Acidosis defense mechanisms in the preimplantation stages of embryos in BALB/c strain mice.

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.

Tramadol Reverses the Effects of Neuropathic Pain on Oocyte Maturation and Copulation Ratio in Mice.

OBJECTIVE: Neuropathic pain (NP) is an inescapable stressor that significantly affects both the nervous and endocrine system functions. In this study, we investigated the effect of NP on female reproductive function using the number of oocytes as an index as well as the copulation rates of female mice, with and without males. We also examined whether NP symptoms stopped after injecting tramadol, an opioid analgesic. MATERIALS AND METHODS: The partial sciatic nerve was tightly ligated to produce neuropathy, and allodynia was assessed using the cold-plate test. A superovulation protocol was applied to control, sham, neuropathy, and neuropathy+tramadol groups. Each group was divided into two subgroups according to two housing conditions: female alone and female with a male. After inducing superovulation, oocytes/zygotes were isolated from the ampulla of female mice. Total number of oocytes, oocyte maturation, and copulation rates were determined. RESULTS: The results showed that allodynia, which is a prominent NP symptom, was detected in all neuropathic mice, but tramadol (50 mg/kg, i.p.) stopped these symptoms. The results also showed that NP decreased oocyte maturation and copulation rates of mice, and tramadol reversed all these effects. CONCLUSION: In conclusion, we suggest that NP affects reproductive performance by altering the regulation of neuroendocrine mechanisms. Prospective studies that determine the levels of cortisol, fertility hormone, cytokine, and other potential endogenous substances in NP animals are needed to clarify the mechanisms.

Evaluation of intracellular pH regulation and alkalosis defense mechanisms in preimplantation embryos.

Intracellular pH (pHi) regulation is an important homeostatic function of cells. There are three major pHi-regulatory mechanisms: HCO3(-)/Cl(-) exchanger (anion exchanger [AE]), which alleviates alkalosis, and the Na(+)/H(+) and Na(+),HCO3(-)/Cl(-) exchangers, both of which alleviate acidosis. We hypothesized that there would be developmental changes in pHi-regulatory activity in preimplantation embryos as conditions in the oviduct are alkaline but acidic in the uterus. This study focused on the AE mechanism in pronuclear (PN) zygotes, two-cell (2-c), four-cell (4-c), morula, and blastocyst stage embryos from Balb/c mice. Microspectrofluorometry was used to monitor changes in pHi in embryos subjected to Cl(-)-free media in presence or absence of an AE inhibitor, DIDS, and in embryos recovering from NH4Cl-induced alkalosis. Real-time polymerase chain reaction was used to identify AE isoforms. The pHi changes were greatest in PN zygotes (0.086 ± 007 pHU/min) but fell as embryos developed to the 2-c, 4-c, morula, and blastocyst stages (0.063 ± 006; 0.035 ± 007; 0.024 ± 004, and 0.014 ± 004 pHU/min, respectively). DIDS significantly reduced the rise in pHi caused by Cl(-) removal in all embryos; the finding pointed out that this pHi changes are due to AE activity. But DIDS only inhibited the recovery responses of PN zygote, 2-c and 4-c embryos but not morula or blastocyst stage embryos. In bicarbonate-containing medium, all embryos recovered from induced alkalosis but only the morula and blastocyst stages could fully compensate from ammonium induced-alkalosis in bicarbonate-free medium. The finding showed that commonly used ammonium pulse method to investigate AE function against alkalosis is not suitable for morula and blastocyst embryonic stages. All embryos expressed SLC4A2 and SLC4A4 coding for AE-2 and AE-4, but none expressed either AE-1 or AE-3. The gradual change in the response to alkalosis in preimplantation embryos may be adaptations to their normal in vivo environment, where the early embryos are located in the alkaline oviduct, whereas the morula and blastocyst move into the acidic uterus.