Vitamin C Synergistically Enhances Protective Effects of Vitamin E Against Preantral Follicle Degeneration of Ovine Vitrified/Warmed Ovarian Tissue.

This study aimed to evaluate whether the addition of vitamins E and C as two conventional antioxidants improves the cryotolerance of preantral follicles enclosed in ovine ovarian tissue slices. For this purpose, ovarian slices were obtained from abattoired juvenile lambs and randomly distributed to the following groups: fresh, toxicity, vitrified (control), and three treatment groups in two experiments. Vitamin E, vitamin C, or vitamin E + C was added to the vitrification media alone in the first experiment and added to all vitrification, warming, and culture media in the second experiment. Finally, the treated tissues were cultured in vitro for 12 hours. The histological analysis showed that single or combined use of vitamins E and C increases intact preantral follicles in comparison to the control in two experiments (p < 0.05), and simultaneous use of vitamins E and C had a synergistic effect on increasing the percentage of normal preantral follicles in experiment 2 (p < 0.05). Due to the better results in Experiment 2, stromal cell density, antioxidant activity, and molecular evaluation were followed only in this experiment. The vitamin E + C group had higher stromal cell density compared with control group (p < 0.05). Vitamin E strengthened antioxidant capacity compared with the control and vitamin C groups (p < 0.05). This effect was exacerbated when used in combination with vitamin C (p < 0.05). The expression of all evaluated genes (BMP4, BMP15, GDF9, and KITLG) was significantly increased in ovarian tissue treated with vitamin E + C compared with the control group (p < 0.05). This increase was also observed in BMP4, GDF9, and KITLG genes compared with the vitamin C group (p < 0.05). In conclusion, this study revealed the positive effects of vitamins E and C on preantral follicle viability and to some extent a synergistic action of vitamin C on the protective effects of vitamin E against preantral follicle degeneration and increasing antioxidant capacity and development of preantral follicles after ovine ovarian tissue vitrification.

Advanced strategies for single embryo selection in assisted human reproduction: A review of clinical practice and research methods.

Among the primary objectives of contemporary assisted reproductive technology research are achieving the births of healthy singletons and improving overall fertility outcomes. Substantial advances have been made in refining the selection of single embryos for transfer, with the aim of maximizing the likelihood of successful implantation. The principal criterion for this selection is embryo morphology. Morphological evaluation systems are based on traditional parameters, including cell count and fragmentation, pronuclear morphology, cleavage rate, blastocyst formation, and various sequential embryonic assessments. To reduce the incidence of multiple pregnancies and to identify the single embryo with the highest potential for growth, invasive techniques such as preimplantation genetic screening are employed in in vitro fertilization clinics. However, new approaches have been suggested for clinical application that do not harm the embryo and that provide consistent, accurate results. Noninvasive technologies, such as time-lapse imaging and omics, leverage morphokinetic parameters and the byproducts of embryo metabolism, respectively, to identify noninvasive prognostic markers for competent single embryo selection. While these technologies have garnered considerable interest in the research community, they are not incorporated into routine clinical practice and still have substantial room for improvement. Currently, the most promising strategies involve integrating multiple methodologies, which together are anticipated to increase the likelihood of successful pregnancy.

In vitro spermatogenesis in artificial testis: current knowledge and clinical implications for male infertility.

Men's reproductive health exclusively depends on the appropriate maturation of certain germ cells known as sperm. Certain illnesses, such as Klinefelter syndrome, cryptorchidism, and syndrome of androgen insensitivity or absence of testis maturation in men, resulting in the loss of germ cells and the removal of essential genes on the Y chromosome, can cause non-obstructive azoospermia. According to laboratory research, preserving, proliferating, differentiating, and transplanting spermatogonial stem cells or testicular tissue could be future methods for preserving the fertility of children with cancer and men with azoospermia. Therefore, new advances in stem cell research may lead to promising therapies for treating male infertility. The rate of progression and breakthrough in the area of in vitro spermatogenesis is lower than that of SSC transplantation, but newer methods are also being developed. In this regard, tissue and cell culture, supplements, and 3D scaffolds have opened new horizons in the differentiation of stem cells in vitro, which could improve the outcomes of male infertility. Various 3D methods have been developed to produce cellular aggregates and mimic the organization and function of the testis. The production of an artificial reproductive organ that supports SSCs differentiation will certainly be a main step in male infertility treatment.

Generation of Haploid Spermatids on Silk Fibroin-Alginate-Laminin-Based Porous 3D Scaffolds.

In vitro production of sperm is a desirable idea for fertility preservation in azoospermic men and prepubertal boys suffering from cancer. In this study, a biocompatible porous scaffold based on a triad mixture of silk fibroin (SF), alginate (Alg), and laminin (LM) is developed to facilitate the differentiation of mouse spermatogonia stem cells (SSCs). Following SF extraction, the content is analyzed by SDS-PAGE and stable porous 3D scaffolds are successfully prepared by merely Alg, SF, and a combination of Alg-SF, or Alg-SF-LM through freeze-drying. Then, the biomimetic scaffolds are characterized regarding the structural and biological properties, water absorption capacity, biocompatibility, biodegradability, and mechanical behavior. Neonatal mice testicular cells are seeded on three-dimensional scaffolds and their differentiation efficiency is evaluated using real-time PCR, flow cytometry, immunohistochemistry. Blend matrices showed uniform porous microstructures with interconnected networks, which maintained long-term stability and mechanical properties better than homogenous structures. Molecular analysis of the cells after 21 days of culture showed that the expression of differentiation-related proteins in cells that are developed in composite scaffolds is significantly higher than in other groups. The application of a composite system can lead to the differentiation of SSCs, paving the way for a novel infertility treatment landscape in the future.

The protective effects of astaxanthin on pre-antral follicle degeneration in ovine vitrified/warmed ovarian tissue.

This study assesses the protective effects of astaxanthin (AST) against vitrification/warming-induced cryoinjuries of ovarian tissue slices in sheep. Cortical slices of slaughterhouse acquired-ovine ovaries were randomly distributed in different groups: fresh, toxicity, and five vitrification groups including vitrification in presence of 0 (control group), 1, 10 and 100 µM astaxanthin or 100 µM vitamin E. After vitrification/warming and 24 h culturing, the samples were subjected to histological studies, antioxidant evaluation by TAC and TBAR assays, and assessment of relative expression of BMP4, BMP15, GDF9 and KITLG genes related to folliculogenesis and follicular growth regulation. According to the results, vitrification reduced the percentage of morphologically intact follicles compared to the fresh and toxicity groups (p < 0.05). In vitrification groups, vitamin E and all three concentrations of AST increased the percentage of intact pre-antral follicles and antioxidant activity relative to the vitrified control (p < 0.05). This enhancement significantly occurred in 10 µM AST group more than vitamin E (p < 0.05). Also, 10 µM concentration of AST enhanced the expression of all the examined genes compared to the control (p < 0.05), while the expression of BMP4, BMP15 and KITLG was higher in the AST than vitamin E (p < 0.05). The latter could increase only the expression of GDF9 compared to the control group (p = 0.011). In conclusion, AST is a highly effective antioxidant for maintaining the survival of pre-antral follicles, retaining cell density, increasing total antioxidant capacity, and increasing the expression of some genes related to follicular development after short-term culture of vitrified/warmed ovarian tissue slices.

Co-culturing or conditioned medium of Sertoli cells: Which one supports in vitro maturation of bovine oocytes and developmental competency of resulting embryos?

BACKGROUND: Sertoli cells (SCs) as supportive cells in the seminiferous tubule play an essential role in the nutrition and development of adjacent cells by secreting several beneficial growth factors, stimulators and cytokines which can be conceived to improve the developmental competency of oocyte or embryo in the co-culture system. OBJECTIVES: This study aimed to improve the maturation of bovine oocytes and consequently the development of resulting embryos in co-culture with SCs and their conditioned medium (CM). METHODS: The retrieved cumulus-oocyte complexes (COCs) from the abattoir-derived ovaries were matured in maturation medium alone (control group), in co-culture with ovine SCs (co-culture group), and in presence of 10% CM prepared in 33°C and 39°C (CM33 and CM39 groups). The nuclear maturation competency and subsequent embryo development rate of cultured COCs in all groups were evaluated. RESULTS: The results of this study showed that SCs and CM increased meiosis resumption from GV to the MII compared to the control group, significantly (p < 0.05). Besides, the degenerated oocytes in the co-culture group were significantly higher than those in the control, CM33 and CM39 groups (p < 0.05), and the lowest cleavage rate belonged to the co-culture group (p < 0.05). The blastocyst rate was also lower in the co-culture group than other groups and there was a significant difference between the control and two CM groups (p < 0.05). CONCLUSIONS: The Sertoli cells can be suitable for co-culturing with oocytes during IVM but detrimental for subsequent embryo development. In turn, Sertoli cell-derived conditioned medium (SC-CM) can provide sufficient bioactive materials for COCs to enhancing oocyte competence and embryo development.

Astaxanthin Relieves Busulfan-Induced Oxidative Apoptosis in Cultured Human Spermatogonial Stem Cells by Activating the Nrf-2/HO-1 pathway.

Many child cancer patients endure anticancer therapy containing alkylating agents before sexual maturity. Busulfan (BU), as an alkylating agent, is a chemotherapy drug, causing DNA damage and cytotoxicity in germ cells. In the present study, we aimed to investigate the protective effect of astaxanthin (AST), as a potent antioxidant and powerful reactive oxygen species (ROS) scavenger, on BU-induced toxicity in human spermatogonial stem cells. For this purpose, testes were obtained from four brain-dead donors. After tissue enzymatic digestions, testicular cells were cultured for 3 weeks for spermatogonial stem cell (SSC) isolation and purification. K562 cell line was cultured to survey the effect of AST on cancer treatment. The cultured SSCs and K562 cell line were finally treated with AST (10µM), BU (0.1nM), and AST+BU. The expression of NRF-2, HO-1, SOD2, SOD3, TP53, and apoptotic genes, including CASP9, CASP3, BCL2, and BAX, were assayed using real-time PCR. Moreover, ROS level in different groups and malondialdehyde level and total antioxidant capacity in cell contraction of SSCs were measured using ELISA. Data showed that AST significantly upregulated the expression of NRF-2 gene (P<0.001) and protein (P<0.005) and also significantly decreased the production of BU-induced ROS (P<0.001). AST activated the NRF-2/HO-1 pathway that could remarkably restrain BU-induced apoptosis in SSCs. Interestingly, AST upregulated the expression level of apoptosis genes in the K562 cell line. The results of this study indicated that AST reduces the side effects of BU on SSCs without interference with its chemotherapy effect on cancerous cells through modulation of the NRF-2/HO-1 and mitochondria-mediated apoptosis pathways.

Cryopreservation and its effects on motility and gene expression patterns and fertilizing potential of bovine epididymal sperm.

Despite encountering new challenges in using epididymal sperm recovered from cauda epididymides, this accessible and, in some species, worthwhile sample makes inevitable the further development of a suitable cryopreservation protocol. In this study, sperm was recovered from the epididymis of 4°C overnight stored slaughtered bulls' testes and the effects of cryopreservation on the bovine epididymal sperm motility (with CASA) and gene expression patterns (with quantitative Real time-PCR) were evaluated. Moreover the fertilizing potential of cryopreserved epididymal sperm was used in in vitro fertilization (IVF). After freezing and thawing of epididymal sperm, total and slow progressive sperm motility, VCL, VAP, MAD, ALH and BCF were significantly decreased (p < .05), while in the parameters of fast progressive motility, VSL, LIN, WOB and STR there were not any significant variations in the frozen sperm compared to fresh (non-frozen) counterpart. The assessment of abundance of transcripts encoding motility (TSSK6) and fertility (PRM1 and PRM2)-related genes in epididymal sperm, showed that these transcripts were affected by freezing especially in slow progressive motility status (p < .01). Furthermore, cleavage and blastocyst rate did not present any significant differences between bovine embryos produced in vitro by fresh or frozen-thawed epididymal sperm. It can be concluded that epididymal sperm has enough freezability after overnight testes storage, and cryopreservation could not affect the percentage of in vitro produced embryos in spite of the changes of relative abundance of some transcripts and direction progressive motility pattern of sperm.

The effect of amniotic membrane stem cells as donor nucleus on gene expression in reconstructed bovine oocytes.

Nuclear reprogramming of a differentiated cell in somatic cell nuclear transfer (SCNT) is a major concern in cloning procedures. Indeed, the nucleus of the donor cell often fails to express the genes which are a prerequisite for normal early embryo development. This study was aimed to evaluate the developmental competence and the expression pattern of some reprogramming related genes in bovine cloned embryos reconstructed with amniotic membrane stem cells (AMSCs) in comparison with those reconstructed with mesenchymal stem cells (MSCs) and adult fibroblasts (AF) as well as with in vitro fertilized (IVF) oocytes. In vitro matured abattoir-derived oocytes were considered as recipients and a hand-made cloning technique was employed for oocyte enucleation and nuclear transfer (NT) procedures. The expression pattern of genes involved in self-renewal and pluripotency (POU5F1, SOX2, NANOG), imprinting (IGF2, IGF2R), DNA methylation (DNMT1, DNMT3A), histone deacetylation (HDAC2), and apoptosis (BAX, BCL2) were evaluated in NT and IVF derived embryos. Despite the insignificant difference in cleavage rate between reconstructed and IVF oocytes, the blastocyst rate in the IVF group was higher than that of other groups. Among reconstructed oocytes, a higher blastocysts rate was observed in MSC-NT and AMSCs-NT derived embryos that were significantly higher than AF-NT derived ones. There were more similarities in the expression pattern of pluripotency and epigenetic modification genes between MSC-NT and IVF derived blastocysts compared with other groups. In conclusion, considering developmental competence, AMSCs, as alternative donors in SCNT procedure, like MSCs, were prone to have more advantage compared with AF.

Expression of ZFX gene correlated with the central features of the neoplastic phenotype in human brain tumors with distinct phenotypes.

BACKGROUND: The zinc finger transcription factor zinc finger protein, X-linked (ZFX) acts as an important director of self-renewal in several stem cell types. Moreover, ZFX expression abnormally increases in various cancers and relates to tumor grade. We performed this study, to examine its role in the pathogenesis of astrocytoma and meningioma. MATERIALS AND METHODS: We used real-time reverse transcription polymerase chain reaction method for evaluation of ZFX expression in 25 astrocytoma tumoral tissue and 25 meningioma tumoral tissues with different WHO grades. Furthermore, the association of gene expression with various clinic-pathological characteristics was examined. RESULTS: We found that there is a significant association between gene expression and different tumor grades, the presence or absence of invasion, forming and nonforming of glomeruloid vessels, the age over or under 50 and the presence or absence of calcification in astrocytomas. This is the first report that shows that ZFX was directly correlated with the central features of the neoplastic phenotype, including the growth of cancer cells, angiogenesis, and invasion. CONCLUSION: Regarding all the above-mentioned studies, it is highly plausible that silencing the expression of ZFX gene in gliomas has a major role in the therapeutic interventions of the disease in future.

Connexin-43: A possible mediator of heat stress effects on ram Sertoli cells.

Sertoli cells are an essential group of cells in seminiferous epithelium which provide nutritional and structural supports for spermatogenic cells via cell junctions. In this study, the gene expression of connexin-43, the most abundantly distributed gap junction protein of cells, was investigated in ram Sertoli cells under mild and severe heat stresses with real-time quantitative PCR. Sertoli cells were isolated from testes of 10 lambs. After culture and 3 passages, they were treated with mild (39 ˚C) and severe (42 ˚C) heat stress for 6 hr. The results showed a significant reduction in the percentage of live cells under severe heat stress compared to the control group (32 ˚C), (p <0.05). Relative quantification analysis revealed significantly higher (3.80 fold increase) values of connexin-43 transcripts in severely heat stressed group than control group (p <0.05). It is concluded that challenging Sertoli cells with 42 ˚C heat could threaten their survival, and overexpression of connexin-43 may cause dysfunction of Sertoli cells due to heat stress. These findings can be useful to identify the molecular mechanisms involved in adverse effects of heat stress on male reproduction and enhance our understanding of its pathogenesis.