Sperm and oocyte as carriers for bovine viral diarrhoea virus biotypes during in vitro fertilization.

Bovine viral diarrhoea virus (BVDV) is an important viral agent causing the reproductive failure in cattle. The objectives of the study were to assess the role of male and female gametes, as carriers of cytopathic (CP) and non-cytopathic (NCP) BVDV to embryonic cells during in vitro fertilization. In this respect, sperm and oocytes were separately exposed to concentrations of 104.5 or 105.5 TCID50 /mL CP and NCP BVDV, for 2 h before fertilization. After washing, the intact gametes with the infected gametes were inseminated. Seven days post-fertilization, the virus-exposed embryos were examined for presence of the viral genome by RT-PCR. One-way anova with post-hoc Tukey's HSD test and an independent samples t-test were used to compare within and between groups, respectively. The results presented a significant decrease in the blastocyst rates for CP-infected groups than NCP-infected groups (p ≤ .01). Compared to the controls and the infected oocyte groups, the cleavage rates of the infected sperm groups (NCP and CP BVDV) were significantly reduced both in low (104.5 TCID50 /mL) and high (105.5 TCID50 /mL) titres of the virus (p ≤ .01). The proportion of embryos which was developed to blastocyst stages was significantly lower for CP and NCP-infected groups than the control groups (p ≤ .001). According to the molecular results, all samples of the retarded/degenerated embryos (at least one blastocyst within each one) in CP and NCP groups, one sample (at least one blastocyst in that) within a CP-infected group, and six samples (at least one blastocyst in each one of those) of NCP-infected groups contained the viral nucleic acid. Likewise, the results of viral enrichment showed all reactions in which RT-PCR were positive induced CPEs in MDBK monolayers. In conclusion, it is clear that CP and NCP BVDV were able to traverse zona pellucida during fertilization, and they had also negative effects on embryo development.

The effects of PFT-µ on spermatogonial stem cell viability and Pluripotency.

INTRODUCTION: Spermatogonial stem cells (SSCs) offer remarkable competencies for animal reproduction and overcoming human disease as a result of their differentiation capability. We evaluated the effect of small molecule pifithrin-mu (PFT-µ) as a well-known inhibitor of P53 on SSC biological processes such as viability, apoptosis, and gene expression pattern. METHODS: The SSCs were isolated from the testes of adult NMRI mice and then cultured in DMEM / F12 medium containing 10% FBS. Then, they were characterized by the immunocytochemistry (ICC) technique by high PLZF and low c-Kit expressions. SSCs colony formation assay was carried out and their viability was estimated by MTT (Methylthiazolyldiphenyl-tetrazolium bromide, or 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay upon exposure to PFT-µ (0, 0.6, 1.2, 2.5, and 5µM). The apoptosis percentages also were measured using FACS analysis, and finally, Oct4 and Stra8 expression at mRNA levels was assessed using real-time quantitative PCR. RESULTS: The 0.6 and 1.2µM PFT-µ improved the viability of SSC based on MTT assay results; however, 2.5 and 5µM PFT-µ reduced SSC viability compared with the control group. Moreover, PFT-µ at lower concentration enhanced the colony size of SSCs and diminished their apoptosis. As well, as exposure to PFT-µ up-regulated Oct4 expression, while down-regulating the meiotic entry marker, Stra8. CONCLUSION: Based on findings, optimized concentrations of PFT-µ can decrease SSCs apoptosis, and conversely potentiate their pluripotency and self-renewal capacities in vitro.

The study of a hermaphroditic sheep caused by a mutation in the promoter of SRY gene.

In mammals, sex-determining region Y (SRY) gene plays vital role as a transcription factor to regulate the expression of the genes contributing to development of male genitals. Any mutation disrupting expression of SRY gene can cause disorders of sex development (DSDs). In this study, the examination of a hermaphroditic (female-like) Shal sheep which was referred for infertility is described. Initially, the reproductive system of the sheep was histologically and anatomically assessed. Karyotyping was used to determine the real gender of the animal. Sex hormones including progesterone, estradiol, and testosterone were measured by enzyme-linked immunosorbent assay (ELISA). Eventually, promoter part and SRY gene were sequenced and aligned to detect any potential mutation using NCBI data base. Although anatomical inspection led to identification of uterus, ovary, and enlarged clitoris as well as testes in the sheep, the karyotyping results interestingly revealed that the animal was genetically a male. Although the sheep had both male and female gonads, there were no overt signs of reproductive behavior and gamete production was not observed. Plasma steroid hormone levels were reported to be at basal levels. Additionally, a mutation was detected on the promoter of the SRY gene. In conclusion, the case implies that mutation on the promoter part of SRY gene could disrupt sexual development of the fetus culminating in DSDs in the sheep.

Effect of bovine viral diarrhea virus biotypes exposure on bovine gametes in early embryonic development in vitro.

Bovine viral diarrhea virus (BVDV) is an important viral agent causing reproductive failure in cattle. The objectives of the current study were to investigate the interaction between two BVDV biotypes, cytopathic (CP) and Non-cytopathic (NCP) and bovine gametes during in vitro fertilization (IVF) processing, the existence of the virus within embryonic cells and early embryonic development rates. Sperm and ova were exposed separately to CP and NCP BVDV at two concentrations of 104.5 and 105.5 tissue culture infectious dose 50.00% (TCID50) mL-1 prior to IVF, respectively. After five days post-IVF, early embryonic development rates of infected groups were assessed. Several embryos of each group, normal and degenerated, were selected for a viral assay using reverse transcription polymerase chain reaction technique. The result showed that the early embryonic development rates were decreased in treatment groups. The rates in the CP groups were lower than the NCP groups. In the CP groups, the proportions were, respectively, 10.00, 6.00 and 11.00, and 6.00% in the infected sperm and oocyte groups (104.5 and 105.5 TCID50 mL-1) that were higher than 50.00% in the control group. In NCP groups, the rates were, respectively, 25.00, 18.00 and 24.00, and 21.00% in the infected groups compared to 48.00% in the control group. In the CP groups, no BVDV was detected in normal embryos, whereas, all degenerated embryos were completely virus-positive. In the NCP groups, the virus was detected in both normal and degenerated embryos. In conclusion, this study supported detrimental impacts of CP and NCP BVDV on early embryonic development and the role of sperm and the zona pellucida layer as carriers of the virus.

The association between TIPARP gene polymorphisms rs2665390 and ovarian cancer susceptibility.

Ovarian cancer is taken as the most typical malignancy among women and the ninth most typical cancer in Iran. Predictive tools are of great importance as ovarian cancer is usually detected in patients at later stages of the disease. In other countries, the TIPARP gene rs2665390 has been reported to be pertinent to ovarian cancer as a risk factor. This study aims to examine if this polymorphism pertains to the risk of ovarian cancer to diagnose suitable biomarkers in the Iranian population. Method: In the present case-control piliot study, peripheral blood samples were gathered from 60 control subjects and 60 patients with ovarian cancer. The gene was determined by Tetra ARMS PCR after DNA extraction. Tetra ARMS PCR is a flexible, rapid, and cost-effective method to detect allele-specific DNA polymorphisms. The data were analyzed by chi-square test. Results: The results indicated that there was a significant association between the T/T and C/C genotypes distribution and C and T allele in ovarian cancer for rs2665390 polymorphism in the two populations. In addition, significant correlations were observed in patients with the (T/T) genotype (p = 0.0048) as frequencies of ovarian cancer decreased. Discussion & Conclusions: Based on the results, rs2665390 polymorphism of TiPARP gene might be pertained to the susceptibility of ovarian cancer in the Iranian pilot population, which can be used as a suitable biomarker for the population and help physicians with their predictions. However, more studies need to be conducted in this area to broaden our horizons on this issue.

Phenotype Compensation in Reproductive ADAM Gene Family: A Case Study with ADAM27 Knockout Mouse.

Background: A disintegrin and metalloproteinase (ADAM) cell surface proteins are expressed in different cells and are involved in biological processes such as cell-cell interactions, cell differentiation, sperm attachment and fertilization. A significant number of ADAM isoforms are expressed in the reproductive tracts of male mice and other mammals, which shows the importance of this gene family in reproduction. Objectives: The role of ADAM27 protein in reproduction was investigated. Materials and Methods: ADAM27 knock-out mutant mice were generated using the blastocyst microinjection technique. The knock-out mice were analyzed genetically and phenotypically to discover any abnormalities. Results: The results of this study revealed that the homozygote mutant male mice were fertile and showed no significant differences compared to wild-type male mice. A histological exam, sperm analysis and in-vitro fertilization experiments showed no statistical differences. Conclusions: We can conclude that the role of deficient ADAM27 protein is probably compensated mainly by other ADAM isoforms which are expressed in the reproductive system.

Cloning and Embryo Splitting in Mammalians: Brief History, Methods, and Achievements.

Embryo splitting is one of the newest developed methods in reproductive biotechnology. In this method, after splitting embryos in 2-, 4-, and even 8-cell stages, every single blastomere can be developed separately, but the embryos are genetically identical. Embryo splitting, as an approach in reproductive cloning, is extensively employed in reproductive medicine studies, such as investigating human diseases, treating sterility, embryo donation, and gene therapy. In the present study, cloning in mammalians and cloning approaches are briefly reviewed. In addition, embryo splitting and the methods commonly used in embryo splitting and recent achievements in this field, as well as the applications of embryo splitting into livestock species, primate animals, and humans, are outlined. Finally, a perspective of embryo splitting is provided as the conclusion.

Evaluation of multi-epitope recombinant protein as a candidate for a contraceptive vaccine.

Contraceptive vaccine (CV) is a valuable, non-invasive, and alternative method for purposeful contraception. Sperm antigens are useful targets for producing CVs due to their specialized expression in sperm. In this study, a recombinant protein containing three main sperm epitopes (IZUMO1, SACA3, and PH-20) was designed and evaluated as CV to control fertility in male mice. The chimeric recombinant protein was expressed and purified in E. coli. Male mice were immunized by 100 µg purified protein and sera were collected to assess IgG antibodies. Evaluating the reproductive performance, immunized male mice mated with normal-fertile female mice and mating rate and the number of newborns was studied. Immunized mice were sacrificed and necropsy and histopathology studies were conducted. The results revealed that the designed chimeric protein stimulated the immune system of the mice effectively. The level of IgG antibody was significantly higher in vaccinated mouse rather than control mouse. Eighty percent of the vaccinated mice became infertile and in the remaining ones, the number of children decreased to 4-6 offspring instead of 10-12 in normal mice. Histopathological studies showed that no organs including heart, brain, lung, liver, kidney and intestine were damaged. However, Normal spermatogenesis has been disrupted and necrotic spermatogonia cells were reported in Seminiferous tubules. We concluded that the designed chimeric protein containing IZUMO1, SACA3, and PH-20 epitopes can stimulate the immune system and cause male contraception without any side effects.

The chicken hypersensitive site-4 insulator cannot fully shield the murine phosphoglycerate kinase-1 promoter from integration site effects in transgenic mice.

Differential expression of transgenes in transgenic animals is one of the main drawbacks of pronuclear injection. To overwhelm this issue, the genetic constructs are equipped with insulators. In this study, the consensus of exerting chicken hypersensitive site-4 (cHS4) insulator was examined on the shield of phosphoglycerate kinase-1 (Pgk-1) promoter from the surrounding chromatin in transgenic mice. The PGK-EGFP cassette was flanked by insertion of three copies of the cHS4 insulators. Mouse zygotes' microinjection by the constructed cassette was resulted in the birth of nine transgenic founders (F0). Copy-number-dependent expression of the EGFP was investigated in the transgenic F1 offspring by fluorometry and real-time PCR. They showed no correlation between the expression level of transgene and gene copy number among the transgenic lines. Moreover, dissection of the EGFP-expressing mice revealed heterogeneous expression of the EGFP in the different organs. In conclusion, for the first time, these findings indicated that the cHS4 sequence is not a perfect insulator to fully protect the Pgk-1 promoter from the side effects of integration site in transgenic mice and it needs probably to some additional elements of the cHS4 locus to reach this goal.

Biochemical Characterization of Recombinant Thermostable Cohnella sp. A01 ß-Glucanase

Background: Typically, non-cellulytic glucanase, including fungi and yeast cell wall hydrolyzing enzymes, are released by some symbiotic fungi and plants during the mycoparasitic fungi attack on plants. These enzymes are known as the defense mechanisms of plants. This study intends to investigate the biochemical properties of ß-1,6-glucanase (bg16M) from native thermophilic bacteria, Cohnella A01. Methods: bg16M gene was cloned and expressed in E. coli BL21 (DE3). The enzyme was purified utilizing Ni-NTA nikcle sepharose column. Pustulan and laminarin were selected as substrates in enzyme assay. The purified bg16M enzyme was treated with different pH, temperature, metal ions, and detergents. Results: The expressed protein, including 639 amino acids, showed a high similarity with the hydrolytic glycosylated family 30. The molecular weight of enzyme was 64 kDa, and purification yield was 46%. The bg16M demonstrated activity as 4.83 U/ml on laminarin and 2.88 U/ml on pustulan. The optimum pH and temperature of the enzyme were 8 and 50 °C, respectively. The enzyme had an appropriate stability at high temperatures and in the pH range of 7 to 9, showing acceptable stability, while it did not lose enzymatic activity completely at acidic or basic pH. None of the studied metal ions and chemical compounds was the activator of bg16M, and urea, SDS, and copper acted as enzyme inhibitors. Conclusion: Biochemical characterization of this enzyme revealed that bg16M can be applied in beverage industries and medical sectors because of its high activity, as well as thermal and alkaline stability.

A Study on Merits and Demerits of Two Main Gene Silencing Techniques in Mice.

BACKGROUND: Transgenic mice are being considered as invaluable tool in biological sciences towards comprehension of the cause of the genetic diseases. Manipulated embryonic stem (ES) cells are used to produce loss-of-function mutant mice. Microinjection of manipulated ES cells into blastocoel cavity, and morula fusion are the two main techniques in producing transgenic mice. So far, no reports have dealt with the comparison of these two methodologies provide. OBJECTIVE: The object of this study was to determine advantages and disadvantages of knockout mouse creation protocols. MATERIALS AND METHODS: Both blastocyst microinjection and morula aggregation were implemented to produce chimeric mice and the advantages and disadvantages of each technique were evaluated. For this, embryonic stem cells were transfected with a GFP-expression vector. In blastocyst microinjection technique, first transfected ES cell were cultured and appropriate colonies were selected. The cells were microinjected to blastocoel cavity of the expanded blastocyst. In morula aggregation technique, the transfected ES cell colonies were sandwiched between two naked morulas. After 16 h incubation in a 5% CO2 at 37 °C the morulas and infected ES cell were aggregated to produce a new morula. All the injected blastocyst and aggregated morulas were transferred to uterus of foster mice. The new born mice were analyzed for chimera confirmation. RESULTS: Five chimeric mice (21.75%) from morula aggregation and eight chimeric mice (63%) from blastocyst microinjection were born. The results indicated that both techniques can be used to generate chimeric mouse, however the success rate was higher in blastocyst microinjection. CONCLUSION: Morula fusion stands out where the required instrumentations are in place. Furthermore, the quality of ES cells plays a prominent role in the success rate. When the cell quality is low the blastocoel microinjection is recommended. The microinjection technique is more effective than morula aggregation.

Influence of heavy nanocrystals on spermatozoa and fertility of mammals.

In recent years, quantum dots (QDs) have been widely used in upcoming nanotechnology-based solar cells, light-emitting diodes and even bioimaging, due to their tunable optical properties and excellent quantum yields. But, such nanostructures are currently constituted by heavy elements which can threat the human health and living environment. Hence, in this work, the in vivo effects of CdTe nanocrystals (NCs) (as one of the promising QDs) on spermatozoa of male mice and subsequently on fertility of female mice were investigated, for the first time. To do this, CdTe NCs were synthesized through an environment-friendly (aqueous-based solution) method. The sperm cells presented a high potential for uptake of the heavy QDs. Meantime, the NCs exhibited concentration-dependent adverse effects on morphology, viability, kinetic characteristics and DNA of the spermatozoa. At low concentration of 0.1µg/mL, the NCs showed a moderate toxicity (~25% reduction in viability and motility of the spermatozoa), while remarkable toxicities were observed at higher concentrations of 1.0-100µg/mL (~67% reduction in viability and motility for 100µg/mL). Furthermore, significant in vitro DNA fragmentation of the spermatozoa was observed at CdTe concentrations ≥10µg/mL. In vivo toxicity of the NCs was found lower than the in vitro toxicity. Nevertheless, the in vivo destructive effects of the NCs still caused ~34% reduction in viability as well as motility and ~5% damages in DNA of male mice spermatozoa. These resulted in ~26% decrease in fertility and gestation of female mice, along with an overall hormone secretion during the pregnancy, and ~39% reduction in viability of pups/pregnant females.