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.

Inactivating KISS1 mutation and hypogonadotropic hypogonadism.

Gonadotropin-releasing hormone (GnRH) is the central regulator of gonadotropins, which stimulate gonadal function. Hypothalamic neurons that produce kisspeptin and neurokinin B stimulate GnRH release. Inactivating mutations in the genes encoding the human kisspeptin receptor (KISS1R, formerly called GPR54), neurokinin B (TAC3), and the neurokinin B receptor (TACR3) result in pubertal failure. However, human kisspeptin loss-of-function mutations have not been described, and contradictory findings have been reported in Kiss1-knockout mice. We describe an inactivating mutation in KISS1 in a large consanguineous family that results in failure of pubertal progression, indicating that functional kisspeptin is important for puberty and reproduction in humans. (Funded by the Scientific and Technological Research Council of Turkey [TÜBITAK] and others.).

Changes in the activity of defense mechanisms against induced acidosis during meiotic maturation in mouse oocytes.

Previous work has indicated that although activity of the HCO3⁻/Cl⁻exchanger (AE), which regulates intracellular alkalosis, is high in the germinal vesicle (GV) stage, the oocyte is inhibited as it progresses through meiotic maturation. In this study, we aimed to investigate the defense mechanisms against acidosis during the meiotic maturation stages. Intracellular pH (pH(i)) was recorded using a microspectrofluorometric technique, and Na(+)/H(+) (NHE) and Na(+)-dependent HCO3⁻/Cl⁻ exchanger (NDCBE) activity were determined by measuring the recovery rate from induced acidosis. Additionally, SLC9A1 (for NHE) and SLC4A8 (for NDCBE) gene transcription levels were determined by real-time PCR. The recovery rate of first meiotic prophase (GV) oocytes was high, but it decreased during the meiotic metaphase II (MII) stage in HCO3⁻-free medium; it became high again at the pronuclear zygote (PN) stage. Recovery rate was significantly inhibited by 5-(N-ethyl-N-isopropyl) amiloride and cariporide or in the absence of extracellular Na(+), implicating NHE, specifically NHE1 activity. Moreover, the level of SLC9A1 transcription correlated with the observed changes in NHE activity. The changes in NHE activity during meiotic maturation displayed a similar pattern to that of AE. The recovery rate from acidosis was significantly higher in MII stage oocytes and PN zygotes in HCO3⁻-containing medium; however, the increase was significantly inhibited in Na(+)-free medium or 4,4'-diisocyanatostilbene-2,2'-disulfonic acid. Furthermore, changes in the transcription of SLC4A8 during meiotic maturation were concordant with the level of exchanger activity. These results indicate that NDCBE activity is present in mouse oocytes and zygotes, and that this activity exhibits a different pattern than that of AE and NHE during meiotic maturation.

Changes of HCO3-/Cl- exchanger activity during meiotic maturation in Balb/c strain mouse oocytes and zygotes.

Intracellular pH-regulatory mechanisms are acquired by growing mouse oocytes with meiotic competence, and these mechanisms become fully active when the oocytes develop to the germinal vesicle (GV) stage as shown in CF1 and Balb/c strains mice. On the other hand, there is some evidence showing that intracellular pH-regulatory mechanisms are inhibited at the stages of Metaphase I (MI) and II (MII) oocytes in the CF1 strain mouse and hamster. Since it has been shown that the intracellular pH regulatory mechanism can be functionally different among mouse strains (e.g., CF1, Balb/c), the aim of this study was to investigate the activity of HCO3-/Cl- exchanger (anion exchanger, AE), which protects cells against alkalosis during the meiotic maturation process, in the GV oocyte up to the pronuclear (PN) zygote derived from the Balb/c strain mouse. Intracellular pH (pHi) was recorded using a microspectrofluorometric technique during meiotic maturation stages. KSOM-based solutions were used as culture and recording solutions. AE activity was determined using a Cl- removal assay and was reported as the change in pHi per minute. AE activity was high in GV stage oocytes but was significantly inhibited at the MI and MII stages. AE activity was higher in the PN zygote stage. This activity was significantly inhibited in all oocyte and zygote stages by 4,4'-Diisocyanatostilbene-2,2'-disulfonic acid disodium salt. After alkalosis induction, the pHi of MI and MII stage oocytes did not completely recover; however, almost complete recovery occurred in the GV stage oocytes and PN zygotes. These results suggest that AE is inhibited during the meiotic maturation process in the Balb/c strain mouse.

Effects of specific dosages of magnesium and zinc on the teratogenicity of cadmium, nickel, and cobalt in Xenopus embryos, as assessed by the FETAX test.

The objective of this study was to determine if exposure to divalent cations, Cd(2+), Ni(2+), and Co(2+) would lead to malformations in Xenopus laevis embryos, and whether addition of Mg(2+) and Zn(2+); separately and in combination, would reduce their toxicity and teratogenicity on the embryos of Xenopus laevis as assessed by 96-h FETAX tests. Results indicate that exposure to Cd(2+), Ni(2+) or Co(2+) lead to an increase in toxicity and teratogenicity in embryos, whereas Mg(2+), Zn(2+), or a combination of them reduced the toxic and teratogenic effects of these divalent cations. Modulation of Cd(2+), Ni(2+) or Co(2+) toxicity and teratogenicity by Mg(2+) and Zn(2+), varied with the metal. Zn(2+) was observed to be a better suppressor of Co(2+) toxicity and teratogenicity than Mg(2+). In contrast, Ni(2+), and Cd(2+) teratogenicity was reduced more prominently by Mg(2+). On the other hand, combination of Mg(2+) and Zn(2+) showed potentialization effect on all divalent cation toxicity and teratogenicity. We concluded that Mg(2+) and Zn(2+) reduced the toxicity and teratogenicity of Cd(2+), Ni(2+), Co(2+).

Effects of a multimodal exercise program for people with ankylosing spondylitis.

BACKGROUND AND PURPOSE: Few randomized controlled studies have examined the effects of exercise in patients with ankylosing spondylitis (AS). This study investigated the effects of a 12-week, multimodal exercise program in patients with AS. SUBJECTS: A convenience sample of 30 patients with AS (18 male, 12 female), with a mean age of 34.9 years (SD=6.28), participated in the study. Twenty-six subjects were classified as having stage I AS and 4 subjects were classified as having stage II AS according to the modified New York Criteria. METHODS: This study was a randomized controlled trial. Subjects were assigned to either a group that received an exercise program or to a control group. The exercise program consisted of 50 minutes of multimodal exercise, including aerobic, stretching, and pulmonary exercises, 3 times a week for 3 months. Subjects in both groups received medical treatment for AS, but the exercise group received the exercise program in addition to the medical treatment. All subjects received a physical examination at baseline and at 12 weeks. The examinations were conducted under the supervision of a physician who specialized in physical medicine and rehabilitation and included the assessment of spinal mobility using 2 methods: clinical measurements (chin-to-chest distance, Modified Schober Flexion Test, occiput-to-wall distance, finger-to-floor distance, and chest expansion) and inclinometer measurements (gross hip flexion, gross lumbar flexion, and gross thoracic flexion). In addition, vital capacity was measured by a physiologist, and physical work capacity was evaluated by a doctorally prepared exercise instructor. RESULTS: The measurements of the exercise group for chest expansion, chin-to-chest distance, Modified Schober Flexion Test, and occiput-to-wall distance were significantly better than those of the control group after the 3-month exercise period. The spinal movements of the exercise group improved significantly at the end of exercise program, but those of the control group showed no significant change. In addition, the results showed that the posttraining value of gross thoracic flexion of the exercise group was significantly higher than that of the control group. Physical work capacity and vital capacity values improved in the exercise group but decreased in the control group. DISCUSSION AND CONCLUSION: In this study, a multimodal exercise program including aerobic, stretching, and pulmonary exercises provided in conjunction with routine medical management yielded greater improvements in spinal mobility, work capacity, and chest expansion.

Mechanisms regulating intracellular pH are activated during growth of the mouse oocyte coincident with acquisition of meiotic competence.

Oocytes grow within ovarian follicles, and only gain the ability to complete meiosis when they are nearly fully grown. We have found that both of the major types of intracellular pH regulatory mechanisms in the mammal-the Na+/H+ and HCO3-/Cl- exchangers-were essentially inactive in mouse oocytes over most of the course of their growth. However, as oocytes approached full size, Na+/H+ and HCO3-/Cl- exchangers became simultaneously active, and, at the same time, the intracellular pH of isolated oocytes increased sharply by about 0.25 pH unit. This activation of intracellular pH regulatory mechanisms and increase in pH occurred coincident with the acquisition of meiotic competence. The activation of pH regulatory mechanisms during oocyte growth represents a previously unknown milestone in the development of the capacity of the oocyte to function independently upon ovulation.

The effects of aerobic exercise on skeletal muscle metabolism, morphology and in situ endurance in diabetic rats.

The effects of aerobic exercise training on skeletal muscle endurance capacity were examined in diabetic rats in situ. Moderate diabetes was induced by iv injection of streptozotocin and an exercise training program on a treadmill was carried out for 8 weeks. The animals randomly assigned to one of the four experimental groups: control-sedentary (CS), control-exercise (CE), diabetic-sedentary (DS) or diabetic-exercise (DE). The changes in the muscle endurance capacity were evaluated through the square wave impulses (supramaximal) of 0.2-ms duration at 1 Hz in the in situ gastrocnemius-soleus muscle complex. Muscle was stimulated continuously until tension development reduced to the half of this maximal value. Time interval between the beginning and the end of stimulation period is defined as contraction duration. Following the training period, blood glucose level reduced significantly in the DE group compared to DS group (p < 0.05). The soles muscle citrate synthase activity was increased significantly in both of the trained groups compared to sedentary animals (p < 0.05). Fatigued muscle lactate values were not significantly different from each other. Ultrastractural abnormality of the skeletal muscle in DS group disappeared with training. Presence of increased lipid droplets, mitochondria clusters and glycogen accumulation was observed in the skeletal muscle of DE group. The contraction duration was longer in the DE group than others (p < 0.001). Fatigue resistance of exercised diabetic animals may be explained by increased intramyocellular lipid droplets, high blood glucose level and muscle citrate synthase activity. Key PointsAerobic training of diabetic animals increased the endurance capacity.Presence of abnormal ultrastructural alterations with diabetes disaapered with regular training.Increased intramyocelluler lipid droplets, high blood glucose level with citrate synthase activity may explain this finding.