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  • Moreover in this study weak expression of MT


    Moreover, in this study, weak expression of MT1 and MT2 was also detected in the oviduct muscular layers. Although there are no reports regarding the role of MT1 and MT2 in oviduct muscular layers, previous studies have demonstrated that melatonin plays an important role in the uterine contractility (Hertz-Eshel and Rahamimoff, 1965). In addition, the MT1 receptor has been identified in the rat myometrium (Zhao et al., 2002) where it was found to decrease uterine contractility (spontaneous and oxytocin-induced) (Ayar et al., 2001). In the human myometrium, MT2 was also detected, and associated signaling resulted in dramatic synergism with oxytocin receptor signaling (Sharkey et al., 2009, 2010), which might provide a critical hormonal trigger for the initiation of labor (Olcese, 2012). The expression of MRs in oviduct muscular layers suggests that melatonin might also play a similar role to that in the myometrium, and could be involved in the regulation of oviduct contractility. In addition, mRNA and protein expression results revealed that MT1 and MT2 levels are significant higher in the oviduct ampulla of the non-ovulating side than that of the ovulating side. Previous studies have demonstrated that the pre-ovulation follicle contains much higher levels of E2 (Tetsuka and Nancarrow, 2007), and that after ovulation, the luminal side (apical compartment) of the oviduct epithelium is temporarily exposed to follicular fluid (Palma-Vera et al., 2017). It has been demonstrated that exogenous E2 induces a significant decrease in melatonin binding to ovarian membranes and down-regulates MT1 in rat ovaries in vivo in rat ovaries (Clemens et al., 2001). Thus, we hypothesize that the change in expression of MRs in the oviduct ampulla, between the non-ovulating and ovulating side, might be regulated by the high level of estradiol in the follicular fluid. Estrogens exert their actions by binding the ER. Classic ERs comprise two forms including ERα and ERβ, which are members of the nuclear receptor superfamily of ligand-inducible transcription factors (Evans, 1998; Heldring et al., 2007). In cows, the function of both ERα and ERβ have been demonstrated in the oviduct (Ulbrich et al., 2003). In this study, we also detected ERs in cultured oviduct epithelial cells. One study showed that the MT1 receptor is upregulated in estrogen receptor-negative 1,4-DPCA pathway (MDA-MB-231) and downregulated in estrogen receptor-positive cells (MCF-7) (Treeck et al., 2006). To explore the effect of E2 on the expression of MRs in oviductal epithelial cells, the non-selective ER antagonist ICI182780 was used. We found that the inhibitory effect of E2 on MT1 and MT2 expression was inhibited by ICI182780, which suggest that this effect is mediated by ERs. However, this study also has several limitations. It has been reported that estrogen may exert early physiological effects that are extremely rapid and mediated via the membrane ER, which has been characterized as the G-protein coupled seven transmembrane receptor, GPR30 (Hall et al., 2001, Björnström and Sjöberg, 2005). Moreover, GPR30 was also detected in ovine oviduct epithelial cells (Wen et al., 2012). Because we primarily focused on the inhibitory effects of E2 in this study, we did not investigate which ER — ERα, ERβ, or GPR30 — participates in this regulatory process. This question can be answered in future studies by using specific commercial ligands of these receptors.
    Introduction The covalent attachment of ubiquitin (Ub) to substrates plays a central role in regulating a broad range of biological processes in eukaryotes [1], [2]. Substrates can be modified with a single Ub (monoubiquitination) or with varying types of polyubiquitin chains (polyubiquitination) that are distinguished by the particular lysine through which one Ub is joined to the next [3], [4]. Ub can also be conjugated to protein N termini to form an N-terminal Ub fusion [5], [6] or a linear polyubiquitin chain [7]. The functional consequences of ubiquitination are determined by the distinct nature and topology of the Ub modification [3], [8], [9], [10]. Ub is conjugated to substrates via the concerted action of Ub-activating enzyme (E1), Ub-conjugating enzyme (E2), and Ub ligase (E3) enzymes [11]. An E1 Ub-activating enzyme is charged with Ub and then transfers its Ub to the active site cysteine of an E2 Ub-conjugating enzyme to yield a charged E2~Ub thioester. The E3 ligase binds to both substrate and the E2~Ub thioester and catalyzes the transfer of Ub to the target substrate, resulting in an isopeptide bond between the C terminus of Ub and the epsilon-amino group of the substrate lysine [3], [9] or, in select cases, a peptide bond with the N-terminal alpha-amino group of the substrate [5], [6], [12]. In the case of really interesting new gene (RING)/U-box E3 ligases, which comprise the largest class of E3s [13], the E3 binds to both E2~Ub and substrate and catalyzes the attack of the substrate lysine on the E2~Ub thioester to yield the ubiquitinated substrate [14]. RING domains bind to E2 enzymes in a conserved manner, with the RING domain contacting a surface that includes the E2 N-terminal helix 1 and loops 4 and 7 [15]. In addition, the RING domain also contacts the donor ubiquitin, stabilizing a conformation of the E2~Ub conjugate [16], [17], [18] that promotes a nucleophilic attack by the substrate lysine on the thioester linkage, yielding an isopeptide bond between the lysine and the Ub C terminus [19]. Most RING E3 ligases can ubiquitinate substrates in conjunction with multiple E2 enzymes. Depending upon the identity of the E2, these modifications can take on a variety of forms. In many cases, the E2 itself appears to govern the nature of the Ub modification, in terms of both the multiplicity of the modifications and, in the case of polyubiquitin chains, the linkage type [3], [9], [14]. While much of the core enzymology and structures of the Ub conjugation machinery are widely conserved, it is clear that individual E2–E3 pairs have evolved an array of mechanisms to generate distinct Ub modifications.