细胞内pH(pHi)的调节是细胞的重要稳态功能。有三种主要的pHi调节机制:HCO3-/Cl-交换剂(AE),减轻碱中毒,和Na+/H+交换剂(NHE)和Na+,HCO3-/Cl-交换器(NDBCE),两者都能抵消酸中毒。NHE活动,在卵母细胞的生发囊泡阶段很高,在减数分裂成熟过程中被抑制,而当卵母细胞达到受精卵的原核(PN)阶段时,这种抑制作用被消除。另一方面,我们以前发现NDBCE在减数分裂成熟过程中对酸中毒进行互补调节。此外,我们发现AE活性,这是一种针对碱中毒的防御机制,在胚胎发育的着床前逐渐减少。考虑到NHE活性在减数分裂成熟过程中受到抑制,而AE活性在胚胎发育阶段逐渐降低,我们调查了NHE和NDBCE活动,两者都对抗酸中毒,从PN受精卵到胚胎囊胚阶段的功能变化,并在Balb/c菌株的小鼠中在分子水平上鉴定了这些pH调节蛋白。PN受精卵,两单元(2-c),四单元(4-c),从5-8周龄,通过使用经典的超排卵程序,性成熟的雌性Balb/c小鼠。通过使用显微荧光光谱法对同时装载有pH敏感荧光团的受精卵和胚胎记录pHi,2\',7'-双(2-羧乙基)-5(6)-羧基荧光素(BCECF)。从无碳酸氢盐和含碳酸氢盐的培养基中诱导酸中毒的恢复曲线确定NHE和NDBCE的活性,分别。特异性抑制剂,如cariporide(1μM),S3226(1和10μM),EIPA(1、5和25μM),和阿米洛利(1mM)用于功能鉴定NHE亚型,和非特异性抑制剂4,4'-二异氰酸根合二苯乙烯-2,2'二磺酸,使用二钠盐(DIDS)来确认NDBCE活性。还使用实时PCR通过分子生物学鉴定了pHi调节蛋白的同种型。我们发现NHE活性在所有胚胎阶段都很高,各阶段之间差异不显著。功能和分子研究结果表明,NHE1和5的同工型存在于胚泡中,而NHE1、3和4的同种型在早期胚胎阶段起作用。尽管在所有胚胎阶段都检测到NDBCE活性对诱导酸中毒恢复的贡献,仅在PN合子和2-c胚胎中显著。分子分析证实了这一发现,在所有胚胎阶段检测到编码NDBCE的SLC4A8的表达。总之,NHE是针对酸中毒的积极且重要的防御机制,并且由Balb/c小鼠品系的所有阶段中的至少两种蛋白质同工型编码。NDBCE在所有胚胎阶段都具有支持功能,尤其是在PN合子和2-c胚胎中。植入前阶段胚胎具有响应于其代谢终产物(酸负荷增加)和子宫内酸性环境而抵抗酸中毒的有效机制。
Regulation of intracellular pH (pHi) is an important homeostatic function of cells. There are three major pHi regulatory mechanisms: the HCO3-/Cl- exchanger (AE), which alleviates alkalosis, and the Na+/H+ exchanger (NHE) and Na+,HCO3-/Cl- exchanger (NDBCE), both of which counteract acidosis. NHE activity, which is high at the germinal vesicle stage of oocyte, is inhibited during meiotic maturation, while this inhibition is abolished when the oocyte reaches the pronuclear (PN) stage of the zygote. On the other hand, we have previously found that NDBCE performs complementary regulation against acidosis during meiotic maturation. Additionally, we found that AE activity, which is a defense mechanism against alkalosis, gradually decreases during preimplantation period of embryonic development. Considering that NHE activity is inhibited during meiotic maturation and AE activity gradually decreases during embryonic development stages, we investigated whether NHE and NDBCE activities, both of which act against acidosis, functionally change from the PN zygote to the blastocyst stage of the embryo and identified these pH-regulating proteins at the molecular level in mice of the Balb/c strain. PN zygotes, two-cell (2-c), four-cell (4-c), morula and blastocyst stage embryos were obtained from 5-8-week-old, sexually mature female Balb/c mice by using the classical superovulation procedure. pHi was recorded by using the microspectrofluorometric technique on zygotes and embryos simultaneously loaded with the pH-sensitive fluorophore, 2\',7\'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The activities of NHE and NDBCE were determined from the recovery curve of induced-acidosis in bicarbonate-free and bicarbonate-containing media, respectively. Specific inhibitors such as cariporide (1 μM), S3226 (1 and 10 μM), EIPA (1, 5, and 25 μM), and amiloride (1 mM) were used to functionally identify NHE isoforms, and the nonspecific inhibitor 4,4\'-diisocyanatostilbene-2,2\' disulphonic acid, disodium salt (DIDS) was used to confirm NDBCE activity. The isoforms of the pHi-regulatory proteins were also identified by molecular biology using real-time PCR. We found that NHE activity was high at all embryonic stages, and differences between stages were not significant. Functional and molecular findings indicated that isoforms of NHE 1 and 5 are present in the blastocyst, whereas isoforms of NHE 1, 3, and 4 are functional at earlier embryonic stages. Although the contribution of NDBCE activity to recovery from induced-acidosis was detected at all embryonic stages, it was significant only in the PN zygote and the 2-c embryo. This finding was confirmed by molecular analysis, which detected the expression of SLC4A8 encoding NDBCE at all embryonic stages. In conclusion, NHE is an active and important defense mechanism against acidosis and is encoded by at least two protein isoforms in all stages of the Balb/c strain of mice. NDBCE has a supportive function in all embryonic stages, especially in the PN zygote and the 2-c embryo. Preimplantation stage embryos have effective mechanisms to defend against acidosis in response to their metabolic end products (increased acid load) and the acidic environment in utero.