Effects of nonylphenol on vitellogenin synthesis in adult males of the spotted ray Torpedo marmorata.

The aim of this investigation was to assess the effects of nonylphenol (NP), an oestrogen-like environmental pollutant, on the vitellogenin (VTG) synthesis in adult males of the aplacental viviparous cartilaginous fish Torpedo marmorata. The VTG recovery in males is considered a biomarker of xeno-oestrogenic pollution as this lipophosphoglycoprotein is physiologically induced by oestrogens only in females of oviparous and ovoviparous vertebrates. Using in situ hybridization and immunohistochemistry, T. marmorata males injected with nonylphenol showed the presence of VTG in the liver and the kidney. In particular, vtg messenger (m)RNA and VTG protein were expressed in the liver, whereas in the kidney cells only the presence of VTG was recorded. By contrast, no expression for VTG was detected in the testis. These results demonstrate that in T. marmorata NP induces the expression of vtg only in the liver; the presence of VTG in the kidney and its absence in the testis are discussed.

Lipovitellin constitutes the protein backbone of glycoproteins involved in sperm-egg interaction in the amphibian Discoglossus pictus.

Our knowledge of the molecules that interact with sperm at the egg membrane is restricted to a short list. In the eggs of Discoglossus pictus, fusion with sperm is limited to a differentiated structure, the dimple, offering several advantages for detecting molecules involved in fertilization. Previous studies have identified fucosylated glycoproteins of 200, 260, and 270 kDa located at the surface of the dimple that are able to bind sperm in vitro. Here, we show that dimple glycoproteins and a protein represented by a 120-kDa band released following gel-into-gel SDS-PAGE of both glycoproteins share the same N-terminal amino acid sequence, which itself is similar to the N-termini of Xenopus liver-synthesized vitellogenin (VTG) and the lipovitellin 1. MALDI/MS mass spectrometry indicated that the 120-kDa band is part of both gps 200 and 270/260. A 117-kDa major protein of the egg lysate exhibits the same MALDI/MS spectrum, and LC-MSMS indicates that this is a lipovitellin 1 (DpLIV) that coincides with the 120-kDa band and is responsible for the formation of the 200-270-kDa dimers. Therefore, lipovitellin 1 constitutes the protein backbone of the dimple glycoconjugates. In vitro assays using polystyrene beads coated with DpLIV or with its dimers indicate that significant sperm binding occurs only with DpLIV dimers. In amphibians, VTG is taken up by the oocyte, where it releases lipovitellins destined to form yolk. In Discoglossus, our data suggest that yolk proteins are also synthesized by the oocyte. The dimple forms in the ovulated oocyte following the exocytosis of vesicles that likely expose DpLIVs at their membrane. Indeed, in whole mounts of immunostained eggs, anti-vitellogenin antibodies label only the surface of the dimple.

Misregulation of poly(ADP-ribose) polymerase-1 activity and cell type-specific loss of poly(ADP-ribose) synthesis in the cerebellum of aged rats.

Protein modification by ADP-ribose polymers is a common regulatory mechanism in eukaryotic cells and is involved in several aspects of brain physiology and physiopathology, including neurotransmission, memory formation, neurotoxicity, ageing and age-associated diseases. Here we show age-related misregulation of poly(ADP-ribose) synthesis in rat cerebellum as revealed by: (i) reduced poly(ADP-ribose) polymerase-1 (PARP-1) activation in response to enzymatic DNA cleavage, (ii) altered protein poly(ADP-ribosyl)ation profiles in isolated nuclei, and (iii) cell type-specific loss of poly(ADP-ribosyl)ation capacity in granule cell layer and Purkinje cells in vivo. In particular, although PARP-1 could be detected in virtually all granule cells, only a fraction of them appeared to be actively engaged in poly(ADP-ribose) synthesis and this fraction was reduced in old rat cerebellum. NAD(+), quantified in tissue homogenates, was essentially the same in the cerebellum of young and old rats suggesting that in vivo factors other than PARP-1 content and/or NAD(+) levels may be responsible for the age-associated lowering of poly(ADP-ribose) synthesis. Moreover, PARP-1 expression was substantially down-regulated in Purkinje cells of senescent rats.

Oocyte plasma membrane proteins and the appearance of vitellogenin binding protein during oocyte growth in the lizard Podarcis sicula.

In the ovary of the lizard Podarcis sicula, the micropinocytotic uptake of the yolk exogenous precursor (i.e., vitellogenin; VTG) occurs only in the reproductive period and involves the plasma membrane of > or =2000-microm oocytes. This paper analyzes the intrinsic proteins extracted from the plasma membrane of growing oocytes to identify the vitellogenin binding protein during the different stages of the annual ovarian cycle of this species. Despite the well-known ultrastructural changes of the oocyte plasma membrane, SDS-PAGE failed to show marked variation in the total number of membrane proteins during the most significant stages of oocyte auxocytosis. Nevertheless, ligand blotting, using homologous VTG and anti-VTG, revealed that an congruent with115-kDa protein of the oocyte plasma membrane bound plasma vitellogenin only in the reproductive period (spring-summer) in both vitellogenic and nonvitellogenic oocytes. During the nonreproductive period, this molecule was never observed. However, it could be induced in the coldest months (winter) by hypophyseal gonadotropins.

Plasma vitellogenin and 17 beta-estradiol levels during the annual reproductive cycle of Podarcis s. sicula Raf.

Plasma vitellogenin and 17 beta-estradiol concentration were determined during the annual reproductive cycle of the female lizard Podarcis s. sicula Raf. living around Naples. Plasma vitellogenin was purified from estrogenized males for characterization and to raise specific immune serum. Using ELISA, plasma vitellogenin titers were determined in relation to ovary weight; plasma 17 beta-estradiol was measured by RIA method. Native vitellogenin was present as two polypeptide bands: alpha and beta. The electrophoretic patterns, studied in normal male and estrogenized male and female, showed vitellogenin to be a protein present in female and in estrogenized male plasma but not in normal males. Lizard monomeric VTG, determined by SDS-PAGE, was about 200 kDa. Correlations between seasonal ovarian weight variations and plasma vitellogenin and 17 beta-estradiol suggest that ovarian development in Podarcis depends on plasma vitellogenin synthesis, which in turn relies on plasma estradiol levels. The two ovulatory waves observed in this study coincided with the two peak values of plasma vitellogenin and 17 beta-estradiol.

Exogenous vitellogenesis and micropinocytosis in the lizard, Podarcis sicula, treated with follicle-stimulating hormone.

The regulation of oocyte growth and of exogenous vitellogenesis by micropinocytosis has been studied in lizard Podarcis sicula kept at 28 degrees during the winter stasis and stimulated by follicle-stimulating hormone (FSH). Under these experimental conditions, oocyte auxocytosis as well as vitellogenesis is stimulated, while the follicular hierarchy is preserved. At the ultrastructural level, the flow of exogenous yolk precursors toward the oocyte increases and the pathway taken by them through intercellular spaces and zona pellucida is the same as that taken by peroxidase (tracer). Yolk precursor endocytosis is found only in oocytes greater than 1500 microns in diameter and takes place through the formation of several coated pits and vesicles. It is suggested that membrane receptors necessary for micropinocytosis are available only in such oocytes. Last, a different permeability of the ovarian follicle to exogenous yolk precursors during the different stages of oocyte growth and endovarian control of vitellogenesis are suggested.

Investigation on the pars tuberalis of the hypophysis of Psammodromus algirus L. (Sauria). An electron microscopy study.

The anatomic-microscopic and cytological aspects of the lateral lobes of adenohypophysis of Psammodromus algirus have been studied. Along the whole of its extension, the 'pars tuberalis' appears to be separated from the hypothalamic region by a space of 1600 A with a winding course filled with fibrillar matrix. The main inclusions of 'pars tuberalis' cells are glycogen granules histochemically characterized by the Thiery's method. A correlation between the electron and light microscopy observations on the lateral lobes of Psammodromus algirus is attempted.