NLRP5 mediates mitochondrial function in mouse oocytes and embryos

Abstract

Unraveling molecular pathways responsible for regulation of early embryonic development is crucial for our understanding of female infertility. Maternal determinants that control the transition from oocyte to embryo are crucial molecules that govern developmental competence of the newly conceived zygote. We describe a series of defects that are triggered by a disruption of maternal lethal effect gene, Nlrp5. Previous studies have shown that Nlrp5 hypomorph embryos fail to develop beyond the two-cell stage. Despite its importance in preimplantation development, the mechanism by which the embryo arrest occurs remains unclear. We confirmed that Nlrp5 mutant and wild-type females possess comparable ovarian germ pool and follicular recruitment rates. However, ovulated oocytes lacking Nlrp5 have abnormal mitochondrial localization and increased activity in order to sustain physiological ATP content. This results in an accumulation of reactive oxygen species and increased cellular stress causing mitochondrial depletion. Compromised cellular state is also accompanied by increased expression of cell death inducer Bax and depletion of cytochrome c. However, neither genetic deletion (Bax/Nlrp5 double knockout) nor mimetic interference (BH4 domain or Bax inhibitory peptide) were sufficient to alleviate embryo demise caused by depletion of Nlrp5. We therefore conclude that lack of Nlrp5 in oocytes triggers premature activation of the mitochondrial pool, causing mitochondrial damage that cannot be rescued by inactivation of Bax.

FIG. 1

Mitochondrial characterization in Nlrp5-deficient oocytes. A) Mitochondrial membrane potential as measured by JC-1 dye in Nlrp5^+/+ and Nlrp5^tm/tm oocytes. A significant increase in mitochondrial membrane potential was observed in Nlrp5^tm/tm oocytes. Values represent ratio of J-aggregate to J-monomer ± SEM. Asterisk (*) indicates significance (P = 0.016). Representative images are shown below the graph. B) Respiring mitochondrial pool measured by Mitotracker Red in Nlrp5 oocytes. Consistent with JC-1 results, a significant increase in respiring mitochondria was observed in Nlrp5^tm/tm oocytes. Values represent mean fluorescence units ± SEM (*P < 0.01). C) MTT assay reflecting reducing activity of mitochondrial enzymes was elevated in mutant oocytes (P = 0.05). Values shown represent colorimetric OD (optical density) reading generated by conversion of dye to colored substrate in pools of 10 oocytes. N indicates number of pools. D) Oxidized FAD⁺⁺ measurement in Nlrp5 oocytes using an FITC filter. Values shown are relative fluorescence units ± SEM. Numbers in parentheses represent number of oocytes/sample sets used per group; P = 0.008. E) Total mitochondrial pool as measured by Mitotracker Green in Nlrp5 hypomorph oocytes. Representative images are shown below the graph. Values represent mean fluorescence units ± SEM (*P < 0.01). F) Decreased expression of mitochondrial protein Tom20 in mutant oocytes corroborates decreased mitochondrial pool. Values represent mean fluorescence units ± SEM (*P < 0.001). N in each graph (with the exception of MTT assay) indicates number of oocytes used for each assay. Original magnification ×200.

FIG. 2

Altered GSH levels result in increased ROS production in Nlrp5-deficient oocytes. A) ROS measurement in Nlrp5^+/+ and Nlrp5^tm/tm oocytes. Values represent relative fluorescence units ± SEM. Increased ROS production was observed in Nlrp5 hypomorph oocytes. Asterisk (*) indicates significance; P < 0.05. Representative images are shown below. B) Baseline GSH content in Nlrp5^+/+ and Nlrp5^tm/tm oocytes. Values represent mean fluorescence units ± SEM. A significant reduction in the baseline GSH content was observed in tm/tm oocytes (*P < 0.05). C) Expression of p66 SHC in oocytes. Relative fluorescent intensity in RFU generated by phospho p66 (green) to total p66Shc (red) was used to show increased expression of phosphorylated isoform (P < 0.001). There was no change in the signal intensity of total p66SHC, and change in the ratio is due to rise of phospho p66SHC signal (green). Numbers in parentheses represent number of oocytes used in each experiment. Original magnification ×200.

FIG. 3

Mitochondrial and molecular changes in two-cell stage embryos. A) Proportion of abnormal embryos retrieved from WT and Nlrp5^tm/tm pregnant females retrieved at Day 1.5 from oviducts. Data are shown per female as percentage of abnormal embryos with fragmented or irregular size blastomeres. B, C) BAX protein (total and activated, NT form respectively) expression in Nlrp5^tm/tm two-cell embryos. Consistent with the transcript levels, a significant increase in BAX protein was observed in two-cell embryos. Values represent mean fluorescence units ± SEM. Asterisk indicates significance; P < 0.05. D) Cytochrome c level in mutant embryos was decreased by two-cell stage. Values represent sum of fluorescent signal (in RFUs) ± SEM. Asterisk indicates significance; P = 0.049. E) Total mitochondrial pool as measured by Mitotracker Green in Nlrp5^tm/tm two-cell embryos. Representative images are shown below the graph. Values represent sum of fluorescent signal (in RFUs) ± SEM. Asterisk indicates significance; P = 0.007. (F) Mitochondrial membrane potential as measured by JC-1 dye in Nlrp5^+/+ and Nlrp5^tm/tm two-cell embryos. Values represent ratio of J-aggregate (red) to J-monomer (green) ± SEM. Asterisk means values are significant, P < 0.05. Representative images are shown below the graph. Original magnification ×200. Numbers in parentheses represent number of oocytes per group. For generation of graphs and statistics, negative control (NC) values were subtracted from total RFUs.

FIG. 4

Developmental progression rate and rescue using TAT-BH4 and BIP-V5 peptides in Nlrp5 embryos. A) Zygotes cultured either in vehicle or vehicle + BH4 and observed for their progression rate. Significant increases in the formation of blastocysts were seen in Nlrp5^+/+ compared to Nlrp5^tm/tm embryos. However, presence of BH4 neither rescued the two-cell arrest in tm/tm embryos nor increased the rate of blastocyst formation in +/+ embryos. Values are expressed in percentages. B) Mean cell number and cell death index in Nlrp5 embryos treated with vehicle only. A significant reduction in mean total cell number along with a significant increase in cell death index was observed in Nlrp5^tm/tm embryos. Cell death index is shown in percentage and is expressed as mean ± SEM. Representative images of both +/+ and Nlrp5^tm/tm blastocysts are shown below. Original magnification ×400. Arrow indicates apoptotic cells. Asterisk indicates values are significant (P < 0.05). Numbers in parentheses represent the number of embryos assessed. C) Nlrp5 zygotes cultured either in vehicle, vehicle + BIP-V5, or vehicle + BIP-NC were assessed for their developmental progression from Day 1.5 to Day 4.5. A significant increase in the formation of blastocysts was seen in Nlrp5^+/+ compared to Nlrp5^tm/tm embryos. However, presence of BIP neither rescued the two-cell arrest of Nlrp5^tm/tm embryos nor increased the rate of blastocyst formation in +/+ embryos. Values are expressed as a percentage of embryos reaching indicated developmental stage.