Betaine is accumulated via transient choline dehydrogenase activation during mouse oocyte meiotic maturation

Abstract

Betaine (N,N,N-trimethylglycine) plays key roles in mouse eggs and preimplantation embryos first in a novel mechanism of cell volume regulation and second as a major methyl donor in blastocysts, but its origin is unknown. Here, we determined that endogenous betaine was present at low levels in germinal vesicle (GV) stage mouse oocytes before ovulation and reached high levels in the mature, ovulated egg. However, no betaine transport into oocytes was detected during meiotic maturation. Because betaine can be synthesized in mammalian cells via choline dehydrogenase (CHDH; EC 1.1.99.1), we assessed whether this enzyme was expressed and active. Chdh transcripts and CHDH protein were expressed in oocytes. No CHDH enzyme activity was detected in GV oocyte lysate, but CHDH became highly active during oocyte meiotic maturation. It was again inactive after fertilization. We then determined whether oocytes synthesized betaine and whether CHDH was required. Isolated maturing oocytes autonomously synthesized betaine in vitro in the presence of choline, whereas this failed to occur in Chdh^-/- oocytes, directly demonstrating a requirement for CHDH for betaine accumulation in oocytes. Overall, betaine accumulation is a previously unsuspected physiological process during mouse oocyte meiotic maturation whose underlying mechanism is the transient activation of CHDH.

Keywords: betaine; choline; choline dehydrogenase; enzyme; gene knockout; meiosis; mouse; oocyte.

Figure 1.

Endogenous betaine in oocytes. Betaine was measured by liquid chromatography tandem mass spectrometry in groups of 250 intact preantral follicles with enclosed oocytes from postnatal (P11) ovaries and groups of 50 cumulus–oocyte complexes with enclosed GV oocytes (COC), GV oocytes (GV), first meiotic metaphase (MI) oocytes undergoing meiotic maturation at 3 (MI 3) and 6 (MI 6) h post-hCG injection; in mature eggs (MII); and in 1-cell embryos (1c). The amount of betaine is expressed as pmol per follicle, COC, oocyte, or embryo. The measured background levels of betaine measured in the final wash have been subtracted from each paired measurement. Betaine was essentially undetectable in all paired background samples (<0.05 pmol per oocyte or embryo) except in the paired background for one MII egg sample (0.35 pmol/oocyte). Each point represents an independent repeat (i.e. a measurement on one group of 250 P11 follicles or 50 oocytes or embryos). Horizontal lines, means ± S.E. (error bars). Means that do not share the same letter are significantly different (p < 0.001 for a or b versus c and p < 0.05 for a versus b by ANOVA with Tukey's test).

Figure 2.

Betaine transport during meiotic maturation. The transport of [³H]-betaine was measured in GV oocytes, oocytes during meiotic maturation through first meiotic metaphase (MI, 3–12 h post-hCG), mature eggs (MII, 16 h post-hCG), and one-cell embryos (1c) that were developed in vivo and collected at the times indicated after ovulation had been induced by hCG. Oocytes, eggs, or embryos were incubated with 1 μm [³H]betaine for 30 min, and the rate of transport was expressed as total betaine (fmol) per oocyte, egg, or embryo min⁻¹. No betaine transport was detectable in oocytes or eggs. As expected, substantial betaine transport was found in 1-cell embryos where the SIT1 transporter is active. Each symbol represents the rate of transport for one of three independent repeats. There was no significant difference between mean rates for oocytes at each stage or eggs (NS within bracket), whereas the mean rate for 1-cell embryos was significantly different (***, p < 0.001 by ANOVA with Tukey's test).

Figure 3.

Expression of choline dehydrogenase. A, Chdh transcripts were detectable by conventional RT-PCR (top right, 40 cycles) in growing oocytes from P11 ovaries, fully grown oocytes from P21, GV oocytes from adult females, COCs containing GV oocytes, and 1-cell embryos. Kidney served as a positive control (+ve), whereas water added to reaction mix was a negative (−ve). Transcripts for H2afz and Ppia controls were detected as expected. The gels shown represent one example of three similar repeats. Markers visible in the left and right lanes are 100, 200, and 300 bp. Quantitative RT-PCR (bottom left) confirmed the presence of Chdh in growing (P11) oocytes and fully grown (P21) oocytes from neonatal ovaries, GV oocytes, and 1-cell embryos, but not in 2-cell (2c), 4-cell (4c), 8-cell (8c), morula (M), or blastocyst (B) stage embryos. Bars, mean ± S.E. (error bars) of five independent repeats, except 10 repeats for GV oocytes. Bars that do not share the same letter are significantly different (p < 0.001 for a or b versus c and p < 0.05 for a versus b by ANOVA with Tukey's test). A fixed amount (1 pg) of Gfp cRNA was added to four sets of selected samples, and Q-RT-PCR was performed to assess the efficiency of recovery. Mean recovery was similar for each repeat and ranged from 79 to 84% (not shown). Thus, the transcript levels reported here probably represent ∼80–85% of the actual levels at each stage. B, Q-RT-PCR was performed to detect H2afz and Ppia as controls. The relative expression levels are essentially as reported previously (32). Transcript numbers were not calculated for these, and the results are expressed in fg of amplicon. C, Western blotting of GV oocytes, MII eggs, and 1-cell (1c), 2-cell (2c), and blastocyst (B) stage embryos showing CHDH (top) and GAPDH as a loading control (bottom). Fifty oocytes or embryos were loaded in each lane. Kidney lysate (250 ng of total protein) from Chdh^+/+ and Chdh^−/− neonates demonstrates antibody specificity. Positions of molecular weight markers are indicated and labeled at the left. The example shown is representative of three independent repeats. D, Western blotting of GV oocytes (60 oocytes/lane) isolated from Chdh^+/+, Chdh^+/−, and Chdh^−/− P21 neonatal ovaries. The oocytes were cultured for 6 h post-isolation in MEMα, at which point maximal CHDH activity would have developed. CHDH (top) and GAPDH loading controls (middle) are shown, with genotyping by PCR (bottom). The positions of the bands were essentially identical to those shown in C, and therefore only the immediate region of the gel is shown. One of three similar independent repeats is shown.

Figure 4.

Choline dehydrogenase enzyme activity assay development. A, kidney inner medulla lysate was used to validate the choline dehydrogenase activity assay. [³H]Betaine production from [³H]choline was measured and expressed as counts/min of ³H eluted from the column. Activity increased with increasing total kidney lysate protein and reached a plateau, whereas 3,3-dimethylbutanol (10 mm) completely eliminated measured activity above the background seen with no lysate added (at 0 mg). Each point represents the mean ± S.E. (error bars) of three independent repeats. B, background in the presence of 3,3-dimethylbutanol was subtracted from total activity (data from A) and fit by linear regression for the linear range (R² = 0.83). C, activity measured in kidney inner medulla lysate (Kidney, 0.9 mg total protein) compared with boiled kidney lysate or lung lysate or when only BSA rather than lysate was added (each at 0.9 mg). Bars, mean ± S.E. of three independent repeats. Bars that do not share the same letter are significantly different (p < 0.001 by ANOVA with Tukey's test).

Figure 5.

Choline dehydrogenase enzyme activity in mouse oocytes and preimplantation embryos. A, choline dehydrogenase activity measured in in vivo-developed oocytes and preimplantation embryos as a function of time post-hCG. Activity in GV oocytes was not significantly different from 0 (p = 0.43, one-sample t test). Activity peaked during meiotic maturation in maturing oocytes (MI) and eggs (MII). Significantly lower activity was present after fertilization in 1-cell (1c), 2-cell (2c), morula (M), and blastocyst (B) stage embryos. Each point represents the mean ± S.E. (error bars) of three independent repeats. Points lying above the dashed line are significantly different from those below (p < 0.05 by ANOVA with Tukey's test). B, the subset of choline dehydrogenase activity data (circles) for oocytes, eggs, and 1-cell embryos in A is replotted with the endogenous betaine content (squares, from Fig. 1), to show the temporal relationship between choline dehydrogenase activity and accumulation of betaine in vivo. C, choline dehydrogenase activity measured in oocytes undergoing spontaneous meiotic maturation in vitro after removal from antral follicles. Each point represents the mean ± S.E. of three independent repeats. Points that do not share the same letter are significantly different (p < 0.05 by ANOVA with Tukey's test). Stages are indicated as in A. D, choline dehydrogenase activity measured in MI oocytes isolated from the ovaries at 9 h post-hCG was equally inhibited by 3,3-dimethylbutanol (3,3-DMB; 10 mm) or betaine aldehyde (BA; 5 mm) or when the oocyte sample was boiled. Bars, mean ± S.E. of three independent repeats. Bars that do not share the same letter are significantly different (p < 0.001 by ANOVA with Tukey's test). For A–C, background in the presence of 10 mm 3,3-dimethylbutanol has been subtracted. Total counts/min are shown in D.

Figure 6.

Dependence of choline dehydrogenase activity development on meiotic progression and protein synthesis. A, choline dehydrogenase activity was measured as a function of time following the removal of GV oocytes from antral follicles, either undergoing spontaneous meiotic maturation in vitro (control) or maintained in GV arrest with Bt₂cAMP (300 μm). Both the effects of Bt₂cAMP and that of time are significant by two-way ANOVA (p = 0.0002), with Bt₂cAMP inhibiting the development of choline dehydrogenase activity. *, p < 0.05; **, p < 0.01 for effect of Bt₂cAMP at the indicated time points by Bonferroni post hoc test. B, inhibition of protein synthesis with cycloheximide (CHX; 50 μm) significantly inhibits the development of choline dehydrogenase activity in oocytes measured at 3 h after removal from the follicle (**, p = 0.01 by t test). In A and B, each symbol or bar represents the mean ± S.E. (error bars) of three independent repeats. C, CHDH was assessed by Western blotting in GV oocytes that had been cultured for 3 h (GV(+3h)), when maximal choline dehydrogenase activity has developed, in the absence (−) or presence (+) of cycloheximide. Mouse kidney lysate (K) was run to indicate the position of the CHDH band. GAPDH served as a loading control. The example shown is representative of two independent repeats. D, CHDH protein did not increase at 6 h post-hCG relative to GV oocytes. The example shown is representative of three independent repeats. E, CHDH protein was not higher in MII eggs than GV oocytes. Lanes contained the lysate of the number of oocytes or MII eggs indicated at the bottom. The example shown is representative of three independent repeats. In C–E, the positions of the bands relative to molecular weight markers (not shown) are essentially identical to those shown in Fig. 3C.

Figure 7.

Dependence of betaine accumulation in oocytes on choline and Chdh. A, GV oocytes were isolated immediately (GV) for analysis or cultured overnight to mature to MII eggs in the presence (+) or absence (−) of choline (0.5 mm) in mKSOM medium and then processed for betaine measurement by LC-MS/MS. Each point represents the measured betaine of a group of 25–30 oocytes or eggs, expressed as pmol/oocyte or egg. Bars indicate means ± S.E. (error bars) (n = 10 for GV, n = 11 for MII); symbols indicate individual measurements. Means that do not share the same letter are significantly different (p < 0.01 by ANOVA with Tukey's test). Eggs cultured in the presence of choline contained significantly higher betaine than GV oocytes, indicating that betaine was synthesized during meiotic maturation. B, GV oocytes from Chdh^+/+, Chdh^+/−, and Chdh^−/− females were in vitro-matured to MII eggs in the presence (+) or absence (−) of choline and then were processed as in A. Indistinguishable levels of betaine were present in wild-type (+/+) and heterozygous (+/−) GV oocytes, whereas betaine was not detected in null (−/−) oocytes. Only wild-type oocytes accumulated betaine during meiotic maturation, whereas there was no significant increase in betaine in null or heterozygous oocytes. Means that do not share the same letter are significantly different (p < 0.05 by ANOVA with Tukey's test). C, the amount of betaine present in GV oocytes and MII eggs that developed in vivo in Chdh^+/+ or Chdh^−/− females was determined. GV oocytes and MII eggs from wild-type females contained significantly higher levels of betaine than those from null females (**, p < 0.01; ***, p < 0.001 by ANOVA with Bonferroni test). Because of the difficulty of obtaining ovulated eggs from these females, only a limited data set was obtained (n = 3 pools of 30 oocytes or eggs for each genotype), and the analysis was restricted to comparing Chdh^+/+ with Chdh^−/−.