Comparative Epigenetic Analysis of Imprinting Genes Involved in Fertility, in Cryopreserved Human Sperms with Rapid Freezing versus Vitrification Methods.

OBJECTIVE: Choosing the optimal method for human sperm cryopreservation seems necessary to reduce cryoinjury. The aim of this study is to compare two cryopreservation methods including rapid-freezing and vitrification, in terms of cellular parameters, epigenetic patterns and expression of paternally imprinted genes (PAX8, PEG3 and RTL1) in human sperm which play a role in male fertility. MATERIALS AND METHODS: In this experimental study, semen samples were collected from 20 normozoospermic men. After washing the sperms, cellular parameters were investigated. DNA methylation and expression of genes were investigated using methylation-specific polymerase chain reaction (PCR) and real-time PCR methods, respectively. RESULTS: The results showed a significant decrease in sperm motility and viability, while a significant increase was observed in DNA fragmentation index of cryopreserved groups in comparison with the fresh group. Moreover, a significant reduction in sperm total motility (TM, P<0.01) and viability (P<0.01) was determined, whereas a significant increase was observed in DNA fragmentation index (P<0.05) of the vitrification group compared to the rapid-freezing group. Our results also showed a significant decrease in expression of PAX8, PEG3 and RTL1 genes in the cryopreserved groups compared to the fresh group. However, expression of PEG3 (P<0.01) and RTL1 (P<0.05) genes were reduced in the vitrification compared to the rapid-freezing group. Moreover, a significant increase in the percentage of PAX8, PEG3 and RTL1 methylation was detected in the rapid-freezing group (P<0.01, P<0.0001 and P<0.001, respectively) and vitrification group (P<0.01, P<0.0001 and P<0.0001, respectively) compared to the fresh group. Additionally, percentage of PEG3 and RTL1 methylation in the vitrification group was significantly increased (P<0.05 and P<0.05, respectively) compared to the rapid-freezing group. CONCLUSION: Our findings showed that rapid-freezing is more suitable method for maintaining sperm cell quality. In addition, due to the role of these genes in fertility, changes in their expression and epigenetic modification may affect fertility.

Female Reproductive Health in SARS-CoV-2 Pandemic Era.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic struck global health systems with overgrowing demands in many fields of health care; yet, reproductive care, particularly pregnancy care remains a special focus of interest. Pregnancy is a major physiologic change that alters temporarily normal function of many organs, and specifically the immune system. Therefore, pregnant women are more susceptible to respiratory pathogens compared to the others. The current pandemic may have serious consequences on pregnancy whether directly or indirectly. In the present review, direct and indirect possible adverse effects of SARS-CoV-2 infection on female reproductive system by focusing on pregnancy and delivery has been discussed in details. In addition, the pregnancy consequences and whether maternal infection can affect infants were deliberated. The adverse impact of luck down and related psychological complications and obesity on pregnant women were discussed as well. Finally, the effects of SARS-CoV-2 vaccination on maternal health and pregnancy outcome was analyzed.

Functional and Flow Cytometric Analysis of Buffalo Cryopreserved Spermatozoa: Comparison of Different Breeds and Incubation Times.

BACKGROUND: The purpose of this research was to compare the functional parameters of frozen-thawed Iranian Azari buffalo spermatozoa with imported semen samples of Italian Mediterranean buffalo (IMB) after the thawing process and 4 hours of incubation. MATERIALS AND METHODS: In this experimental study, a total of twenty-four ejaculates from four Iranian Azari buffalo bulls were collected. Semen samples were diluted in AndroMed extender at a concentration of 50×106 spermatozoa/ ml. The diluted samples were filled in 0.5 ml straws and were frozen in a programmable freezer. For imported semen samples, twenty-four samples of four IMB were used, which were diluted in AndroMed extender and frozen by the same procedure. Frozen-thawed sperm motion patterns, mitochondrial activity, membrane integrity, DNA integrity, reactive oxygen species (ROS), and apoptosis status were evaluated immediately after thawing and 4 hours of incubation. RESULTS: Post-thawed sperm motility, progressive motility (PM), mitochondrial activity, membrane integrity were significantly higher in imported semen samples in compare with Iranian Azari buffalo. After 4 hours of incubation, sperm velocity patterns were superior in Iranian Azari semen samples. Moreover, the percentage of sperm cells with un-damaged DNA was higher in Iranian semen samples compared to imported samples at the time 0 of incubation. Following 4 hours of incubation, a significant increase in intracellular ROS level leads to reduced membrane integrity, mitochondrial activity, and DNA integrity in both buffalo breeds. At time 4, Iranian samples showed significantly lower apoptosis and higher dead spermatozoa compared to imported semen samples. CONCLUSION: Our study showed that the post-thawed quality of Iranian Azari buffalo semen was comparable with imported samples after 4 hours of incubation. Further investigations are recommended to assess the in vitro and in vivo fertility rate of both buffalo breeds.

Effects of Dietary Pomegranate Peel on Antioxidant Gene Expression and DJ-1 Protein Abundance in Ram Testes.

BACKGROUND: Pomegranate is an ancient fruit containing Punicalagin, which has known as an effective antioxidant. Pomegranate peel was recognized as a phenol and tannin source, and pomegranate seed contains unique fatty acid (Punicic acid). Limited information exists about the influences of pomegranate peel and seed on antioxidant enzymes and proteins in the male reproduction system. This study was performed to determine the pomegranate peel and seed effects on the expression of antioxidant genes and DJ-1 protein in ram's testis. MATERIALS AND METHODS: In this experimental study, twenty-one mature Iranian rams were randomly divided into three groups (n=7 in each group), and fed experimental diets consisted of a control diet (C), a diet containing dry pomegranate seed pulp (S), and a diet containing pomegranate peel (P) for 80 days. All rams were offered isoenergetic and isonitrogenous rations. Testicular tissue samples were collected, and expression of Gpx1, Gpx4, Prdx4, Prdx5, and Sod2 genes was quantified by real-time polymerase chain reaction (RT-PCR). In addition, western blotting was used to evaluate DJ-1 expression at the protein level. RESULTS: Gpx1 and Sod2 mRNA levels in the peel group were significantly (P<0.05) higher than control. Prdx5 mRNA level was increased (P<0.05) in the seeds group than in the control group. Gpx4 and Prdx4 expression were statistically not affected significantly by the experimental diet. Data analysis showed a significant (P<0.05) increase (1.5-fold) in the expression level of DJ-1 in peel groups than in control. CONCLUSION: The expression of antioxidant genes and DJ-1 protein in ram testes are more influenced by pomegranate peel than seed.

Sperm Associated Antigens: Vigorous Influencers in Life.

Sperm associated antigens (SPAGs) are specific proteins in terms of performance and evolution, that have common expressions in the testes or sperm cells. Moreover, the humoral immune response against some of SPAGs can result in immunological infertilities. On the other hand, recent studies have explored several new properties of SPAGs and shed light on sperm's function, the impact of anti-sperm antibodies (ASA) in immunological infertility, and some tumors related to SPAGs. This article presents an exhaustive review of SPAGs and their roles in the cell cycle, signaling pathways, fertility, sperm-oocyte cross-talk as well as their unfavorable positions as prognostic factors in many types of cancers.

Possible Male Reproduction Complications after Coronavirus Pandemic.

Coronavirus disease 2019 (COVID-19), as a severe respiratory disease, affects various tissues and organs. The specific SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), is highly expressed in male gonads. Thus, male reproductive tissues could be a potential target for virus colonization. We performed a comprehensive search in PubMed and Google Scholar to retrieve relevant articles published till 15 April 2021. The keywords used were: male fertility, male reproductive health, semen parameters, sex hormones, SARS-CoV-2, and COVID-19. Validated evidence about the adverse effects of the SARS-CoV-2 infection on the male reproductive system is limited and few studies have reported semen analysis results or presence of viral RNA in semen samples of infected men. Nevertheless, alterations in reproductive hormones such as decreased level of testosterone (T) with raised luteinizing hormone (LH) have been reported in some patients. Although the impact of SARS-CoV-2 infection on the male reproduction health remains unclear, evidence suggests that male gonads may be potentially vulnerable to SARS-CoV-2 infection. In this article, we discussed the possible impacts of COVID-19 on male gonads, sex hormones, and semen quality and suggested preventive solutions.

Stress Hormone and Oxidative Stress Biomarkers Link Obesity and Diabetes with Reduced Fertility Potential.

OBJECTIVE: Tilting the balance in favor of antioxidant agents could increase infertility problems in obese and diabetic individuals. The aim of this study was to evaluate oxidative stress status in semen of men with type 2 diabetes and obesity to investigate whether excessive amounts of oxidative stress, as a result of diabetes and obesity, influence infertility potential. MATERIALS AND METHODS: A case-control study was conducted in men (n=150) attending the Infertility Center of Royan Institute between December 2016 and February 2017. Participants were categorized into four groups; normal weight (BMI<25 kg/m2) and non-type-2 diabetic (control=40), obese and non- type-2 diabetic (obese=40), non-obese and type- 2 diabetic (Nob-DM=35), and obese and type-2 diabetic (Ob-DM=35). The semen analysis was performed according to the World Health Organization criteria. Oxidative stress, DNA fragmentation, sperm apoptosis, and total antioxidant capacity (TAC) were evaluated in semen samples of men. Serum glucose, HbA1c, cortisol, and testosterone levels were determined using the enzyme-linked immunosorbent assay (ELISA) method. RESULTS: Compared with the control group, sperm motility, progressive motility, and normal morphology were significantly decreased in the obese, Nob-DM, and Ob-DM groups (P<0.01). The obese, Nob-DM, and Ob-DM groups showed significantly lower levels of TAC and higher amounts of oxidative stress, early apoptotic sperm, and the percentage of DNA fragmentation as compared with the control group (P<0.05). Testosterone concentration was decreased in the obese, Nob-DM, and Ob-DM groups when compared with healthy individuals (P<0.05), whereas the cortisol level was significantly increased in the Nob-DM and Ob-DM groups in comparison to the obese and control group (P<0.01). CONCLUSION: Increased amount of reactive oxygen species (ROS) levels and DNA fragmentation in men affected by either diabetes or obesity could be considered prognostic factors in sub-fertile patients, alerting physicians to an early screen of male patients to avoid the development of infertility in prone patients.

Reduction of truncated Kit Expression in Men with Abnormal Semen Parameters, Globozoospermia and History of Low or Fertilization Failure.

OBJECTIVE: Phospholipase C zeta 1 (PLCζ) is one of the main sperm factor involved in oocyte activation and other factors may assist this factor to induce successful fertilization. Microinjection of recombinant tr-kit, a truncated form of c-kit receptor, into metaphase II-arrested mouse oocytes initiate egg activation. Considering the potential roles of tr- KIT during spermiogenesis and fertilization, we aimed to assess expression of tr-KIT in sperm of men with normal and abnormal parameters and also in infertile men with previous failed fertilization and globozoospermia. MATERIALS AND METHODS: This experimental study was conducted from September 2015 to July 2016 on 30 normozoospermic and 20 abnormozoospermic samples for experiment one, and also was carried out on 10 globozoospermic men, 10 men with a history low or failed fertilization and 13 fertile men for experiment two. Semen parameters and sperm DNA fragmentation were assessed according to WHO protocol, and TUNEL assay. Sperm tr- KIT was evaluated by flow cytometry, immunostaining and western blot. RESULTS: The results show that tr-KIT mainly was detected in post-acrosomal, equatorial and tail regions. Percentage of tr-KIT-positive spermatozoa in abnormozoospermic men was significantly lower than normozoospermic men. Also significant correlations were observed between sperm tr-KIT with sperm count (r=0.8, P<0.001), motility (r=0.31, P=0.03) and abnormal morphology (r=-0.6, P<0.001). Expression of tr-KIT protein was significantly lower in infertile men with low/ failed fertilization and globozoospermia compared to fertile men. The significant correlation was also observed between tr-KIT protein with fertilization rate (r=-0.46, P=0.04). In addition, significant correlations were observed between sperm DNA fragmentation with fertilization rate (r=-0.56, P=0.019) and tr-KIT protein (r=-0.38, P=0.04). CONCLUSION: tr-KIT may play a direct or indirect role in fertilization. Therefore, to increase our insight regarding the role of tr-KIT in fertilization further research is warranted.

Induction of Sublethal Oxidative Stress on Human Sperm before Cryopreservation: A Time-Dependent Response in Post-Thawed Sperm Parameters.

OBJECTIVE: A recent innovative approach, based on induction of sublethal oxidative stress to enhance sperm cryosurvival, has been applied before sperm cryopreservation. The purpose of this study was to investigate the effects of different induction times of sublethal oxidative stress before cryopreservation on human post-thawed sperm quality. MATERIALS AND METHODS: In this experimental study, we selected semen samples (n=20) from normozoospermic men according to 2010 World Health Organization (WHO) guidelines. After processing the samples by the density gradient method, we divided each sample into 5 experimental groups: fresh, control freezing, and 3 groups exposed to 0.01 µM sodium nitroprusside (SNP) [nitric oxide (NO) donor] for 30 (T30), 60 (T60), or 90 minutes (T90) at 37˚C and 5% CO2 before cryopreservation. Motion characteristics [computer-assisted sperm analyser], viability, apoptosis [annexin V/propidium iodide (PI) assay], DNA fragmentation [sperm chromatin structure assay (SCSA)], and caspase 3 activity (FLICA Caspase Detection Kit) were assessed after thawing. The results were analysed by using one-way ANOVA and Tukey's test. The means were significantly different at P<0.05. RESULTS: Cryopreservation significantly decreased sperm viability and motility parameters, and increased the percentage of apoptosis, caspase 3 activity, and DNA fragmentation (P<0.01) compared to the fresh group. The T60 group had a higher significant percentage of total motility (TM) and progressive motility compared with other cryopreserved groups (P<0.05). We observed a significantly lower percentage of apoptotic rate and caspase 3 activity in the T60 group compared to the other cryopreserved groups (P<0.05). DNA integrity was not significantly affected by this time of sublethal stress induction (P>0.05). CONCLUSION: Our results have demonstrated that the application of sublethal oxidative stress by using 0.01 µM NO for 60 minutes before the freezing process can be a beneficial approach to improve post-thawed human sperm quality.

Status of sperm-born oocyte activating factors (PAWP, PLCζ) and sperm chromatin in uncapacitated, capacitated and acrosome-reacted conditions.

The aim of this study was to investigate the value of sperm factors PAWP and PLCζ involved in oocyte activation and sperm chromatin status, as indicators of sperm quality during uncapacitated, capacitated and acrosome-reacted conditions. Semen samples collected from 10 normozoospermic men and sperm factors PAWP, PLCζ, protamine deficiency and DNA fragmentation evaluated in fresh samples, processed sample by DGC (density gradient centrifugation), capacitated and acrosome reacted sperm, using immunofluorescence and flow cytometry. The status of capacitation and the acrosome reaction were confirmed by tyrosine kinase phosphorylation and CD46 markers, respectively. The proportion of sperm-expressing weak or zero levels of PLCζ and PAWP were significantly reduced after DGC processing. There were no significant differences among processed DGC, capacitated and acrosome-reacted samples. The proportions of DNA fragmented and protamine-deficient spermatozoa were decreased after DGC and maintained during capacitation and the acrosome reaction. DGC processing, therefore, improved the quality of sperm in terms of chromatin and DNA integrity, and removed sperm which may have had a lower potential to induce oocyte activation and undergo capacitation and the acrosome reaction.

The Principal Forces of Oocyte Polarity Are Evolutionary Conserved but May Not Affect the Contribution of the First Two Blastomeres to the Blastocyst Development in Mammals.

Oocyte polarity and embryonic patterning are well-established features of development in lower species. Whether a similar form of pre-patterning exists in mammals is currently under hot debate in mice. This study investigated this issue for the first time in ovine as a large mammal model. Microsurgical trisection of unfertilized MII-oocytes revealed that cortical cytoplasm around spindle (S) contained significant amounts of total maternal mRNAs and proteins compared to matched cytoplast hemispheres that were located either near (NS) or far (FS) -to-spindle. RT-qPCR provided striking examples of maternal mRNA localized to subcellular substructures S (NPM2, GMNN, H19, PCAF, DNMT3A, DNMT1, and STELLA), NS (SOX2, NANOG, POU5F1, and TET1), and FS (GCN) of MII oocyte. Immunoblotting revealed that specific maternal proteins DNMT3A and NANOG were asymmetrically enriched in MII-spindle-half of the oocytes. Topological analysis of sperm entry point (SEP) revealed that sperm preferentially entered via the MII-spindle-half of the oocytes. Even though, the topological position of first cleavage plane with regard to SEP was quite stochastic. Spatial comparison of lipid content revealed symmetrical distribution of lipids between 2-cell blastomeres. Lineage tracing using Dil, a fluorescent dye, revealed that while the progeny of leading blastomere of 2-cell embryos contributed to more cells in the developed blastocysts compared to lagging counterpart, the contributions of leading and lagging blastomeres to the embryonic-abembryonic parts of the developed blastocysts were almost unbiased. And finally, separated sister blastomeres of 2-cell embryos had an overall similar probability to arrest at any stage before the blastocyst (2-cell, 4-cell, 8-cell, and morula) or to achieve the blastocyst stage. It was concluded that the localization of maternal mRNAs and proteins at the spindle are evolutionarily conserved between mammals unfertilized ovine oocyte could be considered polar with respect to the spatial regionalization of maternal transcripts and proteins. Even though, the principal forces of this definitive oocyte polarity may not persist during embryonic cleavages.

In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells.

OBJECTIVE: Bone marrow has recently been recognized as a novel source of stem cells for the treatment of wide range of diseases. A number of studies on murine bone mar- row have shown a homogenous population of rare stage-specific embryonic antigen 1 (SSEA-1) positive cells that express markers of pluripotent stem cells. This study focuses on SSEA-1 positive cells isolated from murine bone marrow in an attempt to differentiate them into insulin-secreting cells (ISCs) in order to investigate their differentiation potential for future use in cell therapy. MATERIALS AND METHODS: This study is an experimental research. Mouse SSEA-1 positive cells were isolated by Magnetic-activated cell sorting (MACS) followed by characteriza- tion with flow cytometry. Induced SSEA-1 positive cells were differentiated into ISCs with specific differentiation media. In order to evaluate differentiation quality and analysis, dithizone (DTZ) staining was use, followed by reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry and insulin secretion assay. Statistical results were analyzed by one-way ANOVA. RESULTS: The results achieved in this study reveal that mouse bone marrow contains a population of SSEA-1 positive cells that expresses pluripotent stem cells markers such as SSEA-1, octamer-binding transcription factor 4 (OCT-4) detected by immunocytochem- istry and C-X-C chemokine receptor type 4 (CXCR4) and stem cell antigen-1 (SCA-1) detected by flow cytometric analysis. SSEA-1 positive cells can differentiate into ISCs cell clusters as evidenced by their DTZ positive staining and expression of genes such as Pdx1 (pancreatic transcription factors), Ngn3 (endocrine progenitor marker), Insulin1 and Insulin2 (pancreaticß-cell markers). Additionally, our results demonstrate expression of Pdx1 and Glut2 protein and insulin secretion in response to a glucose challenge in the differentiated cells. CONCLUSION: Our study clearly demonstrates the potential of SSEA-1 positive cells to differentiate into insulin secreting cells in defined culture conditions for clinical ap- plications.

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.

The effects of platelet lysate on maturation, fertilization and embryo development of NMRI mouse oocytes at germinal vesicle stage.

Purpose: Improving in vitro maturation could increase the rate of pregnancy from oocytes matured in vitro. Consequently, patients will be prevented from using gonadotropin with its related side effects. In this study, the maturation medium was enriched by platelet lysate (PL), then maturation and subsequent developments were monitored. Methods: Oocytes at germinal vesicle stage with cumulus cells (cumulus-oocyte complex) and without cumulus cells (denuded oocytes) were obtained from mature female mice. The maturation medium was enriched by 5 and 10 % PL and 5 % PL + 5 % fetal bovine serum (FBS) as experimental groups; the control groups' media consisted of 5 and 10 % FBS. After 18 h, the matured oocytes were collected and, after fertilization, subsequent development was monitored. Results: The rates of maturation, fertilization and 2-cell embryo development for the denuded oocyte groups in experimental media 5 % PL and 5 % PL + 5 % FBS were significantly higher than those of the control groups (P < 0.05), while the results for the cumulus-oocyte complex groups were similar between the experimental groups and control groups. Conclusions: The results of this study indicated that platelet lysate could improve the maturation rate in the absence of granulosa cells compared to media with FBS. This extract also had positive effects on fertilization and embryo development.

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.

A comparative study of saffron aqueous extract and its active ingredient, crocin on the in vitro maturation, in vitro fertilization, and in vitro culture of mouse oocytes.

OBJECTIVE: Reactive oxygen species have effects on gamete quality and gamete interaction; they influence spermatozoa, oocytes, embryos, and their environment. In this study, we evaluated the antioxidant effect of different concentrations of saffron (Crocus sativus L.) aqueous extract (SAE) and its ingredient, crocin, on the improvement of in vitro maturation (IVM) and subsequent in vitro fertilization (IVF) and embryo development of mouse oocytes. MATERIALS AND METHODS: Cumulus oocyte complexes were collected from ovaries, and germinal vesicle oocytes were cultured in the presence of SAE and crocin. SAE was added at dosages of 5 µg/mL, 10 µg/mL, and 40 µg/mL; dosages of crocin were 50 µg/mL, 100 µg/mL, and 400 µg/mL. All dosages were added to maturation medium and a group without SAE or crocin was considered as the control group. Following IVM, metaphase II oocytes were fertilized and cultured in vitro in order to observe embryo development. RESULTS: Both SAE and crocin improved the rate of IVM, IVF, and in vitro culture. Addition of 40 µg/mL SAE to maturation medium significantly increased the rate of IVM, IVF, and in vitro culture (p < 0.05). Furthermore 100 µg/mL crocin significantly increased the IVM rate compared to the control group (p < 0.05). CONCLUSION: Use of SAE during IVM can affect on IVM, IVF, and early embryo development in a dose-dependent manner. SAE appears to have a stronger effect than pure crocin.

Evaluation effects of allopurinol and FSH on reduction of ischemia-reperfusion injury and on preservation of follicle after heterotopic auto-transplantation of ovarian tissue in mouse.

Purpose: Allopurinol and FSH injection are applied to reduce ischemia-reperfusion injury and to increase survival rate for ovarian follicles after ovarian heterotopic autotransplantation in mice. Methods: Ovarian tissues from 6-week-old mice were grafted into back muscle then collected after 3 weeks. A total of five groups were included in this experiment as follows: control group (n = 5), sham-operated group (n = 5), allopurinol treatment group (AP) (n = 5), follicle stimulating hormone (FSH) treatment group (n = 5), as well as, allopurinol and FSH treatment group (APF) (n = 5). We investigated survival, number and development of follicles, vaginal cytology along with plasma malondialdehyde (MDA) concentration in grafted ovary. Results: Total follicles count significantly increased in APF group compared with other treatment groups (p < 0.05). MDA concentration significantly decreased in AP group and APF treatment group compared with sham-operated group. In AP group, vaginal smears showed presence of cornified epithelial cells three-five day after surgery. Conclusions: We demonstrated that allopurinol, as a XO inhibitor, plays an important role in order to decrease ischemia injury and to increase survival rate for follicles. Also, FSH, as a folliculogenesis and angiogenesis factor, increases development of follicles. It seems that allopurinol can cause re-establishing hypothalamus-pituitary axis and finally can restore estrous cycle earlier than for the sham operated group, so it explains the increasing survival rate for follicles.

Production of transgenic goats expressing human coagulation factor IX in the mammary glands after nuclear transfer using transfected fetal fibroblast cells.

There are growing numbers of recombinant proteins that have been expressed in milk. Thus one can consider the placement of any gene of interest under the control of the regulatory elements of a milk protein gene in a dairy farm animal. Among the transgene introducing techniques, only nuclear transfer (NT) allows 100 % efficiency and bypasses the mosaicism associated with counterpart techniques. In this study, in an attempt to produce a transgenic goat carrying the human coagulation factor IX (hFIX) transgene, goat fetal fibroblasts were electroporated with a linearized marker-free construct in which the transgene was juxtaposed to ß-casein promoter designed to secret the recombinant protein in goat milk. Two different lines of transfected cells were used as donors for NT to enucleated oocytes. Two transgenic goats were liveborn. DNA sequencing of the corresponding transgene locus confirmed authenticity of the cloning procedure and the complementary experiments on the whey demonstrated expression of human factor IX in the milk of transgenic goats. In conclusion, our study has provided the groundwork for a prosperous and promising approach for large-scale production and therapeutic application of hFIX expressed in transgenic goats.

The effect of intra-peritoneal administration of Papaver bracteatum Lindl. extract on development of NMRI mice oocytes treated with Doxorubicin.

PURPOSE: The effect of water-alcohol Papaver bracteatum Lindl. extract on development of mice oocytes treated with Doxorubicin (dox) was examined in this study. METHODS: The mice were classified into four groups. Control group, mice injected intraperitoneally (IP) with saline. Extract group alone, mice treated with 200 mg/kg of body weight (bw), IP, twelve consecutive days. Dox group alone, mice were given dox, IP, 10 mg/kg bw. Experimental group treated with extract and dox together. Effect of the extract on the development of mice oocytes treated with dox were evaluated through assisted reproductive technology techniques (ARTs). RESULTS: Developmental rate and blastocyst formation was improved by using the extract. A significant increase in in vitro developmental competence in comparison with dox group (P < 0.05) was observed. CONCLUSIONS: The results of this study indicated that P. bracteatum Lindl. extract could prevent dox toxicity of dox affecting both follicle or oocytes, and therefore it can result in improved embryo development which was observed in mice treated with dox plus P. bracteatum Lindl. compared to mice treated with dox alone.

Somatic cell-induced hyperacetylation, but not hypomethylation, positively and reversibly affects the efficiency of in vitro cloned blastocyst production in cattle.

5-Aza-2'-deoxycytidine (AzC), trichostatin A (TSA), and its natural mimetic, sodium butyrate (NaB), are antineoplastic drugs that can modify the epigenetic status of donor cells prior to somatic cell nuclear transfer (SCNT). In this study, we used fibroblast cells treated with these drugs to investigate the direct and indirect effects of induced changes in DNA methylation and acetylation of the lysine 9 residue of histone H3 (H3K9). Additionally, we assayed cellular characteristics (cell growth, cell proliferation, cell cycle progression, and apoptosis) and SCNT efficiency in response to these drugs as well as monitoring these effects 24 h after removing the drugs. We observed the following: (1) AzC, TSA, and NaB all showed dose-dependent effects on different cellular characteristics; (2) TSA and NaB induced H3K9 hyperacetylation accompanied by DNA hypermethylation, whereas AzC induced DNA hypomethylation with no effect on H3K9 hyperacetylation; (3) TSA and NaB improved cloning efficiency, whereas AzC reduced it; and (4) unlike AzC, the effects of TSA and NaB on cellular characteristics and SCNT efficiency were reversed following drug removal. Our results indicate that somatic cells treated with TSA and NaB show better survival and recovery rates following the removal of these drugs. Moreover, H3K9 hyperacetylation (induced with TSA and NaB), but not DNA hypomethylation (induced with AzC), favors cloning efficiency.