Human follicular fluid and mesenchymal stem cell conditioned medium improves in vitro development of vitrified-warmed mouse oocytes.

BACKGROUND: In vitro maturation (IVM) and oocyte cryopreservation are therapeutic options in assisted reproductive technology which is used to preserve fertility in patients with different causes of infertility. OBJECTIVE: To analyze in vitro development of vitrified-warmed oocytes in the presence of human follicular fluid (FF) and bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) as a rescue strategy in fertility preservation. MATERIALS AND METHODS: BMSC-CM and FF media were used as two natural media. Not only osteogenic and adipogenic differentiation but also flow cytometry was carried out to confirm the nature of mesenchymal stem cells. A total of 327 vitrified-warmed oocytes were randomly assigned to three groups with different maturation media. After 24 h the maturation rate was evaluated. In vitro fertilization and also embryo development were also assessed. RESULTS: Oocytes matured in the BMSC-CM and FF groups showed a significant increase compared to the control group (76.6+/-2.9, 53.2±1.0 , and 40.8+/-6.1, respectively) (P % 0.05). Embryo cleavage rates in the BMSC-CM were dramatically higher than FF and control groups (85.6+/-2.2, 70.5+/-2.2, and 60.7+/-1.5, respectively). Blastocyst formation rates in the BMSC-CM group were statically different compared to FF and control groups (73.6+/-1.0, 58.5+/-1.0, and 45.8+/-4.2, respectively). CONCLUSION: BMSC-CM and FF media not only improve the maturation rate of vitrified warmed oocytes but also significantly increase embryo cleavage and blastocyst rates. DOI: 10.54680/fr23210110512.

In vitro maturation medium supplementation: utilization of repaglinide, L-carnitine, and mesenchymal stem cell-conditioned medium to improve developmental competence of oocytes derived from endometriosis mouse models.

Endometriosis (EMS) is one of the most prevalent causes for female infertility. Herein, we investigated the effect of the repaglinide (RG), L-carnitine (LC), and bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) supplementation during in vitro maturation (IVM) on the quality, maturation, and fertilization rates, as well as embryonic quality and development of oocytes derived from normal and EMS mouse model. Immature oocytes were collected from two groups of normal and EMS-induced female NMRI mice at 6-8 weeks of age. Oocytes were cultured in IVM medium unsupplemented (control group), or supplemented with 1 M RG, 0.3 and 0.6 mg/mL LC, and 25 and 50% BMSC-CM. After 24 h of oocyte incubation, IVM rate and antioxidant status were assessed. Subsequently, the rates of fertilization, cleavage, blastulation, and embryonic development were assessed. Our results demonstrated that supplementation of IVM medium with LC and BMSC-CM, especially 50% BMSC-CM, significantly enhanced IVM and fertilization rates, and markedly improved blastocyst development and total blastocyst cell numbers in EMS-induced mice compared to the control group (53.28±0.24 vs 18.09±0.10%). Additionally, LC and BMSC-CM were able to significantly modulate EMS-induced nitro-oxidative stress by boosting total antioxidant capacity (TAC) and mitigating nitric oxide (NO) levels. Collectively, LC and BMSC-CM supplementation improved oocyte quality and IVM rates, pre-implantation developmental competence of oocytes after in vitro fertilization, and enhanced total blastocyst cell numbers probably by attenuating nitro-oxidative stress and accelerating nuclear maturation of oocytes. These outcomes may provide novel approaches to refining the IVM conditions that can advance the efficiency of assisted reproductive technologies in infertile couples.

In vitro maturation medium supplementation: utilization of repaglinide, L-carnitine, and mesenchymal stem cell-conditioned medium to improve developmental competence of oocytes derived from endometriosis mouse models

Endometriosis (EMS) is one of the most prevalent causes for female infertility. Herein, we investigated the effect of the repaglinide (RG), L-carnitine (LC), and bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) supplementation during in vitro maturation (IVM) on the quality, maturation, and fertilization rates, as well as embryonic quality and development of oocytes derived from normal and EMS mouse model. Immature oocytes were collected from two groups of normal and EMS-induced female NMRI mice at 6-8 weeks of age. Oocytes were cultured in IVM medium unsupplemented (control group), or supplemented with 1 M RG, 0.3 and 0.6 mg/mL LC, and 25 and 50% BMSC-CM. After 24 h of oocyte incubation, IVM rate and antioxidant status were assessed. Subsequently, the rates of fertilization, cleavage, blastulation, and embryonic development were assessed. Our results demonstrated that supplementation of IVM medium with LC and BMSC-CM, especially 50% BMSC-CM, significantly enhanced IVM and fertilization rates, and markedly improved blastocyst development and total blastocyst cell numbers in EMS-induced mice compared to the control group (53.28±0.24 vs 18.09±0.10%). Additionally, LC and BMSC-CM were able to significantly modulate EMS-induced nitro-oxidative stress by boosting total antioxidant capacity (TAC) and mitigating nitric oxide (NO) levels. Collectively, LC and BMSC-CM supplementation improved oocyte quality and IVM rates, pre-implantation developmental competence of oocytes after in vitro fertilization, and enhanced total blastocyst cell numbers probably by attenuating nitro-oxidative stress and accelerating nuclear maturation of oocytes. These outcomes may provide novel approaches to refining the IVM conditions that can advance the efficiency of assisted reproductive technologies in infertile couples.

Mesenchymal stem cell condition medium enhanced cell viability in morphine-treated cells.

Bone marrow mesenchymal stem cells (BM-MSC) have recently been predicted to have a major therapeutic potential due to secretion of soluble factors and the release of cytokines and growth factors, which could mediate the cellular communication to induce cell differentiation/maturity. The aim of the present study was to determine the effect of mBM condition medium on morphine-induced cell death in PC12, U87, AGS and MCF-7 cell lines. The condition media were harvested as mBM soup (mBM soup 24 and mBM soup 48h, respectively). To investigate the effect of mBM soup on cell lines, morphological changes were studied with an inverted microscope, the viability of cells was determined with trypan blue staining and MTT assay, the type of cell death was determined using Hoescht / PI staining, and NO secretion analysis. Viability assay showed that mBM soup (24 and 48 h) in time-dependent manner increased cell viability (pndings suggest that mBM soup can enhance the proliferation and growth of cell lines and can suppress cell death induced by morphine (Fig. 8, Ref. 59). Text in PDF www.elis.sk. Keywords: morphine, BM-MSC soup, cell viability, cell death, NO.

Low concentrations of morphine enhanced the neuroglia-like differentiation.

AIM: The present study was undertaken to evaluate the effects of different concentrations of morphine in chronic manner on neuroglial differentiation in PC12 cells. METHODS: PC12 cells were cultured in RPMI 1640 culture medium including 0.02 % bovine serum albumin together with different concentrations of morphine for 12 days. Cytotoxicity was performed by lactate dehydrogenase assay. Cell death was performed by PI/Hoechst staining assay. Neuroglial differentiation was performed by Nestin, Tuj-1, MAP-2, S-100 and GFAP Immunocytochemistry assay. RESULTS: Data showed that morphine either at low or high concentration activated opioid  receptors, which  resulted in a decrease of cytotoxicity and cell death and induction of Nestin, Tuj-1, MAP-2, Neurofilament-M (NF-M), GFAP and S-100 protein expression as compared in treated cells  with the control (untreated cells) (p<005). CONCLUSION: It can be concluded that low concentrations of morphine in chronic manner stimulate the neuroglial-like differentiation by activating protein expression and survival-promoting signaling in PC12 cells with opioid receptor-dependent mechanism (Fig. 8, Ref. 36). Text in PDF www.elis.sk.

Low concentrations of methamphetamine can suppress inflammation in trophoblast JEG-3 cell line induced by nicotine.

AIM: Nicotine at high concentrations induces apoptosis in trophoblastic cells through induction of cell cytotoxicity and Reactive Oxygen Species (ROS). Methamphetamine in low dose has pharmaceutical properties. It seems that this components in low dose can protect the trophoblastic cells from nicotine-induced cell death. METHOD: Trophoblastic (JEG-3) cells grown in DMEM culture medium. MTT assay test detected the cell viability and Lactate Dehydrogenase test measured the cells cytotoxicity. Griess reaction was used for NO production analysis. Cell migration traced by wounding technique. Human Cytokine Array Focused 13-plex was also used for analysis of IL-1α, IL-1ß, IL-6, INFγ, and TNFα pre-inflammatory cytokines. RESULTS: Methamphetamine, in very low dose (pM), increased the cell viability and NO production, and decreased cell cytotoxicity, IL-1α, IL-1ß, IL-6, INFγ, and TNFα pre-inflammatory cytokines of JEG-3 cell which were exposed to high dose of nicotine, respectively. Cell migration was enhanced by low dose of methamphetamine in JEG-3 cells. CONCLUSION: Methamphetamine in very low dose suppressed the JEG-3 cell death induced by high dose of nicotine (Fig. 5, Ref. 48) Keywords: methamphetamine, nicotine, cell death, NO.

Effects of low-dose morphine suppress methamphetamine-induced cell death by inhibiting the ROS generation and caspase-3 activity.

OBJECTIVE: Methamphetamine in low doses can increase vigilance and power and at high doses has destructive effects that cause toxicity and death of various cell lines and affect the central nervous system. Morphine has also protective properties, which were observed in low concentrations, for nerve cells and also seem to have the ability to reduce cell death in neural cell lines. MATERIALS AND METHODS: In this study, we used PC12 and U87 cell lines, which grew in DMEM culture media. Assays used in this study are listed below: MTT test for cell viability detection, LDH test for cytotoxicity measurement, caspase activity colorimetric assay kit (Bio-techne) for caspase 3 activity diagnosis, Rhodamine 123 for Detection of mitochondrial membrane potential. TUNNEL test for DNA fragmentation, fura-2 for Measurement of (Ca2+) ic and (Ca2+) m. fluorescence microscope for measurement of antioxidant enzyme activities. RESULTS: morphine increased cell viability and the rhodamine-123 absorbance. It reduced cell cytotoxicity, caspase 3 activity, ic (et) m Ca2+ concentration, (.OH) generation, and DNA fragmentation in all concentrations of 1 pM t0 100 nM (p < 0.05) by optimal concentration of 1 pM. CONCLUSION: morphine as a pain mediator can reduce the methamphetamine-induced cell death, may be due to its anti-inflammatory properties (Fig. 7, Ref. 52).

Low concentration of morphine protects against cell death, oxidative stress and calcium accumulation by nicotine in PC12 cells.

OBJECTIVE: Nicotine causes cell death in many cell lines. Morphine at low concentrations has protective effects against cell death. We investigated the effects of low concentration of morphine on nicotine-induced cell death in PC12 cells. MATERIALS AND METHODS: PC12 are cells that grow in DMEM culture medium. Cell viability was detected by MTT test and cells cytotoxicity was measured by LDH test. The activity of caspase-3 was diagnosed by the caspase activity colorimetric assay kit, and detection of mitochondrial membrane potential was confirmed by rhodamine 123 and TUNEL test was performed for DNA fragmentation detection. The fura-2 AM and also rhod 2-AM was used for measurement of intracellular calcium (Ca2+) ic and mitochondrial calcium (Ca2+) m and finally, measurement of antioxidant enzyme activities was assessed. RESULTS: The low concentration of morphine increased cell viability and suppressed cell cytotoxicity, cell death and the formation of mitochondrial membrane potential compared to nicotine treated cells.  It also reduced the intracellular calcium (Ca2+) ic and mitochondrial calcium (Ca2+)m concentration, respectively. CONCLUSION: Morphine as a pain reducer drug, in low concentrations, can protect PC12 cells from nicotine-induced cell death (Fig. 7, Ref. 59).

Phosphoinositid signal pathway mediate neurite outgrowth in PC12 cells by staurosporine.

OBJECTIVES: In previous studies, we showed that staurosporine uses intracellular calcium ions to affect cell death in PC12 cells. The bulk release of intracellular excessive Ca(2+) from intracellular sources into cytosol contributes to neuronal apoptotic events, which in turn results in neuronal cell death. However, the mechanisms of Ca(2+)-induced neuronal cell death or neurite elongation is still unclear. Therefore, we investigated the relation between phosphoinositid signal pathway, intracellular calcium, and reactive oxygen species on one hand, with staurosporine-induced neurite outgrowth in PC12 cells on the other. RESULTS: The inhibition of phospholipase C or IP3 receptor antagonist or phosphoinositid signal transduction antagonist produced cell death and suppressed neurite outgrowth by staurosporine in PC12 cells. The inhibition of these enzymes and pathway results in an increase in intracellular Ca(2+) although subsequent hydroxyl radical (•OH) production began after inhibitors exposure. •OH production was significantly attenuated in inhibitor supplemented medium treatment, and it was dependent on the intracellular Ca(2+) concentration. These data indicate that staurosporine activates phosphoinositid signal pathway while endoplasmic Ca(2+), and subsequent •OH production are critical events in staurosporine-induced neurite outgrowth in PC12 cells. CONCLUSION: We conclude that the fact that staurosporine mobilizes Ca2+, probably via activating the subcellular compartment, is responsible for staurosporine-induced (Ca2+]i increase during neurite outgrowth in PC12 cells (Fig. 7, Ref. 30).