Recently several studies addressed cytological and

Recently, several studies addressed cytological and biochemical features of oocyte aging. It was reported that cytological changes associated with the mammalian post-ovulatory oocyte aging include chromatin disorganization, abnormal meiotic spindle location or its disruption, partial exocytosis of cortical granules, hardening of the zona pellucida, mitochondrial dysfunction, increase in the abundance of large autophagic lysosomes and apoptotic fragmentation (Fissore et al., 2002; Takase et al., 1995). The established biochemical and molecular events of oocyte aging include abnormal calcium response and homeostasis, disruption of multiple protein kinase signaling pathways, decrease in maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity, reduced levels of Emi2, reduced contents of Bcl-2 mRNA and protein, decrease of the mitochondrial membrane potential and intracellular ATP content, elevated levels of reactive oxygen species (ROS), as well as various epigenetic modifications (McGinnis et al., 2014; Miao et al., 2009; Premkumar and Chaube, 2016). These changes manifest at the functional level in decreased rates of fertilization, polyspermy, parthenogenesis and abnormal development of embryos. It was hypothesized that oxidative stress may act as the initiator for a cascade of events that lead to aging of postovulatory oocytes (Lord and Aitken, 2013). Although a number of cytological and molecular changes have been observed in aging oocytes, SA-β-gal activity was not thoroughly investigated in these Piperine so far. This enzyme serves as a biomarker of senescence both in cultured cells and in in vivo, however, SA-β-gal activity is not required for senescence. In fact, at present this is the most extensively utilized marker of senescent cells (Itahana et al., 2013). It should be noted, however, that this marker is not completely specific, and it can be detected in a senescence-independent manner, for example, in cells held at confluence or in some differentiated cells (Dimri et al., 1995). The enzyme was identified as the lysosomal β-D-galactosidase encoded by the GLB1 gene and detectable at suboptimal pH 6.0 (Lee et al., 2006). It was demonstrated that SA-β-gal activity elevates in senescent cells due to increased lysosomal content and/or increased mRNA and protein level (Gary and Kindell, 2005; Kurz et al., 2000). The magnitude of this increase in senescence is sufficient for detection of the lysosomal β-D-galactosidase, which displays maximal activity between pH 4.0 and 4.5, at the suboptimal pH of 6.0. Most often SA-β-gal assay is utilized to detect senescent cells in rodent and primates, however it has also been used in other model organisms, such as Danio rerio and C. elegans (Dmitrieva and Burg, 2007; Kishi et al., 2008). More recently, it was found that the old ovaries of the annual fish N. guentheri are characterized by decreased proportion of mature oocytes, increased proportion of atretic oocytes, and high levels of SA-β-gal activity (Liu et al., 2017). In this work, we investigated SA-β-gal activity in aging oocytes and eggs of the African clawed frog Xenopus laevis. Xenopus oocytes and eggs have been widely employed in cell cycle and reproduction studies due to their large size (>1 mm in diameter) and high biochemical tractability. The oocytes and eggs can be obtained in large numbers from living frogs and treated in vitro like a primary cell culture. Fully grown Xenopus oocytes of the stage VI are arrested in the MI prophase with low activity of the key meiotic regulators, MPF, a complex of cyclin B and Cdk1 kinase, and cytostatic factor (CSF), which includes MAPK pathway. During the hormone-induced ovulation, oocytes progress from the prophase I to the metaphase II in the process of meiotic maturation. In frogs, the term “egg” is conventionally used for the mature ovulated oocytes arrested in the metaphase II by high activity of MPF and CSF. The meiotic arrest allows eggs to await fertilization, preventing cell cycle progression and parthenogenesis after meiosis. It was reported that in the absence of fertilization Xenopus eggs spontaneously exit the meiotic arrest and degrade by an apoptotic process, both in vivo and in vitro, within 48 h after ovulation. On the other hand, fully grown immature Xenopus oocytes remain stable and resistant to apoptosis (Tokmakov et al., 2011; Iguchi et al., 2013). Here, we analyzed activity and intracellular distribution of SA-β-gal in Xenopus eggs and oocytes aged in vitro over 48 h.