Betaine homocysteine methyltransferase is active in the mouse blastocyst and promotes inner cell mass development

Abstract

Methyltransferases are an important group of enzymes with diverse roles that include epigenetic gene regulation. The universal donor of methyl groups for methyltransferases is S-adenosylmethionine (AdoMet), which in most cells is synthesized using methyl groups carried by a derivative of folic acid. Another mechanism for AdoMet synthesis uses betaine as the methyl donor via the enzyme betaine-homocysteine methyltransferase (BHMT, EC 2.1.1.5), but it has been considered to be significant only in liver. Here, we show that mouse preimplantation embryos contain endogenous betaine; Bhmt mRNA is first expressed at the morula stage; BHMT is abundant at the blastocyst stage but not other preimplantation stages, and BHMT activity is similarly detectable in blastocyst homogenates but not those of two-cell or morula stage embryos. Knockdown of BHMT protein levels and reduction of enzyme activity using Bhmt-specific antisense morpholinos or a selective BHMT inhibitor resulted in decreased development of embryos to the blastocyst stage in vitro and a reduction in inner cell mass cell number in blastocysts. The detrimental effects of BHMT knockdown were fully rescued by the immediate methyl-carrying product of BHMT, methionine. A physiological role for betaine and BHMT in blastocyst viability was further indicated by increased fetal resorption following embryo transfer of BHMT knockdown blastocysts versus control. Thus, mouse blastocysts are unusual in being able to generate AdoMet not only by the ubiquitous folate-dependent mechanism but also from betaine metabolized by BHMT, likely a significant pool of methyl groups in blastocysts.

FIGURE 1.

Endogenous betaine in preimplantation embryos. Total betaine was determined at each stage as indicated (1c, one-cell; 2c, two-cell; 8c, eight-cell, M, morula; CB, early cavitating blastocyst; B, fully expanded blastocyst), as a function of time post-hCG administration to induce ovulation. Individual measurements (n = 3 for each stage) are shown as black circles, and means (± S.E.) are shown in gray. Means that do not share the same letter are significantly different (ANOVA with Tukey test; p ≤ 0.05). The one low point at the morula stage is due to a much higher level of background in the wash drop than in any other samples, which was subtracted from the paired measurement. The pre-subtraction measurement was similar to the other two.

FIGURE 2.

Bhmt mRNA expression in oocytes and preimplantation embryos. A, RT-PCR showing the presence of Bhmt mRNA from the four-cell through blastocyst stages, with Ppia mRNA as a control. Oocyte stages are as follows: gO, growing oocyte; GV, germinal vesicle stage; MII, mature egg. Preimplantation embryo stages are indicated similarly to Fig. 1. Liver (L) served as a positive control. End lanes contained markers (250-bp marker is visible). Identity of representative bands was confirmed by sequencing. B, quantitative PCR showing Bhmt transcript numbers at each stage of oocyte and preimplantation embryo (upper graph), with Ppia as a control (lower graph). Bars represent mean ± S.E. of three independent repeats (Bhmt, ***, p < 0.001 compared with other stages not including liver; ANOVA with Tukey test). Oocyte and embryo stages are indicated as in A; W indicates quantitative PCR run with water alone. C, quantitative PCR on different two-cell and morula samples (n = 3) from those in B, confirming significant increase of Bhmt transcripts at the morula stage (left graph, *, p = 0.02 by t test) but lack of significant difference (p = 0.37, NS) in H2afz transcripts (right graph) between the same samples. M, morula;; B, fully expanded blastocyst.

FIGURE 3.

BHMT protein expression and localization in preimplantation embryos. A, Western blot of 200 embryos at the blastocyst (B), two-cell (2c), and morula (M) stages, and 0.5 mg of liver homogenate (L). BHMT band is ∼45 kDa. B, confocal immunofluorescence detection of BHMT. BHMT immunofluorescence in confocal sections of a two-cell embryo (2c), a morula (M), and two blastocysts (B) are shown in green, with appreciable signal evident only at the blastocyst stage. DNA (blue) was labeled with bisbenzimide (Hoechst 33258) and F-actin (red) with phalloidin. The same settings and image adjustments were used for each example. C, knockdown of BHMT with morpholino (MO). Representative whole-mount conventional immunofluorescence images of BHMT, following culture with BhmtMO1 antisense or control (mismatched) morpholino, are shown at top (contrast and intensity were adjusted equally in both images for optimal viewing). Panel at bottom shows the fluorescence intensity in arbitrary units normalized to the mean fluorescence in the control group for each of four independent repeats (5–9 per repeat, totals, 22 control; 27 BhmtMO1 antisense). *, p = 0.027 for paired t test between means of four repeats in each treatment. The fluorescence images shown were chosen to have the approximately same measured intensities as the overall mean fluorescence for each group. D, quantitation of BHMT knockdown by Western. Bands from representative gels following culture with Bhmt-specific morpholinos are shown at the top. The upper set shows an example with BhmtMO1 morpholino probed for BHMT above and GAPDH below (59 blastocysts per lane). The lower set is with BhmtMO2 morpholino (73 blastocysts per lane). For both, the control is at left (mismatched control morpholino for BhmtMO1 or Lipofectin alone for BhmtMO2), and morpholino-treated at right, as indicated by labels at bottom. The graph below shows the mean density of BHMT bands for BhmtMO1-treated blastocysts, each normalized to GAPDH in the same sample. **, p = 0.007 by t test, n = 3 (two Westerns with 59 blastocysts in each lane and one with 64 in each lane). Only the one Western shown was done for BhmtMO2 to confirm knockdown (density normalized to GAPDH was decreased by 65%), and thus no statistical analysis was performed.

FIGURE 4.

BHMT activity in blastocysts. A, validation of BHMT assay with liver homogenate. Activity is indicated in counts/min of ³H eluted from column, taken to indicate total dimethylglycine and methionine produced. The assay was run in parallel with no inhibitor (squares) or CBHcy (50 μm, triangles) added. The inset shows the pseudolinear region on an expanded scale. The line was fit by linear regression (r² = 0.999). Each point is the mean of three independent repeats. The error bars (S.E.) are not visible because they lie within the symbols. B, BHMT activity assay of blastocysts, as a function of blastocyst number (0, 10, 25, 50, and 75). The line was fit by linear regression (r² = 0.956). Error bars (S.E.) lie within the symbols. C, comparison of BHMT activity between blastocysts and morulae. Bars indicate the means ± S.E. of three repeats (50 morulae or blastocysts each per repeat). ***, p = 0.0002 by t test. D, effect of morpholino (BhmtMO1) on BHMT activity. Bars (mean ± S.E.) that do not share the same letter are significantly different (by ANOVA with Tukey test; n = 3–5 repeats for each group). Measured activity was not significantly different between blastocysts that had developed in vivo and those cultured from the eight-cell stage in the presence of control morpholino (Con). The BHMT inhibitor CBHcy decreased measured activity in blastocysts to a level not significantly different from the BSA control (p < 0.001, a versus c). Activity in blastocysts cultured in the presence of Bhmt-specific morpholino BhmtMO1 was significantly decreased (p < 0.001, a versus b) to a level not significantly different from that with CBHcy.

FIGURE 5.

Effect of perturbing BHMT on blastocyst cell allocation. A, effect of BHMT knockdown by BhmtMO1 on blastocyst cell allocation. The top panel shows mean trophectoderm (TE) cell numbers and the bottom panel shows ICM cell numbers for embryos with no morpholino treatment (no MO), control morpholino (Ctrl MO), and Bhmt-specific morpholino (BhmtMO1). The numbers of blastocysts assessed were 29 for no morpholino treatment, 59 for control morpholino, and 65 for BhmtMO1, from at least four independent cultures in each condition. For trophectoderm, NS indicates no significant difference among groups by ANOVA (p = 0.19). For ICM, p < 0.001 for a versus b by ANOVA with Tukey test. B, effect of BhmtMO2 on ICM cell numbers. The mean ICM cell numbers are shown for control (Lipofectin only) and BhmtMO2 morpholino-treated blastocysts. The number of blastocysts assessed were 35 for morpholino and 50 for control from three separate cultures. ***, p < 0.0001 by t test. Trophectoderm cell numbers were not significantly different (p = 0.25; data not shown). C, effect of BHMT inhibitor, CBHcy on blastocyst cell allocation. The top panel shows trophectoderm (TE) cell numbers and the bottom panel shows ICM cell numbers for embryos cultured with no addition (control) or CBHcy (500 μm), as indicated at bottom. The numbers of blastocysts assessed were 37 for control and 43 for CBHcy, from three independent cultures for both treatments. For trophectoderm, NS indicates no significant difference among groups (p = 0.28), and for ICM, ***, p < 0.0001, by t test. D, effect of BHMT knockdown on OCT4 expression. The top panel shows an example of Western blot probed for OCT4 (upper panel) and GAPDH (lower panel) of blastocysts with control or Bhmt-specific morpholino BhmtMO1, quantified in the bottom panel as OCT4 normalized in each sample to GAPDH, relative to the expression for the control morpholino in the same experiment set arbitrarily to 100. The bars represent the means ± S.E. for five independent repeats. Within each repeat, equal numbers of blastocysts were loaded in each lane (22–30). **, p = 0.006 by paired t test. In two repeats, embryos cultured with no morpholino were included (data not shown) and had relative band densities of 96 and 144, similar to control morpholino. E, effect of BHMT knockdown on numbers of OCT4- and NANOG-positive cells. Panels show mean numbers of OCT4-positive (left panel) and NANOG-positive (right panel) nuclei in control (Lipofectin only) and BhmtMO2 morpholino-treated blastocysts. The numbers of blastocysts assessed for OCT4 and NANOG were 23 for control and 14 for BhmtMO2, from three independent cultures. An additional four control and seven BhmtMO2 blastocysts were not visibly stained with the OCT4 antibody and were not included. Significance was assessed by t test, yielding p values indicated within the panels. F, rescue of development to the blastocyst stage by methionine added to the medium. Embryos were treated with BhmtMO1 or control morpholino as indicated at bottom, with no (0) methionine (Met) or 0.2 mm added. The number of embryos developing to the blastocyst stage (black) or arrested or dead (white) was scored for eight separate cultures (7–16 embryos per culture, for 104–105 embryos per group) for each condition, and data were pooled. The rescue by methionine on development with Bhmt morpholino was highly significant (***, p < 0.001 by Fisher's exact test for BhmtMO1 morpholino with methionine versus without). G, rescue of ICM cell number by methionine. Treatment groups were as in D. ICM cell numbers were decreased by BhmtMO1 relative to control in the absence of methionine but were not different from control when methionine (0.2 mm) was present in the medium. 45–50 blastocysts from five independent cultures were assessed per treatment group. p < 0.001 for a versus b by ANOVA with Tukey test.

FIGURE 6.

Effect of BHMT knockdown in blastocysts on fetal development on E10.5. A, fetal development on E10.5 after transfer of blastocysts in which BHMT had been knocked down by specific antisense morpholino (BhmtMO1) compared with control morpholino. Pregnancies (fetuses or resorption sites) were found in 12 recipients in the control morpholino group and 14 in the BhmtMO1 morpholino group. Numbers of fetuses and resorptions were pooled for each group and analyzed in a 2 × 2 table, as recommended (33). The control group had 70 fetuses and 12 resorption sites, and the Bhmt morpholino group had 60 fetuses and 49 resorption sites, shown as percentages of the total. The difference was highly significant (p < 0.0001) by Fisher's exact test. B, alternative analysis by frequency of resorptions in each recipient. For this analysis by recipient, only those recipients that had five or more implantations (fetuses + resorptions) were included (8 out of 12 control morpholino recipients, 12 out of 14 BhmtMO1 morpholino). The means (horizontal gray lines) were significantly different (p = 0.0032 by Mann-Whitney test, used because variances were significantly different by F test, p = 0.007).