Cryopreservation of Unfertilized Mouse Oocytes: The Effect of Replacing Sodium with Choline in the Freezing Medium

Although embryo cryopreservation has become commonplace in many species, effective methods are not available for routine freezing of unfertilized eggs. Cryopreservation-induced damage may be caused by the high concentration of sodium ions in conventional freezing media. This study investigates the effect of a newly developed low-sodium choline-based medium (CJ2) on the ability of unfertilized, metaphase II mouse eggs to survive cryopreservation and develop to the blastocyst stagein vitro.Specifically, the effects of cooling to subzero temperatures, thawing rate, LN₂plunge temperature, and equilibration with a low-sodium medium prior to freezing are examined. In contrast to cooling to 23, 0, or −7.0°C in a sodium-based freezing medium (ETFM), cooling in CJ2 had no significant negative effect on oocyte survival or development. Oocytes frozen in CJ2 survived plunging into LN₂from −10, −20, or −33°C at significantly higher rates than oocytes frozen in ETFM. With the protocol used (1.5 M PrOH, 0.1 M sucrose, −0.3 C/min, plunging at −33°C) rapid thawing by direct submersion in 30°C water was more detrimental to oocyte survival than holding in air for 30 or 120 s prior to transfer to water. Equilibration of unfertilized oocytes with a low-sodium medium prior to cryopreservation in CJ2 significantly increased survival and blastocyst development. These results demonstrate that the high concentration of sodium in conventional freezing media is detrimental to oocyte cryopreservation and show that choline is a promising replacement. Reducing the sodium content of the freezing medium to a very low level or eliminating sodium altogether may allow oocytes and other cells to be frozen more effectively.     

Transforming growth factor-α expands progenitor cells of the basal forebrain,but does not promote cholinergic differentiation

Transforming growth factor-α (TGF-α), a member of the epidermal growth factor (EGF) family, binds to the EGF-receptor (EGF-R). The early expression and widespread distribution of TGF-α and EGF-R in the developing central nervous system (CNS) suggest that TGF-α may play a role in the developing CNS. To study possible effects of TGF-α on cholinergic differentiation in the basal forebrain, we cultured septal nuclei with adjacent basal forebrain from embryonic rat brain in the presence and absence of TGF-α. At the highest dose of TGF-α used (100 ng/mL), activity of choline acetyltransferase (ChAT; EC 2.3.1.6) and the number of cholinergic neurons doubled. However, because protein levels tripled, specific ChAT activity actually declined. To determine the mechanism accounting for the increase in ChAT, we labeled dividing precursors present in the cultures with a replication-deficient retrovirus expressing β-galactosidase in the presence and absence of TGF-α. By staining the cultures for both LacZ and ChAT, we determined that the precursor population expanded in size (individually labeled clones contained more cells), but the percentage of cholinergic neurons present in the clones was unchanged. Therefore, while TGF-α expands the precursor pool, it does not promote cholinergic differentiation. Interleukin-9, included to prompt neuronal differentiation, did not by itself increase ChAT activity, nor did it enhance the action of TGF-α. This was true even when basic fibroblast growth factor was included. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 405–412, 1998     

Methyl-group donors cannot prevent apoptotic death of rat hepatocytes induced by choline-deficiency

Choline-deficiency causes liver cells to die by apoptosis, and it has not been clear whether the effects of choline-deficiency are mediated by methyl-deficiency or by lack of choline moieties. SV40 immortalized CWSV-1 hepatocytes were cultivated in media that were choline-sufficient, choline-deficient, choline-deficient with methyl-donors (betaine or methionine), or choline-deficient with extra folate/vitamin B₁₂. Choline-deficient CWSV-1 hepatocytes were not methyl-deficient as they had increased intracellular S-adenosylmethionine concentrations (132% of control; P < 0.01). Despite increased phosphatidylcholine synthesis via sequential methylation of phosphatidylethanolamine, choline-deficient hepatocytes had significantly decreased (P < 0.01) intracellular concentrations of choline (20% of control), phosphocholine (6% of control), glycerophosphocholine (15% of control), and phosphatidylcholine (55% of control). Methyl-supplementation in choline-deficiency enhanced intracellular methyl-group availability, but did not correct choline-deficiency induced abnormalities in either choline metabolite or phospholipid content in hepatocytes. Methyl-supplemented, choline-deficient cells died by apoptosis. In a rat study, 2 weeks of a choline-deficient diet supplemented with betaine did not prevent the occurrence of fatty liver and the increased DNA strand breakage induced by choline-deficiency. Though dietary supplementation with betaine restored hepatic betaine concentration and increased hepatic S-adenosylmethionine/S-adenosylhomocysteine ratio, it did not correct depleted choline (15% of control), phosphocholine (6% control), or phosphatidylcholine (48% of control) concentrations in deficient livers. These data show that decreased intracellular choline and/or choline metabolite concentrations, and not methyl deficiency, are associated with apoptotic death of hepatocytes. J. Cell. Biochem, 64:196–208. © 1997 Wiley-Liss, Inc.     

Activation of GSK‐3 disrupts cholinergic homoeostasis in nucleus basalis of Meynert and frontal cortex of rats

The cholinergic impairment is an early marker in Alzheimer's disease (AD), while the mechanisms are not fully understood. We investigated here the effects of glycogen synthase kinse‐3 (GSK‐3) activation on the cholinergic homoeostasis in nucleus basalis of Meynert (NBM) and frontal cortex, the cholinergic enriched regions. We activated GSK‐3 by lateral ventricular infusion of wortmannin (WT) and GF‐109203X (GFX), the inhibitors of phosphoinositol‐3 kinase (PI3‐K) and protein kinase C (PKC), respectively, and significantly decreased the acetylcholine (ACh) level via inhibiting choline acetyl transferase (ChAT) rather than regulating acetylcholinesterase (AChE). Neuronal axonal transport was disrupted and ChAT accumulation occurred in NBM and frontal cortex accompanied with hyperphosphorylation of tau and neurofilaments. Moreover, ChAT expression decreased in NBM attributing to cleavage of nuclear factor‐κB/p100 into p52 for translocation into nucleus to lower ChAT mRNA level. The cholinergic dysfunction could be mimicked by overexpression of GSK‐3 and rescued by simultaneous administration of LiCl or SB216763, inhibitors of GSK‐3. Our data reveal the molecular mechanism that may underlie the cholinergic impairments in AD patients.     

辽宁碱蓬SlCMO基因盐胁迫表达分析及盐诱导启动子鉴定

胆碱单加氧酶（choline monooxygenase, CMO）是合成甜菜碱的关键酶,甜菜碱在植物抵抗渗透胁迫中起着重要的作用。本研究室前期克隆了盐生植物辽宁碱蓬CMO（Suaeda liaotungensis CMO）基因及启动子。本研究对SlCMO基因在盐胁迫下的表达及盐诱导启动子进行分析。qRT-PCR分析SlCMO基因在辽宁碱蓬不同器官及盐胁迫下的表达,结果表明,SlCMO基因在根、茎、叶中均有表达,其中茎、叶中的表达量较高,SlCMO基因在根、茎、叶中的表达均受盐胁迫诱导。5′端缺失分析SlCMO启动子的盐诱导区段,结果表明,pC5（-267~+128 bp）是SlCMO启动子的盐诱导功能区段,推测pC5调控SlCMO 基因的盐诱导表达。本研究为SlCMO 基因表达调控研究奠定基础,也为植物抗盐基因工程提供可用的启动子。     

Understanding the muscular dystrophy caused by deletion of choline kinase beta in mice

Choline kinase in mice is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneously occurring genomic deletion in murine Chkb results in neonatal bone deformity and hindlimb muscular dystrophy. We have investigated the mechanism by which a lack of choline kinase β, encoded by Chkb, causes hindlimb muscular dystrophy. The biosynthesis of phosphatidylcholine (PC) is impaired in the hindlimbs of Chkb^−/− mice, with an accumulation of choline and decreased amount of phosphocholine. The activity of CTP:phosphocholine cytidylyltransferase is also decreased in the hindlimb muscle of mutant mice. Concomitantly, the activities of PC phospholipase C and phospholipase A₂ are increased. The mitochondria in Chkb^−/− mice are abnormally large and exhibit decreased inner membrane potential. Despite the muscular dystrophy in Chkb^−/− mice, we observed increased expression of insulin like growth factor 1 and proliferating cell nuclear antigen. However, regeneration of hindlimb muscles of Chkb^−/− mice was impaired when challenged with cardiotoxin. Injection of CDP-choline increased PC content of hindlimb muscle and decreased creatine kinase activity in plasma of Chkb^−/− mice. We conclude that the hindlimb muscular dystrophy in Chkb^−/− mice is due to attenuated PC biosynthesis and enhanced catabolism of PC.     

The superlattice model of lateral organization of membranes and its implications on membrane lipid homeostasis

Most biological membranes are extremely complex structures consisting of hundreds of different lipid and protein molecules. According to the famous fluid-mosaic model lipids and many proteins are free to diffuse very rapidly in the plane of the membrane. While such fast diffusion implies that different membrane lipids would be laterally randomly distributed, accumulating evidence indicates that in model and natural membranes the lipid components tend to adopt regular (superlattice-like) distributions. The superlattice model, put forward based on such evidence, is intriguing because it predicts that 1) there is a limited number of allowed compositions representing local minima in membrane free energy and 2) those energy minima could provide set-points for enzymes regulating membrane lipid compositions. Furthermore, the existence of a discrete number of allowed compositions could help to maintain organelle identity in the face of rapid inter-organelle membrane traffic.     

Choline acetyltransferase activity at different ages in brain of Ts65Dn mice, an animal model for Down's syndrome and related neurodegenerative diseases

Ts65Dn mice, trisomic for a portion of chromosome 16 segmentally homologous to human chromosome 21, are an animal model for Down's syndrome and related neurodegenerative diseases, such as dementia of the Alzheimer type. In these mice, cognitive deficits and alterations in number of basal forebrain cholinergic neurons have been described. We have measured in Ts65Dn mice the catalytic activity of the cholinergic marker, choline acetyltransferase (ChAT), as well as the activity of the acetylcholine-degrading enzyme acetylcholinesterase (AChE), in the hippocampus and in cortical targets of basal forebrain cholinergic neurons. In mice aged 10 months, ChAT activity was significantly higher in Ts65Dn mice, compared to 2N animals, in the hippocampus, olfactory bulb, olfactory cortex, pre-frontal cortex, but not in other neocortical regions. At 19 months of age, on the other hand, no differences in ChAT activity were found. Thus, alterations of ChAT activity in these forebrain areas seem to recapitulate those recently described in patients scored as cases of mild cognitive impairment or mild Alzheimer's disease. Other neurochemical markers putatively associated with the disease progression, such as those implicating astrocytic hyperactivity and overproduction of amyloid precursor protein family, were preferentially found altered in some brain regions at the oldest age examined (19 months).