The demands for oocyte cryopreservation have increased in recent years due to several social and health concerns [
37,
38]. It is widely known that women older than 35 years of age have a higher chance of harboring chromosomal abnormalities and intracellular organelle dysfunctions [
39,
40]. Furthermore, the clinical efficiency of vitrification is significantly lower in oocytes from aged women than those from young women [
41]. Women under 35 years of age have high success rates of ART and demonstrate no significant difference with respect to fertilization and implantation rates between the fresh oocyte and vitrificated oocyte. However, in women older than 38 years of age, vitrified-warmed oocytes show low survival, fertilization, and implantation rates [
2,
41]. Thus, maternal age is an essential parameter for predicting oocyte quality [
42]. Therefore, molecular and cellular changes that might occur in oocytes from older individuals are of important research interest. In the present investigation, we assessed two groups of genes that are associated with lipid biology, i.e. lipid uptake and necroptosis. Vitrification using multiple cryoprotectants and fast cooling inevitably exposed oocytes to chemical and physical stressors. Compared to other cell types, the high surface-to-volume ratio of oocytes may render them more vulnerable to external insults. We previously showed in two independent studies that the vitrification process influences the phospholipid content of mouse oocytes [
7], and that the levels of several classes of phospholipids are reduced in oocytes from aged mice [
10]. This led us to investigate the expression of lipid uptake-associated genes. Among the three genes we studied (Fig.
3a), the expression of
Cd36 showed a slight increase in fresh oocytes from aged mice.
Cd36 is expressed in both mouse and human oocytes [
43], and is implicated in the binding of the sperm to the egg—at the time of fertilization—by recognizing phosphatidylserine on the sperm membrane [
44]. In oocytes from aged mice,
Cd36 expression is higher than oocytes from young mice (Fig.
3a). Considering that the levels of certain phospholipids are reduced in oocytes from older mice [
10], it may be associated with an increased demand for specific lipid uptake. The significance of increased
Cd36 expression in oocytes from aged mice is currently unknown and requires further investigation.
Necroptosis is one of the programmed cell death pathways that involves the action of RIPK1, RIPK3, and MLKL [
35]. The signals for necroptosis include tumor necrosis factor α, FAS ligand, and TNF-related apoptosis-inducing ligand (TRAIL) [
18]. With respect to the role of necroptosis in aging, there is evidence that necroptosis is active in the reproductive system of aged male mice [
45]. Necroptotic activation was also observed in the epidermal white adipose tissue of aged mice [
46]. In the female reproductive organs, necroptosis is suggested to be involved during follicular development in primates [
47]. However, to date, there is no available information as to whether the oocytes are subjected to necroptotic activation under specific conditions. Here we showed that components that are crucial for necroptosis are present in mouse oocytes. Vitrification did not influence the expression of these factors significantly. Effectors of necroptosis, including pMLKL and pRIPK1, generally exhibit cytoplasmic puncta patterns that dynamically respond to external stimuli [
17,
21]. While pMLKL in oocytes were shown as numerous cytoplasmic puncta, specific signals on spindle poles and kinetochore were observed (Fig.
4). pRIPK1 also showed a unique distribution in oocytes before and after vitrification-warming, on MTOCs. Such unique localization patterns suggest that these factors may play non-necroptosis-associated roles in oocytes. pRIPK1, along with caspase 8, was shown to promote chromosome alignment during mitosis but was not shown to be localized near the spindle or kinetochore during this event [
48]. Thus, what we observed in oocytes is unusual and has not been observed in other systems. Nec1 is an inhibitor o RIPK1 activity. Nec1 does not show cytotoxicity in mouse oocytes [
25] or other cell types [
49]. Since Nec1 treatment to block RIPK1 activity during vitrification reduced survival rate after warming, intact RIPK1 activity seems to be required for preserving oocyte quality. Further investigation is warranted to examine the necroptosis-dependent and -independent function of RIPK1 in oocyte biology and aging.