Nutr Res Pract.  2022 Jun;16(3):314-329. 10.4162/nrp.2022.16.3.314.

Reduction of oocyte lipid droplets and meiotic failure due to biotin deficiency was not rescued by restoring the biotin nutritional status

• Tsuji A
• Ikeda Y
• Murakami M
• Kitagishi Y
• Matsuda S

• KMID: 2530393
• DOI: http://doi.org/10.4162/nrp.2022.16.3.314

Abstract

BACKGROUND/OBJECTIVES
Oocyte lipid droplets play a crucial role in meiosis and embryo development. Biotin is associated with fatty acid synthesis and is the coenzyme for acetyl-CoA carboxylase (ACC). The effects of a biotin deficiency on the oocyte lipid metabolism remain unknown. This study examined the effects of a biotin deficiency and its replenishment on murine 1) oocyte lipid droplet levels, 2) ovary lipid metabolism, and 3) oocyte meiosis.
MATERIALS/METHODS
Mice were divided into 3 groups: control, biotin deficient (BD), and recovery groups. The control and BD groups were fed a control diet or BD diet (0.004 or 0 g biotin/kg), respectively. The recovery group mice were fed a BD diet until day 21, and were then fed the control diet from days 22 to 64. This study then quantified the oocyte lipid droplet levels, assessed the oocyte mitochondrial function, and examined the ability of oocytes to undergo meiosis. Ovarian phosphorylated ACC (p-ACC), lipogenesis, β-oxidation, and ATP production-related genes were evaluated.
RESULTS
The BD group showed a decrease in lipid droplets and mitochondrial membrane potential and increased p-ACC levels. In the recovery group, the hepatic biotin concentration, ovarian p-ACC levels, and mitochondrial membrane potential were restored to the control group levels. On the other hand, the quantity of lipid droplets in the recovery group was not restored to the control levels. Furthermore, the percentage of oocytes with meiotic abnormalities was higher in the recovery group than in the control group.
CONCLUSIONS
A biotin deficiency reduced the oocyte lipid droplet levels by downregulating lipogenesis. The decreased lipid droplets and increased oocyte meiosis failure were not fully restored, even though the biotin nutrition status and gene expression of lipid metabolism was resumed. These results suggest that a biotin deficiency remains robust and can be longlasting. Biotin might play a crucial role in maintaining the oocyte quality.

Keyword

Vitamin deficiency; oocyte; lipid metabolism; meiosis; mitochondria

Figure

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  Fig. 1 Experimental design. The mice were divided into 3 groups: Ct group, BD group, Re group. The BD group mice were fed a BD diet. The recovery group mice were fed a BD diet until day 21 and a biotin-containing diet from day 22 to day 64. Some mice from each group were euthanized on days 22, 43, or 64.Ct group, control group; BD group, biotin deficient group; Re group, recovery group.

• 

  Fig. 2 Lipid droplet levels in oocytes on days 22, 43, and 64. The oocytes were stained with Nile Red, and the lipid droplets in oocytes were detected using confocal fluorescence microscopy on days 22 (A), 43 (B), and 64 (C). Dots of orange are lipid droplets in the oocyte (right panels). The bar graphs show the fluorescence intensity of oocytes in the Ct group (blue bar), the BD group (white bar), and the Re group (light blue bar). Ct1, 2 and 3 group, n = 32, 82, and 16, on days 22, 43, and 64, respectively; BD1 and 2 group, n = 39 and 57 on days 22 and 43, respectively; Re1 and 2 group, n = 62 and 27 on days 43 and 64, respectively. Values are means ± SE. The data on days 22 and 64 were analyzed using the Student's t-test. The data on day 43 were analyzed by 1-way analysis of variance. A different letter means a significant difference at P < 0.05.Ct group, control group; BD group, biotin deficient group; Re group, recovery group.*P < 0.05, ***P < 0.001.

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  Fig. 3 Mitochondrial membrane potential in oocytes on days 22, 43, and 64. The oocytes from each group were stained with JC-1 (A). The mitochondrial membrane potential was calculated as the ratio of red fluorescence (corresponding to active mitochondria) to green fluorescence (corresponding to mitochondria with lower activity), on days 22 (B), 43 (C), and 64 (D). The bar graphs show the fluorescence intensity of oocytes in the Ct group (blue bar), the BD group (white bar), and the Re group (light blue bar). C1,2 and 3group, n = 19, 67, and 13 on days 22, 43, 64, respectively; BD1 and 2 group, n = 28 and 36 on days 22 and 43, respectively; Re 11 and 2 group, n = 33 and 25 on days 43 and 64, respectively. The values are the means ± SE. The data on days 22 and 64 were analyzed using the Student's t-test. The data on day 43 were analyzed by 1-way analysis of variance. A different letter means a significant difference at P < 0.05.Ct group, control group; BD group, biotin deficient group; Re group, recovery group.*P < 0.05, **P < 0.01.

• 

  Fig. 4 p-ACC levels in ovaries. ACC, p-ACC, and GAPDH protein expression in the ovaries were analyzed by Western blotting (A). The data intensity of each band was quantified using ImageJ software, and the level of expression of p-ACC was normalized to the ACC level (B). The bar graphs show the p-ACC/ACC rate in the ovaries of mice in the Ct group (blue bar), the BD group (white bar), and the Re group (light blue bar). The values are the means ± SE, n = 3 to 4/group. The data on days 22 and 64 were analyzed using the Student's t-test. The data on day 43 were analyzed using 1-way analysis of variance. A different letter means a significant difference at P < 0.05.p-ACC, phosphorylated ACC; ACC, acetyl CoA carboxylase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Ct group, control group; BD group, biotin deficient group; Re group, recovery group.

• 

  Fig. 5 Gene expression related to biotin-dependent enzymes, lipogenesis metabolism, and energy metabolism in the ovaries on days 22, 43, and 64. The mRNA levels of Acc1, Pc, Pcc1, and Mcc1 (A-D) were analyzed as the biotin-dependent enzymes, mRNAs of Fas (E) as a marker for lipogenesis, of Cpt1α (F) and Acox1 (G) for fatty acid β-oxidation, and Shdβ, Cox4, and Atp5b (H-J) for the energy metabolism. The bar graphs show the gene expression levels in the ovaries of the mice in the Ct group (blue bar), the BD group (white bar), and the Re group (light blue bar). The values are the means ± SE, n = 3 to 4/group. The data on days 22 and 64 were analyzed using the Student's t-test. The data on day 43 were analyzed by 1-way analysis of variance. A different letter means a significant difference at P < 0.05.mRNA, messenger RNA; Acc1, acetyl-CoA carboxylase 1; Pc, pyruvate carboxylase; Pcc1, propionyl-CoA carboxylase 1; Mcc1, β-methylcrotonyl-CoA carboxylase 1; Fas, fatty acid synthase; Cpt1α, carnitine palmitoyl transferase 1α; Acox1, peroxisomal acyl-CoA oxidase 1; Sdhβ, succinate dehydrogenase β; Cox4, cytochrome c oxidase 4; Ct group, control group; BD group, biotin deficient group; Re group, recovery group.**P < 0.01.

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  Fig. 6 Frequency of abnormal oocytes on day 64. Tubulin and chromosomes were stained using an immunofluorescence stain and propidium iodide. The oocytes were classified as normal oocytes or abnormal oocytes (the latter includes those oocytes with spindle defects, chromosomal misalignments, or were immature) (A). The percentage of abnormal oocytes was calculated by division (number of abnormal oocytes/number of total oocytes × 100) (B). The bar graphs show the percentage of abnormal oocytes in mice of the Ct group (blue bar) and the Re group (light blue bar). Ct3 group, n = 42; Re2 group, n = 51. The oocytes were collected from 3 to 4 mice in each group. Data were analyzed using the χ2 test.Ct group, control group; Re group, recovery group.**P < 0.01.

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