Bartosz Kempisty

Association of 677C>T polymorphism of methylenetetrahydrofolate reductase (MTHFR) gene with bipolar disorder and schizophrenia

Methylenetetrahydrofolate reductase (MTHFR) gene polymorphism 677C>T has been shown to be a risk factor for psychiatric disorders. We investigated the genotype and allelic frequencies of MTHFR 677C>T polymorphism in the group of patients with bipolar disorder type I (BDI) (n=200) and schizophrenia (n=200), and in the control group (n=300). Odds ratio (OR) for patients with BD and schizophrenia with 677T allele was 1.988 ((95% CI=1.370-2.883); P=0.0003 (P=0.0006 after Bonferroni correction)) and 1.796 ((95% CI=1.237-2.609); P=0.0020 (P=0.0040 after Bonferroni correction)), respectively. The stratification of patients based on gender revealed significant association of 677T allele with male patients with BDI and schizophrenia (OR=2.393; 95% CI=1.429-4.006; P=0.0008 and OR=2.036; 95% CI=1.207-3.433; P=0.0073, respectively). This finding indicates possible association of BD and schizophrenia with the 1p36.3 MTHFR locus.

Analysis of selected transcript levels in porcine spermatozoa, oocytes, zygotes and two-cell stage embryos

It has been suggested that spermatozoa can deliver mRNAs to the oocyte during fertilisation. Using reverse transcription and real-time quantitative polymerase chain reaction analysis (RQ-PCR), we evaluated the presence of clusterin (CLU), protamine 2 (PRM2), calmegin (CLGN), cAMP-response element modulator protein (CREM), methyltransferase 1 (DNMT1), linker histone 1 (H1), protamine 1 (PRM1), TATA box-binding protein associated factor 1 (TAF1) and TATA box-binding protein (TBP) in porcine mature oocytes, zygotes and two-cell stage embryos. Spermatozoa isolated from semen samples of boars contained all transcripts investigated, whereas oocytes contained only CREM, H1, TAF1, and TBP mRNAs. The zygote and two-cell stage embryos contained CLU, CREM, H1, PRM1, PRM2, TAF1 and TBP transcripts. Our observations suggest that porcine spermatozoa may delivery CLU, PRM1 and PRM2 mRNAs to the oocyte, which may contribute to zygotic and early embryonic development.

The morphology of porcine oocytes is associated with zona pellucida glycoprotein transcript contents

We hypothesized that oocyte morphology may be associated with the accumulation of specific mRNAs encoding proteins responsible for the gamete fertilization ability. Therefore, the aim of the study was to evaluate the transcript levels of porcine zona pellucida (pZP1, pZP2, pZP3 and pZP4) glycoproteins in oocytes classified by a four-grade morphological scale (I-IV) accounting for either a homogeneous cytoplasm and a complete cumulus oophorus (grade I) or a heterogenous cytoplasm and decreased number of cumulus layers in the other grades (II, III and IV). We observed a significant increase of all investigated pZP glycoprotein mRNAs in grade I oocytes as compared to other grades (p<0.05). Our observations suggest that porcine oocyte morphology is associated with pZP transcript contents and may be related to an increased fertilization ability of higher quality oocytes.

MTHFD 1958G>A and MTR 2756A>G polymorphisms are associated with bipolar disorder and schizophrenia.

Background The etiology of bipolar disorder (BD) and schizophrenia is very complex. Polymorphic variants of genes encoding enzymes of the monoaminergic may be involved in development of BD and schizophrenia. Therefore, we examined the prevalence of 1958G>A polymorphism of MTHFD1 gene, encoding trifunctional folate enzyme 5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase and 10-formyltetrahydrofolate synthetase (MTHFD1), and 2756A>G variant of methionine synthase (MTR) gene in patients with BD (n=200), schizophrenia (n=200) and in controls (n=300). Objective We investigated the genotypic and allelic frequencies of MTHFD1 1958G>A (R653Q) and MTR 2756A>G (D919G) gene polymorphisms in a group of bipolar (n=200) and schizophrenic patients (n=200), as well as in controls (n=300). Methods The distributon of genotypes in all groups was tested for deviation from Hardy-Weinberg equilibrium (HWE). The Pearsons chi-square (χ2) test and Fishers exact test were applied to assess differences in the genotypic and allelic (respectively) distribution between groups of patients and controls. Main results We found that MTHFD1 1958AA or 1958AG genotypes constitute risk factors for development of bipolar disorder type I (BDI) or schizophrenia with odds ratios (OR)=1.743 (95% CI=1.211–2.508; P=0.0027; P (corr)=0.0054) and 2.667 (95% CI=1.845–3.854; P=0.0001; P (corr)=0.0002), respectively. In the same groups, the MTR 2756GG or 2756AG genotypes also constitute significant risk factors in occurrence of BDI and schizophrenia with OR=1.621 (95% CI=1.130–2.326; P=0.0086; P (corr)=0.0172) and 1.556 (95% CI=1.085–2.232; P=0.0160; P (corr)=0.032), respectively. Gender classification of patients indicated significant association only of MTHFD1 1958A allele with BDI and schizophrenia in the male patients OR=1.838 (95% CI=1.114–3.031; P=0.0166; P (corr)=0.0332) and OR=3.964 (95% CI=2.358–6.663; P=0.0001 P (corr)=0.0002), respectively. Conclusion Since MTHFD and MTR genes are located in 14q24 and 1q43 loci, our findings support the significance of chromosomes 14q and 1q in etiopathogenesis of bipolar disorder and schizophrenia.

Quantitative assessment of transition proteins 1, 2 spermatid-specific linker histone H1-like protein transcripts in spermatozoa from normozoospermic and asthenozoospermic men.

Spermatid-specific linker histone H1-like protein (HILS1), transition proteins 1 and 2 (TNP1 and TNP2), and protamines 1 and 2 (PRM1 and PRM2) contribute to considerable dense packing of spermatid chromatin during spermiogenesis. We evaluated the HILS1, TNP1, and TNP2 transcript levels in spermatozoa isolated from normozoospermic and asthenozoospermic men. Human ejaculates from normozoospermic (n = 70) and asthenozoospermic (n = 100) donors were purified by centrifugation through a discontinuous Percoll density gradient. RNA was isolated from spermatozoa according to the Chomczyński and Sacchi method, treated with DNase I and reverse-transcribed into cDNA. Quantitative analysis of HILS1, TNP1, and TNP2 transcripts was performed by real-time quantitative (RQ-PCR) SYBR green I analysis. We found significantly lower levels of HILS1, TNP1, and TNP2 transcripts in spermatozoa from asthenozoospermic men compared to normozoospermic men. Our observations suggest that a reduction in HILS1, TNP1, and TNP2 transcripts may be associated with asthenozoospermia.

Analysis of the specificity and selectivity of anti-EpCAM antibodies in breast cancer cell lines

The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that is expressed in most normal human epithelia and overexpressed in most carcinomas. Molecule is responsible for cell-to-cell adhesion and additionally participates in signaling, cell migration, proliferation and differentiation. Therefore, EpCAM has been the target of immunotherapy in clinical trials of several solid tumors. It appears to play an important role as a target for circulating tumor cells (CTCs) capturing. The aim of this study was to investigate and compare the specificity and selectivity of different anti-EpCAM antibodies in order to their usefulness for CTCs capturing. All experiments were performed in six different types of breast cancer cell lines (MCF-7, SkBr-3, T47D, CAMA-1, MDAMB-231, BT-20) and with use of three different anti-EpCAM antibodies (EBA-1, AUA-1, 9C4). The experiments revealed that investigated antibodies differ significantly regarding the specificity of EpCAM antigen binding. The most significant role in the circulating tumor cells capturing can play the EBA-1 and 9C4 anti-EpCAM antibodies as they revealed the most specific signal. The strength and specificity of reaction was dependent not only on the type of antibody but also on the type of breast cancer cell line. On the basis of the present outcomes it can be assumed that the best solution for obtaining the most specific results could be the use of mixture of different anti-EpCAM antibodies simultaneously. In conclusion, the proper selection of anti-EpCAM antibody is crucial especially when this antigen is considered as a marker for detection of circulating tumor cells.

Methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms and age of onset in schizophrenia: A combined analysis of independent samples†

Methylenetetrahydrofolate reductase (MTHFR) is involved in the one‐carbon cycle, which is of importance for nucleotide synthesis and methylation of DNA, membranes, proteins and lipids. The MTHFR gene includes two common polymorphisms (rs1801133 or C677T; rs1801131 or A1298C) which both alter enzyme activity. The T‐allele of the C677T polymorphism has recently been associated with earlier age at onset of schizophrenia. In the present study we examined the association between the MTHFR C677T and A1298C polymorphisms and age at onset of schizophrenia in twelve samples consisting of 3,213 unrelated schizophrenia patients, including the original Scandinavian sample. There was no consistent relationship between MTHFR C677T, A1298C or combined 677T/1298C carriers and age of onset in schizophrenia when the results of each study were combined using meta‐analysis. The present results suggest that the investigated MTHFR polymorphisms do not influence age of onset in schizophrenia.

Zona pellucida glycoprotein 3 (pZP3) and integrin β2 (ITGB2) mRNA and protein expression in porcine oocytes after single and double exposure to brilliant cresyl blue test

Brilliant cresyl blues (BCB) staining test is a useful tool in assessing the competence of cumulus-oocyte-complexes (COCs) in several mammalian species. It is mostly used to select gametes after they are recovered from the ovary or before and after IVM to isolate those oocytes that reach developmental competency. However, there is evidence that double exposure to BCB test may lead to impaired fertilization or even have a toxic effect on cells. The aim of the present study was to investigate the expression pattern of sperm-egg interaction molecules in oocytes after single and double exposure to BCB test. Follicles were dissected from porcine ovaries after slaughter and aspirated COCs were cultured in standard porcine IVM culture medium (TCM 199) for 44 h. The BCB test was applied to COCs before and after IVM. In developmentally competent oocytes, assessed by determining the activity of glucose-6-phosphate dehydrogenase (G6PDH; BCB test), real-time quantitative PCR reaction methods, western blot and confocal microscopy analysis were applied to determine the transcript levels of porcine zona pellucida glycoprotein 3 (pZP3), and integrin beta 2 (ITGB2), as well as the levels of pZP3 and ITGB2 proteins. In the control group, assessment of the expression of the investigated genes was performed before and after IVM without BCB test. We observed a significantly higher level of pZP3 mRNA in oocytes after single exposure to BCB test compared to control before and after IVM (P < 0.001), and to double staining (P < 0.05). The level of ITGB2 mRNA was also increased in gametes after single exposure to BCB test as compared to control before and after IVM (P < 0.001, P < 0.01, respectively), and double staining (P < 0.05). Western blot analysis demonstrated a higher level of pZP3 protein in oocytes after single staining with BCB as compared to control both before and after IVM (P < 0.001, P < 0.05, respectively) and double staining (P < 0.05). Confocal microscopic observations have revealed the same pattern of increased level of pZP3 and ITGB2 expression after single exposure to BCB test. In both cases we detected specific cytoplasmic localization of both proteins. The ITGB2 protein has zona pellucida and membrane localization in control oocytes before IVM. After IVM and after single exposure to BCB, ITGB2 was also strongly detected in the cytoplasm. In both cases, after double exposure to BCB both proteins were detected only partially in the cytoplasm. Our results suggest that (i) single exposure to BCB increased the expression of sperm-oocyte interaction genes, (ii) double exposure to BCB leads to only partial expression of pZP3 and ITGB2 in oocyte cytoplasm, (iii) the BCB staining test itself may be a cause of specific pZP3 translocation from the zona pellucida to the cytoplasm, and that (iv) in vitro maturation of oocytes may increase ITGB2 expression and translocation from the zona pellucida to the cytoplasm.

Assessment of zona pellucida glycoprotein and integrin transcript contents in porcine oocytes

Using reverse transcription and real-time quantitative PCR analysis we evaluated the transcript levels of integrins (alphaL, alphaM, beta1, and beta6), CD9 and CD18 antigens as well as zona pellucida glycoproteins (pZP1, pZP2, pZP3 and pZP3alpha) in oocytes isolated from puberal gilts (n=20) and multiparous sows (n=20). We found significantly (p<0.05) higher transcript contents of alphaL, alphaM, beta1, and beta integrins, CD9 antigen, and pZP2 and pZP3 in puberal gilt oocytes compared to multiparous sow oocytes. Our results suggest that a decrease in the level of oocyte transcripts encoding essential proteins involved in oocyte fertilization may be associated with increased porcine female age.

The expression of genes encoding zona pellucida glycoproteins in canine cumulus-oocyte complexes cultured in vitro in media supplemented with progesterone and estradiol

The role of progesterone (P4) and estradiol-17beta (E2) on the efficiency of canine oocyte maturation in vitro is recognized, but little is known about the influence of both steroids on the expression of zona pellucida (ZP) glycoproteins. It has been shown that E2 and P4 used in the IVC significantly influenced canine oocytes meiotic competence, although the effect is specifically related to the combination of hormones used in the experiment. Because both of these steroids may stimulate or inhibit maturation competence of oocytes in a dose-dependent manner, there is a high possibility that they also influence the fertilization ability of canine oocytes. Our study was aimed to analyze whether genes, encoding ZP glycoproteins, are regulated by P4 or E2. Canine cumulus oocyte complexes (COCs) were recovered from anestrous mongrel bitches after ovariohysterectomy and cultured in serum-free tissue culture medium 199. The expression pattern of ZP glycoproteins 2 and 3 (ZP2 and ZP3) mRNAs, using quantitative real-time polymerase chain reaction (RQ-PCR), and of ZP3 and ZP4 proteins, using Western blot analyses, was examined in oocytes after the supplementation of the culture medium with (1) 0.5 μg/mL, 1.0 μg/mL, and 2.0 μg/mL of P4 (experiment 1), or with (2) 2.0 μg/mL E2, and with (3) a combination of E2 (2.0 μg/mL) and P4 (0.5, 1.0, or 2.0 μg/mL, respectively; experiment 2). The analysis revealed an inhibited expression of ZP2 mRNA in oocytes after in vitro maturation (IVM) with different P4 supplementations as compared with oocytes before IVM. The expression of ZP3 mRNA was stimulated (P < 0.01) by the supplementation of 1.0 μg/mL P4. The expression of both ZP3 and ZP4 proteins was also stimulated after the treatment with 1.0 μg/mL P4. On the other hand, the level of ZP2 mRNA was inhibited (P < 0.01) after the supplementation with E2 or with combinations of E2 and P4 as compared with control oocytes. The expression of ZP3 mRNA was significantly higher after the supplementation with E2 and 0.5 μg/mL P4. Similarly, ZP3 and ZP4 proteins were highly expressed (P < 0.01) after such hormone supplementation. The results clearly show that in vitro, P4 regulates the expression of ZP glycoproteins in a dose-dependent manner. We demonstrated that E2 used alone and in combination with P4 upregulates the expression of ZP3 mRNA as well as ZP3 and ZP4 protein in canine oocytes. ZP2 mRNA is downregulated by E2 alone and in combination with E2 and P4. Furthermore, ZP glycoproteins expression is regulated by E2 alone or in combination with P4, and such synergistic or adverse effect is P4 concentration-dependent.