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

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. 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. Correction: Betaine is accumulated via transient choline dehydrogenase activation during mouse oocyte meiotic maturation.Journal of Biological ChemistryVol. 295Issue 31PreviewVOLUME 292 (2017) PAGES 13784–13794 Full-Text PDF Open Access Betaine (N,N,N-trimethylglycine, glycine betaine) is present in mouse preimplantation embryos at very substantial intracellular concentrations (5–10 mm) that are much higher than in most other cells (1.Lee M.B. Kooistra M. Zhang B. Slow S. Fortier A.L. Garrow T.A. Lever M. Trasler J.M. Baltz J.M. Betaine homocysteine methyltransferase is active in the mouse blastocyst and promotes inner cell mass development.J. Biol. Chem. 2012; 287: 33094-33103Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). It serves at least two established roles in embryos, first as a key contributor to cell volume regulation and second as a major source of methyl groups in the blastocyst (1.Lee M.B. Kooistra M. Zhang B. Slow S. Fortier A.L. Garrow T.A. Lever M. Trasler J.M. Baltz J.M. Betaine homocysteine methyltransferase is active in the mouse blastocyst and promotes inner cell mass development.J. Biol. Chem. 2012; 287: 33094-33103Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 2.Zhang B. Denomme M.M. White C.R. Leung K.Y. Lee M.B. Greene N.D. Mann M.R. Trasler J.M. Baltz J.M. Both the folate cycle and betaine-homocysteine methyltransferase contribute methyl groups for DNA methylation in mouse blastocysts.FASEB J. 2015; 29: 1069-1079Crossref PubMed Scopus (32) Google Scholar, 3.Anas M.K. Lee M.B. Zhou C. Hammer M.A. Slow S. Karmouch J. Liu X.J. Bröer S. Lever M. Baltz J.M. SIT1 is a betaine/proline transporter that is activated in mouse eggs after fertilization and functions until the 2-cell stage.Development. 2008; 135: 4123-4130Crossref PubMed Scopus (40) Google Scholar, 4.Anas M.K.I. Hammer M.A. Lever M. Stanton J.A. Baltz J.M. 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Living with water stress: evolution of osmolyte systems.Science. 1982; 217: 1214-1222Crossref PubMed Scopus (3002) Google Scholar). Preimplantation embryos similarly rely on organic osmolytes for cell volume regulation, although they employ entirely different mechanisms of osmolyte accumulation from those of somatic cells (8.Baltz J.M. Zhou C. Cell volume regulation in mammalian oocytes and preimplantation embryos.Mol. Reprod. Dev. 2012; 79: 821-831Crossref PubMed Scopus (28) Google Scholar). Betaine is one of two major organic osmolytes present at high levels in early mouse embryos (3.Anas M.K. Lee M.B. Zhou C. Hammer M.A. Slow S. Karmouch J. Liu X.J. Bröer S. Lever M. Baltz J.M. SIT1 is a betaine/proline transporter that is activated in mouse eggs after fertilization and functions until the 2-cell stage.Development. 2008; 135: 4123-4130Crossref PubMed Scopus (40) Google Scholar, 4.Anas M.K.I. Hammer M.A. Lever M. Stanton J.A. Baltz J.M. 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Schultz R.M. Effect of sodium and betaine in culture media on development and relative rates of protein synthesis in preimplantation mouse embryos in vitro.Mol. Reprod. Dev. 1993; 35: 24-28Crossref PubMed Scopus (75) Google Scholar). Whereas somatic cells can regulate intracellular betaine to function as an organic osmolyte via BGT1 (SLC6A12 protein) or system A (SLC38A2) transporters (13.Yamauchi A. Uchida S. Kwon H.M. Preston A.S. Robey R.B. Garcia-Perez A. Burg M.B. Handler J.S. Cloning of a Na+- and Cl−-dependent betaine transporter that is regulated by hypertonicity.J. Biol. Chem. 1992; 267: 649-652Abstract Full Text PDF PubMed Google Scholar, 14.Petronini P.G. De Angelis E. Borghetti A.F. Wheeler K.P. Osmotically inducible uptake of betaine via amino acid transport system A in SV-3T3 cells.Biochem. J. 1994; 300: 45-50Crossref PubMed Scopus (35) Google Scholar), early preimplantation embryos instead control betaine levels in response to cell volume perturbations via SIT1 3The abbreviations used are: SIT1, betaine transport mechanism (SLC6A20 protein); ALDH, aldehyde dehydrogenase; BHMT, betaine-homocysteine methyltransferase; CHDH, choline dehydrogenase; COC, cumulus–oocyte complex; Bt2cAMP, dibutyryl cyclic AMP; eCG, equine chorionic gonadotropin; GV, germinal vesicle; hCG, human chorionic gonadotropin; ICM, inner cell mass of blastocyst; KSOM, potassium-supplemented simplex-optimized medium for embryo culture; mKSOM, modified KSOM; MI, first meiotic metaphase; MII, second meiotic metaphase; P, postnatal day; Q-RT-PCR, quantitative PCR after reverse transcription; mHEPES-KSOM, modified HEPES-KSOM; ANOVA, analysis of variance; MEM, minimum Eagle's medium. 3The abbreviations used are: SIT1, betaine transport mechanism (SLC6A20 protein); ALDH, aldehyde dehydrogenase; BHMT, betaine-homocysteine methyltransferase; CHDH, choline dehydrogenase; COC, cumulus–oocyte complex; Bt2cAMP, dibutyryl cyclic AMP; eCG, equine chorionic gonadotropin; GV, germinal vesicle; hCG, human chorionic gonadotropin; ICM, inner cell mass of blastocyst; KSOM, potassium-supplemented simplex-optimized medium for embryo culture; mKSOM, modified KSOM; MI, first meiotic metaphase; MII, second meiotic metaphase; P, postnatal day; Q-RT-PCR, quantitative PCR after reverse transcription; mHEPES-KSOM, modified HEPES-KSOM; ANOVA, analysis of variance; MEM, minimum Eagle's medium. (SLC6A20). SIT1-mediated betaine transport is not present in unfertilized eggs but becomes activated several hours after fertilization and persists through the 2-cell stage (3.Anas M.K. Lee M.B. Zhou C. Hammer M.A. Slow S. Karmouch J. Liu X.J. Bröer S. Lever M. Baltz J.M. SIT1 is a betaine/proline transporter that is activated in mouse eggs after fertilization and functions until the 2-cell stage.Development. 2008; 135: 4123-4130Crossref PubMed Scopus (40) Google Scholar, 4.Anas M.K.I. Hammer M.A. Lever M. Stanton J.A. Baltz J.M. The organic osmolytes betaine and proline are transported by a shared system in early preimplantation mouse embryos.J. Cell Physiol. 2007; 210: 266-277Crossref PubMed Scopus (34) Google Scholar). The second well-documented role of betaine is as a methyl donor (15.Lever M. Slow S. The clinical significance of betaine, an osmolyte with a key role in methyl group metabolism.Clin. Biochem. 2010; 43: 732-744Crossref PubMed Scopus (331) Google Scholar). The enzyme betaine-homocysteine methyltransferase (BHMT) catalyzes the transfer of a methyl group from betaine to homocysteine to form methionine that is then complexed with adenosine to produce S-adenosylmethionine, the universal methyl donor used by an array of methyltransferases (16.Ikeda S. Koyama H. Sugimoto M. Kume S. Roles of one-carbon metabolism in preimplantation period: effects on short-term development and long-term programming.J. Reprod. Dev. 2012; 58: 38-43Crossref PubMed Scopus (29) Google Scholar, 17.Garrow T.A. Purification, kinetic properties, and cDNA cloning of mammalian betaine-homocysteine methyltransferase.J. Biol. Chem. 1996; 271: 22831-22838Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Although methyl group generation from betaine via BHMT had been considered to be restricted to the liver in rodents (18.Delgado-Reyes C.V. Wallig M.A. Garrow T.A. Immunohistochemical detection of betaine-homocysteine S-methyltransferase in human, pig, and rat liver and kidney.Arch. Biochem. Biophys. 2001; 393: 184-186Crossref PubMed Scopus (85) Google Scholar), BHMT was unexpectedly found to be active at the blastocyst stage of mouse embryos and highly expressed in the inner cell mass (ICM) that gives rise to the fetus, implying that betaine has a role as a methyl donor specifically in this lineage (1.Lee M.B. Kooistra M. Zhang B. Slow S. Fortier A.L. Garrow T.A. Lever M. Trasler J.M. Baltz J.M. Betaine homocysteine methyltransferase is active in the mouse blastocyst and promotes inner cell mass development.J. Biol. Chem. 2012; 287: 33094-33103Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Suppression of BHMT activity in cultured embryos impairs their ICM development and increases fetal resorptions (1.Lee M.B. Kooistra M. Zhang B. Slow S. Fortier A.L. Garrow T.A. Lever M. Trasler J.M. Baltz J.M. Betaine homocysteine methyltransferase is active in the mouse blastocyst and promotes inner cell mass development.J. Biol. Chem. 2012; 287: 33094-33103Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 2.Zhang B. Denomme M.M. White C.R. Leung K.Y. Lee M.B. Greene N.D. Mann M.R. Trasler J.M. Baltz J.M. Both the folate cycle and betaine-homocysteine methyltransferase contribute methyl groups for DNA methylation in mouse blastocysts.FASEB J. 2015; 29: 1069-1079Crossref PubMed Scopus (32) Google Scholar). This is probably related, at least in part, to a requirement for methyl groups for establishing embryonic DNA methylation patterns following the erasure of gamete-specific DNA methylation earlier in preimplantation embryogenesis (19.Denomme M.M. Mann M.R.W. Genomic imprints as a model for the analysis of epigenetic stability during assisted reproductive technologies.Reproduction. 2012; 144: 393-409Crossref PubMed Scopus (99) Google Scholar). Suppressing BHMT in conjunction with the parallel methyl-generating folate cycle prevented the normally observed increase in nuclear 5-methylcytosine detected in the ICM, whereas BHMT alone, even with the folate cycle inhibited, was sufficient to support the 5-methylcytosine increase (2.Zhang B. Denomme M.M. White C.R. Leung K.Y. Lee M.B. Greene N.D. Mann M.R. Trasler J.M. Baltz J.M. Both the folate cycle and betaine-homocysteine methyltransferase contribute methyl groups for DNA methylation in mouse blastocysts.FASEB J. 2015; 29: 1069-1079Crossref PubMed Scopus (32) Google Scholar). Thus, betaine plays important roles in the preimplantation embryo. Although the betaine present in preimplantation embryos could potentially be supplied by SIT1-mediated transport during the 1- and 2-cell stages (3.Anas M.K. Lee M.B. Zhou C. Hammer M.A. Slow S. Karmouch J. Liu X.J. Bröer S. Lever M. Baltz J.M. SIT1 is a betaine/proline transporter that is activated in mouse eggs after fertilization and functions until the 2-cell stage.Development. 2008; 135: 4123-4130Crossref PubMed Scopus (40) Google Scholar), we recently found that endogenous betaine is already present at equivalently high levels in unfertilized mouse eggs (20.Corbett H.E. Dubé C.D. Slow S. Lever M. Trasler J.M. Baltz J.M. Uptake of betaine into mouse cumulus-oocyte complexes via the SLC7A6 isoform of y+L transporter.Biol. Reprod. 2014; 90: 81Crossref PubMed Scopus (17) Google Scholar). Because any betaine in eggs had been accumulated before SIT1 is activated after fertilization (3.Anas M.K. Lee M.B. Zhou C. Hammer M.A. Slow S. Karmouch J. Liu X.J. Bröer S. Lever M. Baltz J.M. SIT1 is a betaine/proline transporter that is activated in mouse eggs after fertilization and functions until the 2-cell stage.Development. 2008; 135: 4123-4130Crossref PubMed Scopus (40) Google Scholar), a mechanism other than SIT1 must be responsible. Fully grown oocytes in ovarian follicles are arrested at the germinal vesicle (GV) stage in prophase I of meiosis. When ovulation is triggered, GV oocytes are released from arrest and progress through first meiotic metaphase (MI), become uncoupled from granulosa cells, and are re-arrested in second meiotic metaphase (MII) as mature eggs awaiting fertilization (21.Eppig J.J. The relationship between cumulus cell-oocyte coupling, oocyte meiotic maturation, and cumulus expansion.Dev. Biol. 1982; 89: 268-272Crossref PubMed Scopus (217) Google Scholar, 22.Salustri A. Siracusa G. Metabolic coupling, cumulus expansion and meiotic resumption in mouse cumuli oophori cultured in vitro in the presence of FSH or dcAMP, or stimulated in vivo by hCG.J. Reprod. Fertil. 1983; 68: 335-341Crossref PubMed Scopus (71) Google Scholar, 23.Gilula N.B. Epstein M.L. Beers W.H. Cell-to-cell communication and ovulation: a study of the cumulus-oocyte complex.J. Cell Biol. 1978; 78: 58-75Crossref PubMed Scopus (401) Google Scholar), a process that is collectively termed meiotic maturation. Although these nuclear changes have been extensively studied, much less is known about other physiological processes during oocyte meiotic maturation that are important for producing a mature MII egg. As we show in the present study, GV oocytes and early to mid-MI oocytes contain lower endogenous levels of betaine in contrast to the high levels in MII eggs, demonstrating that betaine accumulation must occur during meiotic maturation. Although we recently showed that granulosa cells can take up betaine via the y+LAT2 transporter (SLC7A6 protein) and transfer some to the enclosed GV oocyte via gap junctions (20.Corbett H.E. Dubé C.D. Slow S. Lever M. Trasler J.M. Baltz J.M. Uptake of betaine into mouse cumulus-oocyte complexes via the SLC7A6 isoform of y+L transporter.Biol. Reprod. 2014; 90: 81Crossref PubMed Scopus (17) Google Scholar), we show here that this is not a likely source of the increased concentration of betaine in MII eggs. Another possible source of betaine in mature eggs is through its de novo synthesis. The only known mechanism for betaine synthesis in mammalian cells uses choline as the substrate in a two-step reaction where the first and rate-limiting step is catalyzed by choline dehydrogenase (CHDH; EC 1.1.99.1) (24.Haubrich D.R. Gerber N.H. Choline dehydrogenase: assay, properties and inhibitors.Biochem. Pharmacol. 1981; 30: 2993-3000Crossref PubMed Scopus (71) Google Scholar, 25.Johnson A.R. 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Betaine aldehyde produced by CHDH is then oxidized to betaine by an aldehyde dehydrogenase (ALDH; EC 1.2.1.8) identified as ALDH9A1, which accepts a broad range of aminoaldehyde substrates (27.Chern M.K. Pietruszko R. Human aldehyde dehydrogenase E3 isozyme is a betaine aldehyde dehydrogenase.Biochem. Biophys. Res. Commun. 1995; 213: 561-568Crossref PubMed Scopus (68) Google Scholar, 28.Pietruszko R. Chern M. Betaine aldehyde dehydrogenase from rat liver mitochondrial matrix.Chem. Biol. Interact. 2001; 130: 193-199Crossref PubMed Scopus (17) Google Scholar, 29.Riveros-Rosas H. González-Segura L. Julián-Sánchez A. Díaz-Sánchez A.G. Muñoz-Clares R.A. Structural determinants of substrate specificity in aldehyde dehydrogenases.Chem. Biol. Interact. 2013; 202: 51-61Crossref PubMed Scopus (39) Google Scholar). CHDH is localized to mitochondria, where betaine aldehyde synthesis occurs. To investigate the mechanism underlying the production of the high levels of betaine that are present in MII mouse eggs, which are important during early embryonic development, we have tested the hypotheses that CHDH is present and active in oocytes during meiotic maturation, that betaine is synthesized autonomously by oocytes, and that CHDH is required for betaine accumulation. Substantial betaine is present in preimplantation embryos and ovulated MII eggs of mice (1.Lee M.B. Kooistra M. Zhang B. Slow S. Fortier A.L. Garrow T.A. Lever M. Trasler J.M. Baltz J.M. Betaine homocysteine methyltransferase is active in the mouse blastocyst and promotes inner cell mass development.J. Biol. Chem. 2012; 287: 33094-33103Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 20.Corbett H.E. Dubé C.D. Slow S. Lever M. Trasler J.M. Baltz J.M. Uptake of betaine into mouse cumulus-oocyte complexes via the SLC7A6 isoform of y+L transporter.Biol. Reprod. 2014; 90: 81Crossref PubMed Scopus (17) Google Scholar), but whether betaine is present in oocytes during meiotic maturation was unknown. We measured endogenous betaine by LC-MS/MS in oocytes obtained from CF1 female mice before and during in vivo meiotic maturation by collecting them at the stages and times indicated (Fig. 1). Only lower levels of betaine were detectable in preantral follicles containing growing oocytes from postnatal day 11 (P11) ovaries or in fully grown GV oocytes from adult antral follicles. Cumulus–oocyte complexes (COCs) isolated from antral follicles did not contain more betaine than GV oocytes alone, implying that there is no significant reservoir of betaine in cumulus cells. Betaine levels had not increased in MI oocytes at 3 h after ovulation was induced with human chorionic gonadotropin (hCG), and only a small increase (that did not reach significance) was seen in MI oocytes at 6 h. However, significantly higher levels of betaine were present in MII eggs and 1-cell embryos. Thus, betaine is accumulated during meiotic maturation in vivo. Although we previously showed that there was no detectable betaine transport into GV oocytes, MII eggs, or MI oocytes at 4 h after induction of ovulation (3.Anas M.K. Lee M.B. Zhou C. Hammer M.A. Slow S. Karmouch J. Liu X.J. Bröer S. Lever M. Baltz J.M. SIT1 is a betaine/proline transporter that is activated in mouse eggs after fertilization and functions until the 2-cell stage.Development. 2008; 135: 4123-4130Crossref PubMed Scopus (40) Google Scholar), it was possible that betaine transport could be transiently activated later in MI, which ends 10–12 h after ovulation is triggered in mice (30.Edwards R.G. Gates A.H. Timing of the stages of the maturation divisions, ovulation, fertilization and the first cleavage of eggs of adult mice treated with gonadotrophins.J. Endocrinol. 1959; 18: 292-304Crossref PubMed Scopus (153) Google Scholar). Therefore, we measured the transport of [3H]betaine in GV oocytes; in MI oocytes collected during in vivo meiotic maturation at 3, 6, 9, and 12 h after ovulation was induced with hCG; and in MII eggs collected at 16 h post-hCG. No saturable betaine transport was detected in oocytes during meiotic maturation (Fig. 2). This implies that betaine transport by oocytes during meiotic maturation did not account for the high levels of betaine in MII eggs. Because betaine was accumulated in oocytes during meiotic maturation despite the apparent absence of betaine transport, we next examined whether betaine is synthesized in oocytes during meiotic maturation, which is assumed to require CHDH. Chdh mRNA was present at each oocyte stage tested (Fig. 3A), including growing oocytes, GV oocytes, and 1-cell embryos (fertilized eggs). The identity of representative Chdh cDNA bands was confirmed by sequencing (not shown). Q-RT-PCR (Fig. 3A) showed the highest numbers of Chdh transcripts in P11 oocytes, which are ∼70% of the diameter of fully grown oocytes (31.Erdogan S. FitzHarris G. Tartia A.P. Baltz J.M. Mechanisms regulating intracellular pH are activated during growth of the mouse oocyte coincident with acquisition of meiotic competence.Dev. Biol. 2005; 286: 352-360Crossref PubMed Scopus (34) Google Scholar), and in fully grown oocytes from P21 ovaries. Fully grown GV oocytes from adult females also had a high level of transcripts, which persisted in the 1-cell embryo (fertilized egg) but not in subsequent preimplantation embryo stages. COCs contained an amount of Chdh transcript essentially equal to that in GV oocytes, implying that the transcript is restricted to the oocyte rather than cumulus cells. The expected expression patterns of control housekeeping genes H2afz and Ppia (32.Mamo S. Gal A.B. Bodo S. Dinnyes A. Quantitative evaluation and selection of reference genes in mouse oocytes and embryos cultured in vivo and in vitro.BMC Dev. Biol. 2007; 7: 14Crossref PubMed Scopus (170) Google Scholar) were confirmed in these samples (Fig. 3B). CHDH protein was detected by Western blot analysis as a single band near the predicted size of 66 kDa (UniProtKB Q8BJ64) in GV oocytes and MII eggs as well as in all stages of preimplantation embryo analyzed (Fig. 3C). The band detected in oocytes and embryos was at the same position as the single band in Chdh+/+ mouse kidney lysate, which was absent from Chdh−/− kidney lysate (Fig. 3C). We further confirmed the identity of the band detected in oocytes as CHDH using GV oocytes obtained from Chdh+/+, Chdh+/−, and Chdh−/− ovaries (Fig. 3D). To determine whether CHDH was active in oocytes, we adapted and validated a CHDH enzyme activity assay (24.Haubrich D.R. Gerber N.H. Choline dehydrogenase: assay, properties and inhibitors.Biochem. Pharmacol. 1981; 30: 2993-3000Crossref PubMed Scopus (71) Google Scholar, 33.Grossman E.B. Hebert S.C. Renal inner medullary choline dehydrogenase activity: characterization and modulation.Am. J. Physiol. 1989; 256: F107-F112Crossref PubMed Google Scholar) for use with small samples. The modified assay exhibited increasing activity with increasing kidney inner medulla protein until saturation (Fig. 4A). The CHDH inhibitor 3,3-dimethylbutanol completely blocked activity above background, where background was determined as the counts/min obtained when reaction mix to which no kidney lysate had been added was processed. After subtracting paired 3,3-dimethylbutanol controls, the assay was linear up to ∼0.15 mg of total kidney protein (Fig. 4B). Using a saturating amount of kidney protein (0.9 mg), CHDH activity was detected in kidney lysate but not above the background level in negative controls consisting of the same amount of boiled kidney lysate, lung lysate (which has <1% of the activity of kidney) (24.Haubrich D.R. Gerber N.H. Choline dehydrogenase: assay, properties and inhibitors.Biochem. Pharmacol. 1981; 30: 2993-3000Crossref PubMed Scopus (71) Google Scholar), or BSA instead of tissue lysate (Fig. 4C). We then assessed whether CHDH activity was present in oocyte and embryo lysates (100 oocytes or embryos pooled). CHDH activity began to increase in oocytes as meiotic maturation progressed in vivo with maximal activity present in MI and MII oocytes from 3–16 h after meiotic maturation was induced with hCG (Fig. 5A). CHDH activity then decreased after fertilization with only low levels of CHDH activity remaining in 1-cell embryos through blastocysts. The period of maximal CHDH activity coincided with the period during which endogenous betaine was accumulated (Fig. 5B). A similar activation was evident with GV oocytes removed from the ovary and allowed to undergo spontaneous meiotic maturation in vitro (Fig. 5C). Intact COCs exhibited the same lack of CHDH activity as denuded GV oocytes (109 ± 178 cpm for COCs versus 67 ± 69 for GVs, mean ± S.E., n = 3, p = 0.83 by t test), indicating that cumulus cells do not possess CHDH activity and thus are not likely to be a site of betaine synthesis from choline. To confirm that the measured activity in lysate from maturing oocytes was indeed due to CHDH, we used MI oocytes obtained 9 h after meiotic maturation had been triggered in vivo with hCG. As predicted, activity was inhibited by either 3