<Emphasis Type="SmallCaps">d</Emphasis>-Aspartate affects secretory activity in rat Harderian gland: molecular mechanism and functional significance

In this paper, the role of d-aspartate in the rat Harderian gland (HG) was investigated by histochemical, ultrastructural, and biochemical analyses. In this gland, substantial amounts of endogenous d-Asp were detected, along with aspartate racemases that convert d-Asp to l-Asp and vice versa. We found that the gland was capable of uptaking and accumulating exogenously administered d-Asp. d-Asp acute treatment markedly increased lipid and porphyrin secretion and induced a powerful hyperaemia in inter-acinar interstitial tissue. Since d-Asp is known to be recognized by NMDA receptors, the expression of such receptors in rat HG led us to the hypothesis that d-Asp acute treatment induced the activation of the extracellular signal-regulated protein kinase (ERK) and nitric oxide synthase (NOS) pathways mediated by NMDA. Interestingly, as a result of enhanced oxidative stress due to increased porphyrin secretion, the revealed activation of the stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) pro-apoptotic pathway was probably triggered by the gland itself to preserve its cellular integrity.     

Current knowledge of d-aspartate in glandular tissues

Free d-aspartate (d-Asp) occurs in substantial amounts in glandular tissues. This paper reviews the existing work on d-Asp in vertebrate exocrine and endocrine glands, with emphasis on functional roles. Endogenous d-Asp was detected in salivary glands. High d-Asp levels in the parotid gland during development suggest an involvement of the amino acid in the regulation of early developmental phases and/or differentiation processes. d-Asp has a prominent role in the Harderian gland, where it elicits exocrine secretion through activation of the ERK1/2 pathway. Interestingly, the increase in NOS activity associated with d-Asp administration in the Harderian gland suggests a potential capability of d-Asp to induce vasodilatation. In mammals, an increase in local concentrations of d-Asp facilitates the secretion of anterior pituitary hormones, i.e., PRL, LH and GH, whereas it inhibits the secretion of POMC/α-MSH from the intermediate pituitary and of oxytocin from the posterior pituitary. d-Asp also acts as a negative regulator for melatonin synthesis in the pineal gland. Further, d-Asp can stereo-specifically modulate the production of sex steroids, thus taking part in the endocrine control of reproductive activity. Although d-Asp receptors remain to be characterized, gene expression of NR1 and NR2 subunits of NMDAr responds to d-Asp in the testis.     

The centrosomal kinase NEK2 is a novel splicing factor kinase involved in cell survival

NEK2 is a serine/threonine kinase that promotes centrosome splitting and ensures correct chromosome segregation during the G2/M phase of the cell cycle, through phosphorylation of specific substrates. Aberrant expression and activity of NEK2 in cancer cells lead to dysregulation of the centrosome cycle and aneuploidy. Thus, a tight regulation of NEK2 function is needed during cell cycle progression. In this study, we found that NEK2 localizes in the nucleus of cancer cells derived from several tissues. In particular, NEK2 co-localizes in splicing speckles with SRSF1 and SRSF2. Moreover, NEK2 interacts with several splicing factors and phosphorylates some of them, including the oncogenic SRSF1 protein. Overexpression of NEK2 induces phosphorylation of endogenous SR proteins and affects the splicing activity of SRSF1 toward reporter minigenes and endogenous targets, independently of SRPK1. Conversely, knockdown of NEK2, like that of SRSF1, induces expression of pro-apoptotic variants from SRSF1-target genes and sensitizes cells to apoptosis. Our results identify NEK2 as a novel splicing factor kinase and suggest that part of its oncogenic activity may be ascribed to its ability to modulate alternative splicing, a key step in gene expression regulation that is frequently altered in cancer cells.     

Oestrogen control of the sexual dimorphism in the harderian gland of Xenopus laevis

Xenopus laevis shows a sexual dimorphism of the electrophoretic pattern of Harderian gland (HG) proteins. The male pattern displays three protein fractions whose molecular sizes are approx. 205, 180 and 78 kDa, respectively, and which are absent in the female pattern. Conversely, the female pattern displays two protein fractions of approx. 190 and 76 kDa, respectively. This sexual dimorphism led us to hypothesize a sex steroid control of the HG. Administration of 17β-oestradiol to male Xenopus converts the male protein pattern into the female one, while the administration of testosterone to the female has no effect. In this respect neither Northern analysis nor the RNase-protection assay performed using a 213 bp encoding for the androgen-binding domain reveals the presence of an androgen receptor mRNA in Xenopus HG. Conversely, Northern analysis has shown an oestrogen receptor mRNA whose size is approx. 6.5 kb and the RNase-protection assay performed by using a 197 bp encoding for the oestrogen-binding domain has also displayed the presence of an oestrogen receptor mRNA in the female HG but not in the male one. In addition, the oestrogen administration to male Xenopus induces the appearance of an oestrogen receptor mRNA. Androgen administration to female toad is ineffective. Taken together, all these findings suggest that in Xenopus laevis oestrogens are involved into the HG physiology. The appearance of an oestrogen receptor mRNA in the oestradiol treated males supports the hypothesis of the occurrence of autoinduction of oestrogen receptor mRNA expression in the HG.     

Ovarian activity and reproduction in the frog, Rana esculenta

Rana esculenta specimens were collected, during the last 13 years, in well-defined areas around Naples. The annual ovarian cycle shows distinct phases of recrudescence (starting September; vitellogenesis), breeding (late March-early July; egg deposition and active oogenesis) and quiescence (July-August; no follicular growth). Previtellogenic follicles are recruited for vitellogenesis in early September and in between two successive ovulatory waves. Breeding congregations are generally formed after a heavy rain fall and eggs are laid in standing waters, temporary or permanent. A maximum of three clutches of eggs is produced during the breeding season, at roughly monthly intervals. All mature females reproduce to some extent. Ovarian weight and clutch size are positively correlated to body weight. Depending upon the body size, the potential clutch size ranges from 1000 to 3500 eggs during the first wave of ovulation and it is notably smaller in the successive wave(s) of ovulation. Egg masses and tadpoles are left unprotected and mortality is high. The life cycle from the fertilized egg to completion of metamorphosis is 2 months and oogenesis in the ovary starts in the larva before the onset of metamorphic climax. Young females hatching from the first clutch of eggs may reach sexual maturity and breed in May the following year; those hatching from the last clutch require nearly 20 months to reach sexual maturity. The importance of some endocrine and exocrine factors for the regulation of ovarian activity and reproduction is discussed.     

Biological activity of some steroidal compounds in the adult and larval frogs.

Four steroids were tested for their biological activity, using the sex-steroid-dependent redevelopment of the secondary sex characteristics in adult frogs and gonadal sex differentiation in larval frogs as end points. In adult frogs, 19-norprogesterone and 6-chloro-17alpha-hydroxy-4, 6-pregnadiene-3,20-dione had antiandrogenic and antiestrogenic effects. 2alpha, 17alpha-Dimethyl-DHT and 2alpha-methyl-DHT were potent androgens and effective antiestrogens. In the larval frogs, all four compounds had a masculinizing effect upon the undifferentiated gonads.