reported taking into account the in vitro experiments. E2 and T exposure did not affect ovarian cell size and behaviour nor MYP expression. The obtained results suggest that these hormones are not directly involved in either gamete maturation, as demonstrated for vertebrates, or in vitellogenesis processes, as reported for several asteroid species. However a possible involvement of steroids in echinoid physiology cannot be completely excluded and their role in the regulation of lipid metabolism and protein synthesis during the different reproductive stages should be strongly considered as suggested by several authors. Further specific research on steroid hormone mode of action, physiological function and metabolism are therefore needed to completely understand echinoid reproduction and endocrinology.