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.

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.

Spherical bodies present within the germinal vesicle of Podarcis sicula previtellogenic oocyte derive from the temporaneous inactivation of ribosomal genes.

In the present paper we have investigated the origin of the spherical bodies (SBs) present within the germinal vesicle of about 400 microm previtellogenic oocytes in the lizard Podarcis sicula. In particular, we have attempted to clarify whether they derive from the single, large nucleolus present in early diplotenic oocyte as a consequence of ribosomal gene inactivation. We have, therefore, experimentally induced a decrease in rRNA synthesis by injecting animals with D-galactosamine or by exposing them to low temperatures. The investigations carried out have demonstrated that both treatments induce significant ultrastructural changes in the nucleolar apparatus and in particular fragmentation and the formation of SBs comparable to those observed in germinal vesicle under physiological conditions. These results indicate that the germinal vesicle of Podarcis sicula has a nucleolar apparatus that significantly changes its aspect according to its functional status and reveal that in this species, the time course of rRNA synthesis is peculiar with respect to any other vertebrate oocyte studies so far.

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.

Ultrastructural studies on developing follicles of the spotted ray Torpedo marmorata.

Light and ultrastructural investigations on sub-adult and adult sexually mature females, demonstrates that in Torpedo marmorata folliculogenesis starts in the early embryo and that the two ovaries in the adult contain developing follicles of various sizes and morphology. Initially, the follicle is constituted by a small oocyte, surrounded by a single layer of squamous follicle cells. The organization is completed by a basal lamina and, more externally, by a theca, that at this stage is composed by a network of collagen fibers. As the oocyte growth goes on, during previtellogenesis and vitellogenesis, the organization of the basal lamina and of the oocyte nucleus does not change significantly. The basal lamina, in fact, remains acellular and constituted by fibrils intermingled in an amorphous matrix; the nucleus always shows an extended network of chromatin due to the lampbrush chromosomes, and one or two large nucleoli. By contrast, the granulosa (or follicular epithelium), the ooplasm, and the theca cells significantly change. The granulosa shows the most relevant modifications becoming multi-layered and polymorphic for the progressive appearance of intermediate and pyriform-like cells, located respectively next to the vitelline envelope, or spanning the whole granulosa. The appearance of intermediate cells follows that of intercellular bridges between small follicle cells and the oocyte so that one can postulate that, as in other vertebrates, small cells differentiate into intermediate, and then pyriform-like cells, once they have fused their plasma membrane with that of the oocyte. Regarding the ooplasm, one can observe as in previtellogenic follicles, it is characterized by the presence of intermediate vacuoles containing glycogen, while in vitellogenic follicles by an increasing number of yolk globules. The theca also undergoes significant changes: initially, it is constituted by a network of collagen fibers, but later, an outermost theca esterna containing cuboidal cells and an interna, with flattened cells, can be recognized. The role of the different constituents of the ovarian follicle in the oocyte growth is discussed.