Time and Cost-Effective Genome Editing Protocol for Simultaneous Caspase 8 Associated Protein 2 Gene Knock in/out in Chinese Hamster Ovary Cells Using CRISPR-Cas9 System.

Background: CHO cells are preferred for producing biopharmaceuticals, and genome editing technologies offer opportunities to enhance recombinant protein production. Targeting apoptosis-related genes, such as Caspases 8-Associated Protein 2 (CASP8AP2), improves CHO cell viability and productivity. Integrating robust strategies with the CRISPR-Cas9 system enables its application in CHO cell engineering. Objectives: This study was performed to develop a cost-effective protocol using the CRISPR-Cas9 system combined with the HITI strategy for simultaneous CASP8AP2 gene deletion/insertion in CHO cells and to assess its impact on cell viability and protein expression. Materials and Methods: We developed an efficient protocol for CHO cell engineering by combining CRISPR/Cas9 with the HITI strategy. Two distinct sgRNA sequences were designed to target the 3' UTR region of the CASP8AP2 gene using CHOPCHOP software. The gRNAs were cloned into PX459 and PX460-1 vectors and transfected into CHO cells using the cost-effective PEI reagent. A manual selection system was employed to streamline the process of single-cell cloning. MTT assays assessed gene silencing and cell viability at 24, 48, and 72 hours. Flow cytometry evaluated protein expression in CASP8AP2-silenced CHO cells. Results: The study confirmed the robustness of combining CRISPR-Cas9 with the HITI strategy, achieving a high 60% efficiency in generating knockout clones. PEI transfection successfully delivered the constructs to nearly 65% of the clones, with the majority being homozygous. The protocol proved feasible for resource-limited labs, requiring only an inverted fluorescent microscope. CASP8AP2 knockout (CHO-KO) cells exhibited significantly extended cell viability compared to CHO-K1 cells when treated with NaBu, with IC50 values of 7.28 mM and 14.25 mM at 48 hours, respectively (P-value 24 hours ≤ 0.0001, 48 hours ≤ 0.0001, P-value 72 hours = 0.0007). CHO CASP8AP2-silenced cells showed a 1.3-fold increase in JRed expression compared to native cells. Conclusions: CRISPR-Cas9 and HITI strategy was used to efficiently engineer CHO cells for simultaneous CASP8AP2 gene deletion/insertion, which improved cell viability and protein expression.

Gene Expression Patterns of Colorectal Cancer Stem Cells Following Ibuprofen and Hyperthermia Treatment.

Background: Cancer stem cells (CSCs) substantially influence the development of colorectal cancer (CRC), metastasis, relapse, and resistance to therapy. Ibuprofen and hyperthermia can be effective in the treatment of cancer. Herein, we evaluated the effects of hyperthermia and ibuprofen on the isolated-CSCs of CRC. Methods: This experimental study was conducted between Sep 2020 and Jan 2022 at the Department of Pathology, School of Medicine, Shiraz University of Medical Sciences, Iran. A non-adhesive culture system was used to isolate CSCs from HT-29 cells. To confirm the stemness nature of isolated-CSCs, the expression of stemness genes and protein markers was evaluated by quantitative Real-time PCR (qRT-PCR) and flow cytometry assay. The isolated-CSCs were treated with hyperthermia and ibuprofen. The cell viability was determined by MTT assay and trypan blue staining. The expression of stemness, proliferation, Wnt signaling pathway and apoptosis genes was assessed by qRT-PCR. Results: CSCs were isolated within 14 days. The expression of CD-133 marker and OCT3/4, C-MYC, KLF4, and NANOG genes in isolated-CSCs was higher than HT-29 cells (P<0.05). Cell viability of treated-CSCs were considerably reduced (P<0.05). Hperthermia reduced the expression of OCT3/4, NANOG, PCNA, WNT1 and CTNNB1 genes and increased the expression of P53, BAX, and KLF4 genes (P<0.05). Ibuprofen decreased the expression of OCT3/4, BCL2, NANOG, PCNA, WNT1, and CTNNB1 genes and increased the expression of P53, BAX, and KLF4 genes in treated-CSCs (P<0.05). Conclusion: Hyperthermia and ibuprofen treatment demonstrate an inhibitory effect on colorectal CSCs. However, using combination therapy is remaining to be tested.

Crucial role of corticotropin-releasing hormone, corticotropin-releasing hormone -binding protein, mir-200c, and mir-181a in preterm delivery: A case-control study.

Background: Preterm birth before 37th wk of gestation is called premature birth. Corticotropin-releasing hormone (CRH) and CRH-binding protein (BP) act on various maternal and fetal tissues during pregnancy, such as the myometrium, which regulates the transition from the dormant phase of the uterus to the active phase. Studies have shown that mir-200c and mir-181a interact with CRH and CRH-BP. Objective: The present study aimed to investigate the expression of mir-200c, mir-181a, CRH, and CRH-BP in women with a history of preterm birth. Materials and Methods: In this case-control study, the gene expression level of mir-200c, mir-181a, CRH, and CRH-BP in placental tissue samples obtained from 48 women with a history of preterm labor was assessed in the Mojibian hospital of Yazd, Iran, from January to March 2023. Differences between mir-200c, mir-181a CRH, and CRH-BP gene expressions among cases and controls were assessed. Results: The outcomes indicated that the expression of CRH increased with going on to the regular parturition time (p < 0.001). While outcomes indicated, CRH-BP decreased with going on to the regular parturition time (p < 0.001). In addition, the results showed that the expression of mir-181a increased and mir-200c decreased with approaching the normal delivery time (p < 0.001). Conclusion: In conclusion, the expressions of mir-200c, mir-181a, CRH, and CRH-BP were dissimilar in different weeks of gestation. It could be proposed to use mir-200c, mir-181a, CRH, and CRH-BP as biomarkers to weigh the exact delivery time, which could minimize the side effects of preterm labor for the mother and fetus.

Impaired CD4+ Cytotoxic T Lymphocyte Activity in Polyomavirus BK Infected Kidney Transplant Recipients.

The reactivation of polyomavirus BK (BKPyV) contributes to increased morbidity and mortality rates of transplant patients, especially kidney transplant recipients (KTRs). CD4+ T cells are important immune cells active during BKPyV infection in KTRs. This research tried to examine the phenotype of CD4+ T cells in the stage of BKPyV activation in KTRs.The re cipients were separated into 2 groups of BKPyV-active and nonactive KTRs (10 patients in each group) and were compared with 10 healthy control subjects. The viral load was evaluated by Taq-man quantitative real-time PCR. The frequency of different CD4+ T cell subsets was determined by analyzing markers such as CD45RO, CCR7, CD27, CD107a, perforin, and granzyme B using flow cytometry. The gene expression levels of transcription factors, including TBX21, GATA3, STAT3, and STAT6, contributing to CD4+ T cell activation, were also assessed. A significantly higher proportion in CCR7+CD27+CD45RO-CD4+ T cell (naive Tcell) subsets was detected in BKPyV-active KTRs compared to nonactive ones. A significant increase was detected in the frequency of CD107a+, perforin+, and granzyme B+ CD4+ T cells in the BKPyV-active group compared to the nonactive group. In CD4+ T cells of KTRs, the mRNA expression of TBX21  and GATA3 was significantly increased in KTRs without BKPyV reactivation compared to BKPyV-active ones. This investigation focused on the CD4+ T cell as an immunodominant T cell type with potential cytotoxicity. Based on these results, BKPyV may have a direct influence on the repertoire of CD4+ T cell subsets. Particularly, cytotoxic CD4+ T cells need further investigation to be considered as a therapeutic approach for BKPyV infection.

Allergen-specific immunotherapy by recombinant Der P1 allergen-derived peptide-based vaccine in an allergic mouse model.

AIM: Increased IgE levels have made house dust mite allergens one of the most frequent causes of allergies worldwide. Treatment reduces the IgE antibodies and types two cytokines, namely interleukin-4 (IL-4) and IL-13. Although existing treatments significantly reduce IgE or IL-4/IL-13, they are very costly. This study aimed to construct a recombinant protein derived from rDer p1 peptides in the form of an immunotherapy approach and to measure the response of IgE and IgG antibodies. METHODS: The proteins were isolated, purified, and evaluated using the SDS-PAGE and Bradford test and confirmed by using Western blot. To evaluate immunotherapy efficiency, 24 BALB/C mice were sensitized intraperitoneally with house dust mites (HDM) adsorbed to Aluminum hydroxide (Alum) and randomly divided into four groups of six: control sensitized, HDM extract, rDer p1, and DpTTDp vaccine. To immunization, four groups of random mice were each treated with phosphate-buffered saline, 100 µg of rDer p1 protein, DpTTDp, or HDM extract, every 3 days. Direct ELISA determined HDM-specific IgG and IgE subclasses. Data were analyzed in SPSS and Graph pad prism software. Values of p < .05 were considered significant. RESULTS: After immunization of mice, the rDer P1 and recombinant vaccine like HDM extract increased IgG antibody titer and decreased IgE-dependent reactivity in allergic mice to rDer P1. Also, the levels of inflammatory IL-4 and IL-13 cytokines as allergic stimulants decreased. CONCLUSION: The use of present available recombinant proteins is considered a viable, cost-effective, and long-term option for providing effective HDM allergy immunotherapy vaccines without side effects.

Corrigendum to "Protective effects of Ceratonia siliqua extract on protamine gene expression, testicular function, and testicular histology in doxorubicin-treated adult rats: An experimental study" [Int J Reprod BioMed 2020; 18: 667-682].

[This corrects the article DOI: 10.18502/ijrm.v13i8.7507.].

A Comprehensive Overview of Allergen-specific Immunotherapy Types, Recombinant and Natural Extract Allergens in the Diagnosis and Treatment of Allergies.

Allergen-specific immunotherapy (AIT) involves administering allergen extracts. It is used to desensitize allergic patients. Herbal allergen extracts that are optimum in efficacy and fewest in side effects are still challenging to produce. To overcome these limitations, oral immunotherapy, epicutaneous immunotherapy, intralymphatic immunotherapy, and artificial recombinant allergen preparations have been evaluated. Recombinant allergens have become more popular with the development of molecular diagnostics and therapeutics. Besides food and drug allergens, pollen, fungal spores, and other allergens have been studied. Based on related clinical studies, this comprehensive overview will present the latest perspectives on AIT methods and available allergenic products, as well as discuss the challenges and opportunities for treating allergic disorders.

Toxic effects of polyethylene microplastics on transcriptional changes, biochemical response, and oxidative stress in common carp (Cyprinus carpio).

Aquatic ecosystems have become a place for accumulating microplastics (MPs). MPs can directly or indirectly damage organisms. Although studies of the toxicity of MPs, there are insufficient literature reports on the effects of MPs on freshwater aquatic life. Therefore, this study aimed to evaluate the effect of MPs toxicity on Cyprinus carpio. In this study, biochemical parameters, oxidative biomarkers, and gene expression were assayed in fish exposed to 0, 175, 350, 700, and 1400 µg L-1 of MPs for 30 days. MPs were detected in the liver and intestine of fish using FTIR-analysis. Mt1, Ces2, and P450 mRNA expression were enhanced in the hepatocytes of fish exposed to MPs, while Mt2 gene expression was significantly decreased. After exposure to MPs, MDA and carbonyl protein levels were higher than those of the reference group. The antioxidant capacity and glycogen contents in the hepatocytes significantly declined. MPs significantly inhibited glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH), and catalase (CAT) activities. However, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities increased. MPs decreased the total protein, globulin levels, and butyrylcholinesterase (BChE) activity in blood. In contrast, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and creatine phosphokinase (CPK) activities increased in treated-fish with MPs. Glucose, creatinine, cholesterol and triglyceride concentrations in fish exposed to MPs were significantly higher than that of the reference group. Consequently, MPs exposure could disrupt biochemical homeostasis, oxidative stress and alter the expression of genes involved in detoxification.

Ameliorate effects of resveratrol and l-carnitine on the testicular tissue and sex hormones level in busulfan induced azoospermia rats.

Busulfan (Bus), is an alkylating agent widely used in chemotherapy which has been proven to possess toxic side effects on testicles. This study was carried out to compare the probable treatment effects of resveratrol (Res) or/and l-carnitine (Lca), as strong antioxidants, on the testicular tissue as well as on the level of sex hormones in busulfan-induced azoospermic rat models. A total of 78 adult male rats, were divided into six different experimental groups including: 1) Control; 2) Lca + Res; 3) BUS; 4) Bus + Lca; 5) BUS + Res and 6) Bus + Lca + Res. Busulfan was intraperitoneally administered in a single dose (10 mg/kg b.w), while resveratrol (20 mg/kg b.w/day) and l-carnitine (200 mg/kg b.w/day) were orally administered by gavage during 48 consecutive days to the rats. At the end of the experiment in all groups the level of LH, FSH, and testosterone were biochemically analyzed by ELISA and the testicular tissue evaluated histologically using stereological technique. Results showed that Lca or/and Res, increased the body and testis weight, the volume of the testis, interstitial tissue, germinal epithelium, and seminiferous tubule, the number of the different cells of germinal epithelium and the level of testosterone. On the other hand, Lca, Res and their combination decreased the concentration of LH and FSH compared to the group treated with Bus. In conclusion, these results suggested that l-carnitine or/and resveratrol treatment significantly attenuated busulfan -induced changes of the rat reproductive system led to the recovery of both testis and sperm parameters. However, co-administration of L-ca and Res was more effective than their individual treatment. This combination may alleviate the side effects of alkylating drugs, such as busulfan and may be beneficial for spermatogenesis.

Vitamin D and calcium, together and separately, play roles in female reproductive performance.

Vitamin D (VD) deficiency reduces the chances of successful fertilization; however, it remains to be validated whether this effect is dependent or not on calcium. To address this question, we generated several situation using a mouse model in which VD content was either increased or decreased in a normo or hypocalcemia context. After the measurement of serum 25-hydroxyvitamin D2, calcium and phosphorus levels, an analysis was carried out in terms of oocytes maturation as well as reproductive performance. VD overdose, despite the fact that it resulted in an increased number of mature oocytes, reduced developmental competence and offspring survival. VD deficiency (VDD), on the contrary, reduced the number and percentage of mature oocytes, blastocyst rate, as well as fertility rate and offspring survival. Hypo-calcemia when VD levels were normal, had a similar effect than VDD. The effects of VDD were reversed by a diet that corrected calcium level. Therefore, both VD overdose (in a context of normal calcium level) VD deficiency as well as hypo-calcemia have an effect on female reproductive function. In conclusion, although closely related, VD and calcium act in part independently of each other in defining the "optimum" for female reproductive performance.

Effect of curcumin, and nano-curcumin on sperm function in varicocele rat model.

Varicocele is one of the most important causes of infertility in men which gradually leads to testicular dysfunction. Testicular heat stress-induced oxidative stress is considered the main cause of pathology in these individuals. In this study, the effects of curcumin and nano-curcumin, as natural antioxidants, were investigated on spermatogenesis and sperm function in varicocele-induced rats. Seventy Wistar rats were randomly divided into seven groups; sham, control, varicocele, varicocele + curcumin 50 mg, varicocele + curcumin 100 mg, varicocele + nano-curcumin 4 mg and varicocele + nano-curcumin 8 mg. After 2 months of antioxidant therapy, all the rats were sacrificed. The results demonstrated that the mean sperm concentration and motility were significantly lower while the mean of abnormal morphology, lipid peroxidation, intracytoplasmic ROS and DNA damage was significantly higher in varicocelised rats compared to control and sham groups (p < .05). Both doses of curcumin and also nano-curcumin were significantly effective in improving the aforementioned parameters except for abnormal sperm morphology, and motility where nano-curcumin (4 mg) was significantly more effective than other groups (p < .05). The results of the current study suggest the application of nano-curcumin is more preferable to curcumin in infertile individuals with varicocele.

Supplementation of Estradiol Into Semen Extender Improved Goat Sperm Cryopreservation.

Estradiol is a steroid hormone excreted from the female gonads, mainly during the pre-estrus. However, the potential effects of estradiol are yet to be explored on sperm parameters through cryopreservation. In this study, we supplemented estradiol, 3 and 5 µM, in the goat semen extender and assessed the sperm parameters after a freeze-thawing process. Sperm motility was assessed using the computer-assisted sperm analysis system. Sperm viability and membrane integrity improved using both 3 and 5 µM concentrations of estradiol. The highest rate of progressive motility was observed in the 3 µM estradiol group. However, a higher concentration of estradiol (5 µM) reduced the progressive motility. Then, we were interested to see if the supportive effect of estradiol on sperm motility is mediated through the intracellular concentration of calcium ionophore. We supplemented the semen extender with 1 and 10 mM ethylenediaminetetraacetic acid (EDTA) and showed that 1 mM has no adverse effect on progressive sperm motility. Then, estradiol (3 µM) was supplemented with or without EDTA (1 mM) into the semen extender. Individual EDTA treatment improved the progressive sperm motility compared to the control group. However, in the presence of estradiol, EDTA treatment reduced the progressive motility compared to the individual estradiol group. This indicated a considerable interaction between estradiol and EDTA for progressive sperm motility. Indeed, EDTA reduced the supportive effects of estradiol on sperm cryopreservation parameters. These results indicated that induction of higher progressive sperm motility in response to estradiol is a calcium-dependent process, as the EDTA did completely abrogate the estradiol-mediated effect.

The Effect of Recombinant Human Follicle-Stimulating Hormone on Sperm Quality, Chromatin Status and Clinical Outcomes of Infertile Oligozoospermic Men Candidate for Intracytoplasmic Sperm Injection: A Randomized Clinical Trial.

BACKGROUND: Follicle-stimulating hormone (FSH) plays a crucial role in spermatogenesis; in this study, we assessed the effect of recombinant human FSH (rhFSH) on sperm parameters, chromatin status and clinical outcomes of infertile oligozoospermic men candidates for intracytoplasmic sperm injection (ICSI). MATERIALS AND METHODS: This interventional randomized clinical trials (IRCT) included 40 infertile oligozoospermic men undergoing ICSI. These individuals were randomized into two groups: 20 men received rhFSH drug for three months and the other 20 men who did not receive rhFSH drug were considered the control group. Before and 3 months after treatment initiation, sperm parameters (using computer-assisted semen analysis) and chromatin status [using chromomycin A3, aniline blue, and sperm chromatin dispersion (SCD) tests] were assessed in these individuals. Furthermore, hormonal profile was assessed using enzyme-linked immunosorbent assay (ELISA). Clinical outcomes of ICSI were also compared between the two groups. RESULTS: The rhFSH treated group showed a significant increase in the level of FSH, luteinizing hormone (LH), testosterone (T) and prolactin (PRL), as well as significant improvements in sperm parameters compared to the control group. Also, after administration of rhFSH, there was asignificant reduction in the percentage of sperm DNA damage, protamine deficiency and chromatin immaturity, while such a reduction in these parameters was not observed in the control group. Moreover, the percentage of embryos with grade Aquality, was significantly higher in the rhFSH group compared to the control group. The pregnancy rate in the rhFSH group was higher than the control group but the difference was insignificant. CONCLUSION: Administration of rhFSH improves sperm quality in infertile oligozoospermic men and results in higher rates of good quality embryos post-ICSI (Registration number: IRCT20170923036334N2).

Protective effects of Ceratonia siliqua extract on protamine gene expression, testicular function, and testicular histology in doxorubicin-treated adult rats: An experimental study.

BACKGROUND: Spermatogenesis is a complex process that takes place under the influence of many different genes. OBJECTIVE: The aim of this study was to investigate the possible effects of Ceratonia siliqua hydroalcoholic extract (CSHAE) on protamine gene expression, testicular function, and testicular histology in doxorubicin-treated rats. MATERIALS AND METHODS: 56 adult male rats with a age range of 2.5 to 3 months (210 ± 10 gr) were divided into seven groups (n = 8/each). A) Control group was left untreated; B) Sham group received 0.3 ml distilled water intraperitoneally, C) Negative control group received 3 mg/kg doxorubicin, intraperitoneally once a week for 28 days; and D) Positive control group received 600 mg/kg of CSHAE orally for 48 days; E, F, G) the experimental groups 1, 2, and 3 received 150, 300, and 600 mg/kg of CSHAE respectively orally, for 48 days, as well as 3 mg/kg doxorubicin once a week for 28 days. Hematoxylin-eosin staining was used in the histological study of testes, and enzyme-linked immunosorbent assay method was used in measuring serum levels of testosterone. Protamine gene expression was determined by real-Time PCR method. RESULTS: The mean body weight, testicular weight, testicular volume, testosterone level (p = 0.022), the count of Leydig, spermatogonia, spermatocyte, and spermatid cells, as well as protamine gene expression (p = 0.008) were significantly increased in the experimental group 2 compared to the negative control group. The regeneration of testicular tissue was observed in the experimental group 2. CONCLUSION: CSHAE has protective effect on doxorubicin-induced testicular injuries.

ROCK Inhibitor During Hypothermic Storage Improves Re-Expansion Rate and Quality of Goat Blastocysts.

Embryos can be produced in nonheat stress conditions and transported by air to designated destinations using hypothermic and chilled storage procedures. Therefore, improvement of efficiency of these processes may have an important impact on the dairy industry. The aim of this study was to evaluate the viability of embryos treated with ROCK inhibitor (Y-27632) during 4 days of hypothermic storage of goat blastocysts. In this study, vitrification was used as a standard model of cryopreservation. Treatment with ROCK inhibitor (Y-27632+) significantly improved the re-expansion rate of blastocyst postwarming compared with treatment with Y-26732-, but it was lower than the re-expansion rate in vitrified/warmed blastocysts. The quality of blastocysts in terms of total cell number (TCN) was significantly higher in the control group than in treatment groups, but it was similar between Y-26732+, Y-27632-, and vitrification groups. The relative expression of BAX, as a proapoptotic marker, was similar between all the groups, whereas the relative expression of BCL2 as an antiapoptotic marker was significantly higher in the Y-26732- group. The rate of TUNEL positive cells was similar between Y-26732+ and Y-27632- groups. Thus, our results reveal that addition of Y-27632 improves the re-expansion rate of hypothermic stored embryos possibly through stabilizing the cytoskeleton structure such as intermediate filaments, which prevents the formation of cell membrane blebbing and cytoplasmic fragmentation.

Dietary flax seed oil and/or Vitamin E improve sperm parameters of cloned goats following freezing-thawing.

Semen cryopreservation is affected by individual differences and use of clones animal from the same source is the main tool to eliminate genetic variation. Among many nutrients that are necessary for fertility, essential fatty acids and antioxidants are vital for production of healthy sperm by improving sperm membrane integrity and protecting sperm from oxidative stress. The goal of the current study was to investigate whether a flax seed oil or/and Vitamin E dietary supplementation could improve semen quality of cloned bucks following semen cryopreservation. Accordingly, eight adult cloned Bakhtiari bucks were divided randomly into four groups. Bucks were offered a base diet of hay and concentrate. The concentrate was enriched with flax seed oil, 30 gr/kg body weight/day (OIL), Vitamin E (VIT), 3 gr/kg body weight/day, or combined flax seed oil and the vitamin E (OIL-VIT). The concentrate with no supplements was considered as control group (CONT). Both flax seed oil and Vitamin E supplements were added to the total diet. The bucks were fed with their corresponding diets for a total of 9 weeks while sperm collection was carried out within 10-14 weeks. Ejaculates were diluted with Andromed® and were frozen in liquid nitrogen. Sperm parameters and reactive oxygen species (ROS) contents were evaluated following freezing/thawing. According to the results of our study, dietary supplementation with flax seed oil, or/and Vitamin E can improve sperm motility, vitality and number of sperm with intact plasma membrane following freezing-thawing. But the degree of improvement in these parameters was significantly higher when Flax seed oil and vitamin E were co-supplemented.

Epigenetic modification with trichostatin A does not correct specific errors of somatic cell nuclear transfer at the transcriptomic level; highlighting the non-random nature of oocyte-mediated reprogramming errors.

BACKGROUND: The limited duration and compromised efficiency of oocyte-mediated reprogramming, which occurs during the early hours following somatic cell nuclear transfer (SCNT), may significantly interfere with epigenetic reprogramming, contributing to the high incidence of ill/fatal transcriptional phenotypes and physiological anomalies occurring later during pre- and post-implantation events. A potent histone deacetylase inhibitor, trichostatin A (TSA), was used to understand the effects of assisted epigenetic modifications on transcriptional profiles of SCNT blastocysts and to identify specific or categories of genes affected. RESULTS: TSA improved the yield and quality of in vitro embryo development compared to control (CTR-NT). Significance analysis of microarray results revealed that of 37,238 targeted gene transcripts represented on the microarray slide, a relatively small number of genes were differentially expressed in CTR-NT (1592 = 4.3 %) and TSA-NT (1907 = 5.1 %) compared to IVF embryos. For both SCNT groups, the majority of downregulated and more than half of upregulated genes were common and as much as 15 % of all deregulated transcripts were located on chromosome X. Correspondence analysis clustered CTR-NT and IVF transcriptomes close together regardless of the embryo production method, whereas TSA changed SCNT transcriptome to a very clearly separated cluster. Ontological classification of deregulated genes using IPA uncovered a variety of functional categories similarly affected in both SCNT groups with a preponderance of genes required for biological processes. Examination of genes involved in different canonical pathways revealed that the WNT and FGF pathways were similarly affected in both SCNT groups. Although TSA markedly changed epigenetic reprogramming of donor cells (DNA-methylation, H3K9 acetylation), reconstituted oocytes (5mC, 5hmC), and blastocysts (DNA-methylation, H3K9 acetylation), these changes did not recapitulate parallel marked changes in chromatin remodeling, and nascent mRNA and OCT4-EGFP expression of TSA-NT vs. CRT-NT embryos. CONCLUSIONS: The results obtained suggest that despite the extensive reprogramming of donor cells that occurred by the blastocyst stage, SCNT-specific errors are of a non-random nature in bovine and are not responsive to epigenetic modifications by TSA.

Contrasting effects of G1.2/G2.2 and SOF1/SOF2 embryo culture media on pre- and post-implantation development of non-transgenic and transgenic cloned goat embryos.

This study compared the efficiency of two embryo culture media (SOF1/SOF2 and G1.2/G2.2) for pre- and post-implantation development of somatic cell nuclear transfer goat embryos derived from non-transgenic and transgenic (for htPA and hrcfIX genes) fibroblasts. Despite similar cleavage rates, G1.2/G2.2 supported significantly higher blastocyst development than SOF1/SOF2 (30-35% versus 21%; P < 0.05), irrespective of cell transgenesis. However, following embryo transfer, pregnancy outcomes (establishment, full-term development and live birth) were all significantly higher (P < 0.05) for embryos developed in SOF1/SOF2 versus G1.2/G2.2. Gene expression profiling of 17 developmentally important genes revealed that: (i) SOX2, FOXD3, IFNT, FZD, FGFR4, ERK1, GCN5, PCAF, BMPR1, SMAD5, ALK4, CDC25 and LIFR were significantly induced in blastocysts developed in SOF1/SOF2 but not G1.2/G2.2; (ii) OCT4, CTNNB and CDX2 were similarly expressed in both groups; and (iii) AKT was significantly higher in G1.2/G2.2 than SOF1/SOF2 (P < 0.05). Following IVF, although blastocyst development in G1.2/G2.2 was significantly higher than SOF1/SOF2 counterparts, the majority of assessed genes were similarly expressed in blastocysts developed in both groups. It was concluded that the long-term programming effects of embryo culture medium and/or embryo production method may irreversibly affect post-implantation development of cloned embryos through defined molecular pathways.

The effect of superoxide dismutase mimetic and catalase on the quality of postthawed goat semen.

Manganese(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin chloride (MnTE) is a cell-permeable superoxide dismutase mimetic agent which can convert superoxide to hydrogen peroxide (H2O2). Supplementation of MnTE to a commercial semen extender can protect sperm from superoxide but not H2O2. Therefore, we proposed that addition of catalase (0.0, 200, or 400 IU/mL) in combination with MnTE (0.1 µM) may further improve the cryopreservation efficiency of goat semen in commercially optimized freezing media such as Andromed. Therefore, ejaculates were obtained from three adult bucks twice a week during the breeding season and diluted with Andromed supplemented with or without MnTE and catalase and were frozen in liquid nitrogen. Sperm parameters and reactive oxygen species contents were evaluated 2 hours after dilution (before freezing) and after freezing/thawing. The results revealed that all the treatments significantly (P ≤ 0.05) improved sperm motility, viability, and membrane integrity after freezing and reduced reactive oxygen species content compared with the control group, but maximum improvement was obtained in MnTE + 400 IU/mL catalase. In addition, supplementation with these antioxidants significantly (P ≤ 0.05) increases the cleavage rate after IVF. In conclusion, the results of present study suggest that addition of antioxidant MnTE or catalase to commercial optimized media, such as Andromed, improves total motility, membrane integrity, and viability of goat semen samples after thawing. But the degree of improvement for these parameters significantly (P ≤ 0.05) higher when MnTE and catalase were simultaneously added to the cryopreservation media.

Supplementation of sperm cryopreservation media with cell permeable superoxide dismutase mimetic agent (MnTE) improves goat blastocyst formation.

The aim of this study was to assess whether a cell permeable superoxide dismutase agent such as MnTE, can further improve the quality of frozen/thawed semen sample using a commercially optimized sperm cryopreservation media (Bioxcell). Bioxcell was supplemented with different concentration of MnTE. Sperm membrane integrity, motility, viability and acrosomal status were assessed after freezing. Optimized concentration of MnTE was defined and used to assess fertilization and developmental potential. 0.1 µM MnTE significantly improved membrane integrity while 0.01 µM MnTE significantly improved acrosomal integrity post thawing. Addition of 0.01 µM MnTE also improved blastocyst formation rate. Supplementation of commercially optimized cryopreservation media with MnTE further improves the quality of goat frozen semen sample and may have important consequence of future embryo development. This effect may be attributed to cell permeable behavior of this antioxidant which may protect sperm genome from ROS-induced DNA damage.