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.

alpha and beta spectrin distribution during the differentiation of pyriform cells in follicles of lizard Podarcis sicula.

Using alpha and beta spectrin mammalian antibodies on Western blotting, we demonstrated that lizard ovarian follicles contain two isoforms of alpha spectrin, Mr 94 and 134 kDa, and a 230 kDa beta spectrin, and that their pattern modifies in relation to pyriform cell differentiation. In fact, a positive immunoreaction is firstly evident within follicular epithelium of previtellogenic follicles when small cells differentiate into pyriform cells via intermediate cells. Later on, immunostain is present in pyriform cells and in the oocyte cortex that previously appears unstained. It is noteworthy that immunostain is also present on small cells located in contact with the oocyte membrane, but not on those located under the basal lamina and among pyriform cells, not engaged in pyriform cell differentiation. During the subsequent stages of previtellogenic phase, spectrin immunostain over the follicular epithelium and in the oocyte cortex does not change. By contrast, in vitellogenic follicles, when the follicular epithelium is constituted only by small cells, immunostain is evident at the level of the oocyte cortex and the cytoplasm of regressing pyriform cells. The present data strongly suggest that the alpha and beta spectrin pattern put in evidence during the different phases of lizard oocyte growth is related to the differentiation of small into pyriform cells, where such protein may guarantee a relationship between surface glycoproteins (Andreuccetti et al., 2001: Anat Rec 263:1-9), and the cytoskeleton distribution (Maurizii et al., 2000: Raf Mol Reprod Dev 57:159-166). Furthermore, the distribution of spectrin mRNA, similar to that observed for the protein, demonstrates that spectrin, once synthesized within pyriform cells, is transferred through intercellular bridges in the oocyte cortex, thus confirming that pyriform cells are nurse that significantly are involved in the oocyte growth. Finally, the present data demonstrate that alpha spectrin of lizard ovarian follicles has Mr quite different from those so far reported and may constitute a new group of isoforms. This important result will be the focus of future experiments. Mol. Reprod. Dev. 67: 101-107, 2004.

Role of apoptosis and Fas/FasL system in the oogenesis of the spotted ray Torpedo marmorata.

In the present paper we investigated the role played by apoptosis during oogenesis in the cartilaginous fish Torpedo marmorata. TEM, TUNEL and immunohistochemical techniques were employed to specifically reveal morphological and biochemical hallmarks of apoptosis in specimens from birth to sexual maturity. Data obtained demonstrate that apoptosis occurs in prefollicular oocyte selection, in maintaining the homeostasis of granulosa in healthy growing oocyte and in resorbing atretic follicles. In this respect, the involvement of apoptosis in Torpedo marmorata oogenesis closely parallels that found in mammals, thus confirming that strategies of germ cell selection among vertebrates have been evolutionarily preserved.

Fine structure of leydig and sertoli cells in the testis of immature and mature spotted ray Torpedo marmorata.

An ultrastructural investigation revealed the presence of true Leydig cells in the testis of sexually mature specimens of Torpedo marmorata. They showed the typical organization of steroid-hormone-producing cells, which, however, changed as spermatocysts approached maturity. In fact, they appeared as active cells among spermatocysts engaged in spermatogenesis, while in regions where spermiation occurred, they progressively regressed resuming the fibroblastic organization typically present in the testis of immature specimens. Such observations strongly suggest that these cells might be engaged in steroidogenesis and actively control spermatogenesis. Sertoli cells, too, appeared to play a role in spermatogenesis control, since, like Leydig cells, they showed the typical aspect of steroidogenic cells. In addition, the presence of gap junctions between Sertoli cells suggests that their activity might be coordinated. After sperm release, most Sertoli cells were modified and, finally, degenerated, but few of them changed into round cells (cytoplasts) or round cell remnants, which continued their steroidogenic activity within the spermatocyst and the genital duct lumen. From the present observations, it can be reasonably concluded that, in T. marmorata, spermatogenesis depends on both Leydig and Sertoli cells, and, as postulated by Callard (1991), in cartilaginous fish, the function of the Leydig cells as producers of steroids might be more recent and subsequent to that of Sertoli cells. In this regard, it is noteworthy that, in immature males, when Leydig cells showed a fibroblastic organization, Sertoli cells already displayed the typical organization of a steroidogenic cell.