Effect of 17beta-estradiol and progesterone on vitellogenesis in the spotted ray Torpedo marmorata Risso 1810 (Elasmobranchii: Torpediniformes): studies on females and on estrogen-treated males.

The influence of 17beta-estradiol (E(2)) on vertebrate vitellogenesis is well ascertained. The aim of the present paper is to study the involvement of E(2) and progesterone (P) in the induction and regulation of vitellogenesis in females and experimental E(2)-treated males of Torpedo marmorata. We analyzed females in various stages of the reproductive cycle and E(2) experimentally treated males. The presence of vitellogenin was investigated in the plasma and in the liver by western blot and immunohistochemistry; its site of synthesis was investigated by in situ hybridization. The steroid levels in the plasma were measured by Enzyme Immunoassay. In treated males, E(2) induces in the liver the synthesis of VTG which is then secreted into the bloodstream as a 205-kDa polypeptide, the same that is found in the plasma of non-pregnant vitellogenic females. In females, E(2) is naturally present in the plasma and its level is correlated with VTG synthesis in the liver and with the female reproductive cycle. Indeed, large amounts of E(2) are only found in mature vitellogenic females, whose liver is involved in VTG synthesis and secretion. By contrast, small amounts of E(2) are evident in juveniles whose ovaries are lacking in vitellogenic follicles and in females preparing for ovulation. Low titers are also found in gravid females, whose liver is not engaged in VTG synthesis. We show that P, which is absent in untreated males and juvenile females, is evident in the blood serum of E(2)-treated males and sexually mature females. Interestingly, in treated males P appears in the plasma just 24h after the first injection of E(2) and its titer increases; a week after the last injections, the P level is similar to that recorded in non-gravid vitellogenic females. Finally, it is noteworthy that the highest titer of P was recorded in pregnant females. We demonstrate that in Torpedo vitellogenin synthesis, as in other vertebrates, is under the control of E(2) but also that this synthesis is probably under the control of progesterone.

Ovarian follicle cells in torpedo marmorata synthesize vitellogenin.

The pattern of vitellogenesis is similar in all non-mammalian vertebrates: the liver, under oestrogenic stimulus, synthesizes vitellogenin (VTG) that, via the maternal circulation, is delivered to the oocyte and here internalized by receptor-mediated endocytosis (Wallace, 1985: Development Biology. A comprehensive synthesis. Vol. 1 Oogenesis:127-177; Schneider, 1996: Int Rev Cytol 166:103-134; LaFleur, 1999: Encyclopedia of Reproduction Vol. 4:985-992). The contribution to vitellogenesis of different components of the ovarian follicle has also been reported in amphibians (Wallace, 1985), squamate reptiles (Ghiara and Limatola, 1980: Acta Embryol Morphol Exper 1:5-6; Andreuccetti, 1992: J Morphol 212:1-11), and recently, supporting previous reports (Chieffi and Pierantoni, 1987: Hormones and Reproduction in Fishes, Amphibians and Reptiles Single vol.:117-144), in Torpedo marmorata (Prisco et al., 2001: Perspective in comparative endocrinology: Unity and diversity Single vol.:1197-1201; Prisco et al., 2002b: Gen Comp Endocrinol 128:171-179). The present investigation, performed with immunoblotting, immunohistochemical, and in situ hybridization techniques during different stages of follicular growth in T. marmorata, shows that, as previously supposed (Prisco et al., 2002b), granulosa cells in both previtellogenic and vitellogenic phases actively synthesize VTG. This is the first time among vertebrates that the synthesis of this protein has been found to occur also within the ovarian follicle. The present data also demonstrate that the contribution of granulosa cells becomes particularly evident during vitellogenesis. Indeed, in vitellogenic follicles, small, intermediate, and pyriform-like cells cross-react with an anti-VTG antibody and are positive to a hybridization signal with a VTG mRNA probe. By contrast, in previtellogenesis only the enlarged cells, i.e., intermediate and pyriform-like cells, are involved in VTG synthesis.