Interplay Between Non-Coding RNAs and Programmed Cell Death Proteins.

Programmed cell death (PDCD) family of proteins includes at least 12 members, function of seven of them being more investigated. These members are PDCD1, PDCD2, PDCD4, PDCD5, PDCD6, PDCD7 and PDCD10. Consistent with the important roles of these proteins in the regulation of apoptosis, dysregulation of PDCDs is associated with diverse disorders ranging from intervertebral disc degeneration, amyotrophic lateral sclerosis, immune thrombocytopenia, type 1 diabetes, congenital hypothyroidism, Alzheimer's disease to different types of cancers. More recently, the interaction between non-coding RNAs and different members of PDCD family is being discovered. In the current study, we described the functional interactions between PDCDs and two classes of non-coding RNAs, namely microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). miR-21 and miR-183 are two miRNAs whose interactions with PDCDs have been assessed in different contexts. The lncRNAs interaction with PDCDs is mainly assessed in the context of neoplasia indicating the role of MALAT1, MEG3, SNHG14 and LINC00473 in this process.

Exploring the role of non-coding RNAs in autophagy.

As a self-degradative mechanism, macroautophagy/autophagy has a role in the maintenance of energy homeostasis during critical periods in the development of cells. It also controls cellular damage through the eradication of damaged proteins and organelles. This process is accomplished by tens of ATG (autophagy-related) proteins. Recent studies have shown the involvement of non-coding RNAs in the regulation of autophagy. These transcripts mostly modulate the expression of ATG genes. Both long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been shown to modulate the autophagy mechanism. Levels of several lncRNAs and miRNAs are altered in this process. In the present review, we discuss the role of lncRNAs and miRNAs in the regulation of autophagy in diverse contexts such as cancer, deep vein thrombosis, spinal cord injury, diabetes and its complications, acute myocardial infarction, osteoarthritis, pre-eclampsia and epilepsy.Abbreviations: AMI: acute myocardial infarction; ATG: autophagy-related; lncRNA: long non-coding RNA; miRNA: microRNA.

Advanced bioengineering of male germ stem cells to preserve fertility.

In modern life, several factors such as genetics, exposure to toxins, and aging have resulted in significant levels of male infertility, estimated to be approximately 18% worldwide. In response, substantial progress has been made to improve in vitro fertilization treatments (e.g. microsurgical testicular sperm extraction (m-TESE), intra-cytoplasmic sperm injection (ICSI), and round spermatid injection (ROSI)). Mimicking the structure of testicular natural extracellular matrices (ECM) outside of the body is one clear route toward complete in vitro spermatogenesis and male fertility preservation. Here, a new wave of technological innovations is underway applying regenerative medicine strategies to cell-tissue culture on natural or synthetic scaffolds supplemented with bioactive factors. The emergence of advanced bioengineered systems suggests new hope for male fertility preservation through development of functional male germ cells. To date, few studies aimed at in vitro spermatogenesis have resulted in relevant numbers of mature gametes. However, a substantial body of knowledge on conditions that are required to maintain and mature male germ cells in vitro is now in place. This review focuses on advanced bioengineering methods such as microfluidic systems, bio-fabricated scaffolds, and 3D organ culture applied to the germline for fertility preservation through in vitro spermatogenesis.

Effects of chemotherapeutic agents on male germ cells and possible ameliorating impact of antioxidants.

Treatment of cancer in young adults is associated with several side effects, particularly in the reproductive system. Detrimental effects of chemotherapy on the germ cells depend on many factors including primary semen parameters, the way of drug administration, the kind and dose of chemotherapeutic regimens, and the phase of spermatogenesis during the time of drug administration. Lack of appropriate fertility preservation treatments particularly in the affected children necessitates the introduction of methods to amend the harmful effects of chemotherapeutic agents on male germ cells. Several studies have assessed the toxic effects of chemotherapeutic agents in rodent models and tested a number of antioxidants to evaluate their possible impact on the preservation of sperm cells. In the present manuscript, we describe the effects of the mostly investigated chemotherapeutic drugs in this regard i.e., cisplatin, doxorubicin, paclitaxel, 5-fluorouracil, and cyclophosphamide. As several in vivo and in vitro studies have shown the impact of antioxidants on chemotherapy-induced damage of sperms, we also describe the protective effects of antioxidants in this regard.

Interaction between non-coding RNAs and Toll-like receptors.

Toll-like receptors (TLRs) are a large group of pattern recognition receptors which are involved in the regulation of innate immune responses. Based on the interplay between TLRs and adapter molecules, two distinctive signaling cascades, namely the MyD88-dependent and TRIF-dependent pathways have been recognized. TLRs are involved in the development of a wide variety of diseases including cancer and autoimmune disorders. A large body of evidence has shown interaction between two classes of non-coding RNAs, namely microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). These interactions have prominent roles in the pathogenesis of several disorders including infectious disorders, autoimmune conditions and neoplastic disorders. This review aims at description of the interaction between these non-coding RNAs and TLRs.

The Impact of Non-coding RNAs in the Epithelial to Mesenchymal Transition.

Epithelial to mesenchymal transition (EMT) is a course of action that enables a polarized epithelial cell to undertake numerous biochemical alterations that allow it to adopt features of mesenchymal cells such as high migratory ability, invasive properties, resistance to apoptosis, and importantly higher-order formation of extracellular matrix elements. EMT has important roles in implantation and gastrulation of the embryo, inflammatory reactions and fibrosis, and transformation of cancer cells, their invasiveness and metastatic ability. Regarding the importance of EMT in the invasive progression of cancer, this process has been well studies in in this context. Non-coding RNAs (ncRNAs) have been shown to exert critical function in the regulation of cellular processes that are involved in the EMT. These processes include regulation of some transcription factors namely SNAI1 and SNAI2, ZEB1 and ZEB2, Twist, and E12/E47, modulation of chromatin configuration, alternative splicing, and protein stability and subcellular location of proteins. In the present paper, we describe the influence of ncRNAs including microRNAs and long non-coding RNAs in the EMT process and their application as biomarkers for this process and cancer progression and their potential as therapeutic targets.

The Interplay Between Non-coding RNAs and Insulin-Like Growth Factor Signaling in the Pathogenesis of Neoplasia.

The insulin-like growth factors (IGFs) are polypeptides with similar sequences with insulin. These factors regulate cell growth, development, maturation, and aging via different processes including the interplay with MAPK, Akt, and PI3K. IGF signaling participates in the pathogenesis of neoplasia, insulin resistance, diabetes mellitus, polycystic ovarian syndrome, cerebral ischemic injury, fatty liver disease, and several other conditions. Recent investigations have demonstrated the interplay between non-coding RNAs and IGF signaling. This interplay has fundamental roles in the development of the mentioned disorders. We designed the current study to search the available data about the role of IGF-associated non-coding RNAs in the evolution of neoplasia and other conditions. As novel therapeutic strategies have been designed for modification of IGF signaling, identification of the impact of non-coding RNAs in this pathway is necessary for the prediction of response to these modalities.

Counteracting effects of heavy metals and antioxidants on male fertility.

Infertility is regarded as a global health problem affecting 8-12% of couples. Male factors are regarded as the main cause of infertility in 40% of infertile couples and contribute to this condition in combination with female factors in another 20% of cases. Abnormal sperm parameters such as oligospermia, asthenospermia, and teratozoospermia result in male factor infertility. Several studies have shown the deteriorative impact of heavy metals on sperm parameters and fertility in human subjects or animal models. Other studies have pointed to the role of antioxidants in counteracting the detrimental effects of heavy metals. In the currents study, we summarize the main outcomes of studies that assessed the counteracting impacts of heavy metal and antioxidants on male fertility. Based on the provided data from animal studies, it seems rational to administrate appropriate antioxidants in persons who suffer from abnormal sperm parameters and infertility due to exposure to toxic elements. Yet, further human studies are needed to approve the beneficial effects of these antioxidants.

Interaction between non-coding RNAs and JNK in human disorders.

Jun N-terminal Kinase (JNK) signaling pathway is a conserved cascade among species with particular roles in diverse processes during embryogenesis and normal life. These kinases regulate functions of neurons and the immune system by affecting the expression of genes, modulating the arrangement of cytoskeletal proteins, and regulating apoptosis/survival pathways. They are also involved in carcinogenesis. Several miRNAs and lncRNAs have a functional relationship with JNKs. This interaction contributes to the pathogenesis of traumatic brain injury, ulcerative colitis, hepatic ischemia/ reperfusion injury, acute myocardial infarction, and a number of other disorders. Lung cancer, hepatocellular carcinoma, gall bladder cancer, melanoma, and colon cancer are among malignant conditions in which JNK-related miRNAs/ lncRNAs contribute. The current review aims at depicting the functional interaction between JNKs and lncRNAs/ miRNAs and describing the role of these regulatory transcripts in the pathobiology of human disorders.

The role of different compounds on the integrity of blood-testis barrier: A concise review based on in vitro and in vivo studies.

Sertoli cells are "nurturing cells'' in the seminiferous tubules of the testis which have essential roles in the development, proliferation and differentiation of germ cells. These cells also divide the seminiferous epithelium into a basal and an adluminal compartment and establish the blood-testis barrier (BTB). BTB shields haploid germ cells from recognition by the innate immune system. Moreover, after translocation of germ cells into the adluminal compartment their nutritional source is separated from the circulatory system being only supplied by the Sertoli cells. The integrity of BTB is influenced by several organic/ organometallic, hormonal and inflammatory substances. Moreover, several environmental contaminants such as BPA have hazardous effects on the integrity of BTB. In the current review, we summarize the results of studies that assessed the impact of these agents on the integrity of BTB. These studies have implications in understanding the molecular mechanism of male infertility and also in the male contraception.

Non-coding RNAs modulate function of extracellular matrix proteins.

The extracellular matrix (ECM) creates a multifaceted system for the interaction of diverse structural proteins, matricellular molecules, proteoglycans, hyaluronan, and various glycoproteins that collaborate and bind with each other to produce a bioactive polymer. Alterations in the composition and configuration of ECM elements influence the cellular phenotype, thus participating in the pathogenesis of several human disorders. Recent studies indicate the crucial roles of non-coding RNAs in the modulation of ECM. Several miRNAs such as miR-21, miR-26, miR-19, miR-140, miR-29, miR-30, miR-133 have been dysregulated in disorders that are associated with disruption or breakdown of the ECM. Moreover, expression of MALAT1, PVT1, SRA1, n379519, RMRP, PFL, TUG1, TM1P3, FAS-AS1, PART1, XIST, and expression of other lncRNAs is altered in disorders associated with the modification of ECM components. In the current review, we discuss the role of lncRNAs and miRNAs in the modification of ECM and their relevance with the pathophysiology of human disorders such as cardiac/ lung fibrosis, cardiomyopathy, heart failure, asthma, osteoarthritis, and cancers.

The mouse testis tissue culture could resume spermatogenesis as same as in vivo condition after human spermatogonial stem cells transplantation.

OBJECTIVE: The introduction of alternative systems in vivo is very important for cancer patients who are treated with gonadotoxic treatment. In this study, we examine the progression of the spermatogenesis process after human spermatogonial stem cell (SSCs) transplantation in vivo and in tissue culture conditions. MATERIALS AND METHODS: Human SSCs were obtained from a Testicular Sperm Extractions (TESE) sample, and characterization of these cells was confirmed by detecting the promyelocytic leukemia zinc finger (PLZF) protein. These cells, after being labeled with Di-alkyl Indocarbocyanine (DiI), were transplanted to adult azoospermia mouse testes treated with Busulfan 40mg/kg. The host testicular tissue culture was then considered a test group and in vivo transplant a control group. After 8 weeks, immunohistochemical, morphometric and molecular studies were performed. RESULTS: The results of morphometric studies indicated that the mean number of spermatogonia, spermatocytes, and spermatids in the test groups was significantly lower than in the control group (P<0.05) and most of the cells responded positively to DiI tracing. Immunohistochemical study in both groups revealed expression of PLZF, Synaptonemal complex protein 3 (SCP3) and Acrosin Binding Protein (ACRBP) proteins in spermatogonial cells, spermatocyte and spermatozoa, respectively. Also, PLZF, Transition Protein 1 (TP1) and Tektin-1 (Tekt1) human-specific genes had a significant difference in the between test groups and control groups (P<0.05) in molecular studies. CONCLUSION: These results suggest that the conditions of testicular tissue culture after transplantation of SSCs can support spermatogenesis resumption, as well as in an in vivo condition.

Characterization of embryonic stem-like cells derived from mouse spermatogonial stem cells following low-intensity ultrasound treatment.

OBJECTIVE: Spermatogonial stem cells (SSCs) are able to form embryonic stem-like cells (ES-like cells) and embryonic bodies (EBs). Low-intensity ultrasound stimulation (LIUS) has positive effects on the growth and differentiation of the different cells. In this study, we tried to investigate the effects of LIUS on SSC differentiation to ES-like cells. MATERIALS AND METHODS: SSCs were isolated from neonatal mice and their identification was confirmed by tracking of PLZF, Oct-4, and C-Kit proteins. The SSCs and Sertoli cells were co-cultured in DMEM/F12 supplemented with 15% FBS and LIF. SSCs stimulated by LIUS with 200mW/CM2 intensity. Characterization of obtained ES-like cells was confirmed with Sox2, Oct-4, and SSEA-1 immunofluorescence staining. Also, real-time PCR was performed to analyse the expression of c-Myc and Nanog genes in ES-Like Cells and Stra8, Piwil2 and Plzf genes in SSCs after 21 days of the in vitro culture. RESULTS: Our results showed c-Kit, PLZF and Oct-4 proteins were expressed positively in SSCs and Sox2, Oct-4, SSEA-1 in the ES-like cells by immunocytochemistry. The results of flow cytometry showed a significant increase in expression of c-Myc and Nanog in ES-like cells compared to SSCs (p<.05), whereas the Stra8, Piwil2, and Plzf became down-regulated during 21 days of culture. ES-like markers cell SSEA-1, Sox2 and Oct-4 were increased in the LIUS group compared to the control group (p<.05). CONCLUSION: The results indicated that ES-like cells with pluripotency characteristics were derived from SSCs.

Regulatory role of microRNAs on PTEN signaling.

Phosphatase and tensin homolog (PTEN) gene encodes a tumor suppressor protein which is altered in several malignancies. This protein is a negative regulator of the PI3K/AKT signaling. Several transcription factors regulate the expression of PTEN in positive or negative directions. Moreover, numerous microRNAs (miRNAs) have functional interactions with PTEN and inhibit its expression. Suppression of PTEN can attenuate the response of cancer cells to chemotherapeutic agents. Based on the critical role of this tumor suppressor gene, the identification of negative regulators of its expression has practical significance particularly in the prevention and management of cancer. Meanwhile, the interaction between miRNAs and PTEN has functional consequences in non-malignant disorders including myocardial infarction, osteoporosis, cerebral ischemic stroke, and recurrent abortion. In the present review, we describe the role of miRNAs in the regulation of expression and activity of PTEN.

Non-coding RNAs regulate angiogenic processes.

Angiogenesis has critical roles in numerous physiologic processes during embryonic and adult life such as wound healing and tissue regeneration. However, aberrant angiogenic processes have also been involved in the pathogenesis of several disorders such as cancer and diabetes mellitus. Vascular endothelial growth factor (VEGF) is implicated in the regulation of this process in several physiologic and pathologic conditions. Notably, several non-coding RNAs (ncRNAs) have been shown to influence angiogenesis through modulation of expression of VEGF or other angiogenic factors. In the current review, we summarize the function and characteristics of microRNAs and long non-coding RNAs which regulate angiogenic processes. Understanding the role of these transcripts in the angiogenesis can facilitate design of therapeutic strategies to defeat the pathogenic events during this process especially in the human malignancies. Besides, angiogenesis-related mechanisms can improve tissue regeneration after conditions such as arteriosclerosis, myocardial infarction and limb ischemia. Thus, ncRNA-regulated angiogenesis can be involved in the pathogenesis of several disorders.

Upregulation of FSHR and PCNA by administration of coenzyme Q10 on cyclophosphamide-induced premature ovarian failure in a mouse model.

Cyclophosphamide (CTX) has been broadly used in the clinic for the treatment of autoimmune disorders and ovarian cancer. The process of chemotherapy has significant toxicity in the reproductive system as it has detrimental effects on folliculogenesis, which leads to an irreversible premature ovarian failure (POF). Coenzyme Q10 (CoQ10) has positive impacts on the reproductive system due to its antioxidant properties, protecting the cells from free-radical oxidative damage and apoptosis. However, little is known about the possible synergistic effect of CTX and CoQ10 on the expression of genes involved in folliculogenesis, such as proliferation cell nuclear antigen (PCNA) and follicle-stimulating hormone receptor (FSHR). A total of 32 NMRI mice were applied and divided into four groups, including healthy control, CTX, CTX + CoQ10, and CoQ10 groups. The effects of CoQ10 on CTX-induced ovarian injury and folliculogenesis were examined by histopathological and real-time quantitative reverse transcription-polymerase chain reaction analyses. The rates of fertilization (in vitro fertilization), embryo development, as well as the level of reactive oxygen species (ROS) in metaphase II (MII) mouse oocytes after PMSG/HCC treatment were also assessed. Results showed that the treatment with CTX decreased the mRNA expression of PCNA and FSHR, IVF rate, and embryo development whereas the application of CoQ10 successfully reversed those factors. CoQ10 administration significantly enhanced histological morphology and decreased ROS levels and the number of atretic follicles in the ovary of CTX-treated mice. In conclusion, it seems that the protective effect of CoQ10 is exerted via the antioxidant and proliferative properties of this substance on CTX-induced ovarian damage.

In vitro transplantation of spermatogonial stem cells isolated from human frozen-thawed testis tissue can induce spermatogenesis under 3-dimensional tissue culture conditions.

BACKGROUND: Sperm production is one of the most complex biological processes in the body. In vitro production of sperm is one of the most important goals of researches in the field of male infertility treatment, which is very important in male cancer patients treated with gonadotoxic methods and drugs. In this study, we examine the progression of spermatogenesis after transplantation of spermatogonial stem cells under conditions of testicular tissue culture. RESULTS: Testicular tissue samples from azoospermic patients were obtained and then these were freeze-thawed. Spermatogonial stem cells were isolated by two enzymatic digestion steps and the identification of these cells was confirmed by detecting the PLZF protein. These cells, after being labeled with DiI, were transplanted in azoospermia adult mice model. The host testes were placed on agarose gel as tissue culture system. After 8 weeks, histomorphometric, immunohistochemical and molecular studies were performed. The results of histomorphometric studies showed that the mean number of spermatogonial cells, spermatocytes and spermatids in the experimental group was significantly more than the control group (without transplantation) (P < 0.05) and most of the cells responded positively to the detection of DiI. Immunohistochemical studies in host testes fragments in the experimental group express the PLZF, SCP3 and ACRBP proteins in spermatogonial cells, spermatocyte and spermatozoa, respectively, which confirmed the human nature of these cells. Also, in molecular studies of PLZF, Tekt1 and TP1, the results indicated that the genes were positive in the test group, while not in the control group. CONCLUSION: These results suggest that the slow freezing of SSCs can support the induction of spermatogenesis to produce haploid cells under the 3-dimensional testicular tissue culture.

Successful Human Spermatogonial Stem Cells Homing in Recipient Mouse Testis after In Vitro Transplantation and Organ Culture.

OBJECTIVE: In vitro transplantation (IVT) of spermatogonial stem cells (SSCs) is one of the most recent methods in transplantation in recent decades. In this study, IVT and SSCs homing on seminiferous tubules of host testis in organ culture have been studied. MATERIALS AND METHODS: In this experimental study, human SSCs were isolated and their identities were confirmed by tracking their promyelocytic leukemia zinc finger (PLZF) protein. These cells were transplanted to adult azoospermia mouse testes using two methods, namely, IVT and in vivo transplantation as transplantation groups, and testes without transplantation of cells were assigned in the control group. Then histomorphometric, immunohistochemical and molecular studies were done after 2 weeks. RESULTS: After two weeks, histomorphometric studies revealed that the number of subsided spermatogonial cells (SCs) and the percentage of tubules with subsided SCs in IVT and in vivo groups were significantly more than those in the control group (P<0.05). Immunohistochemical studies in the transplantation groups confirmed that the PLZF protein was expressed in the cells subsided on the seminiferous tubule. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) demonstrated that the PLZF gene expression was only positive in the transplantation groups, but it was not significantly different between the IVT group and the in vivo group (P>0.05). CONCLUSION: Testicular tissue culture conditions after SSC transplantation can help these cells subside on the seminiferous tubule basement membrane.

In vitro transplantation of spermatogonial stem cells isolated from human frozen-thawed testis tissue can induce spermatogenesis under 3-dimensional tissue culture conditions

BACKGROUND: Sperm production is one of the most complex biological processes in the body. In vitro production of sperm is one of the most important goals of researches in the field of male infertility treatment, which is very important in male cancer patients treated with gonadotoxic methods and drugs. In this study, we examine the progression of spermatogenesis after transplantation of spermatogonial stem cells under conditions of testicular tissue culture. RESULTS: Testicular tissue samples from azoospermic patients were obtained and then these were freeze-thawed. Spermatogonial stem cells were isolated by two enzymatic digestion steps and the identification of these cells was confirmed by detecting the PLZF protein. These cells, after being labeled with DiI, were transplanted in azoospermia adult mice model. The host testes were placed on agarose gel as tissue culture system. After 8 weeks, histomorphometric, immunohistochemical and molecular studies were performed. The results of histomorphometric studies showed that the mean number of spermatogonial cells, spermatocytes and spermatids in the experimental group was significantly more than the control group (without transplantation) (P < 0.05) and most of the cells responded positively to the detection of DiI. Immunohistochemical studies in host testes fragments in the experimental group express the PLZF, SCP3 and ACRBP proteins in spermatogonial cells, spermatocyte and spermatozoa, respectively, which confirmed the human nature of these cells. Also, in molecular studies of PLZF, Tekt1 and TP1, the results indicated that the genes were positive in the test group, while not in the control group. CONCLUSION: These results suggest that the slow freezing of SSCs can support the induction of spermatogenesis to produce haploid cells under the 3-dimensional testicular tissue culture.

Upregulation of Integrin-α6 and Integrin-ß1 Gene Expressions in Mouse Spermatogonial Stem Cells after Continues and Pulsed Low Intensity Ultrasound Stimulation.

OBJECTIVES: low intensity ultrasound (continues and pulsed) is a form of energy. Spermatogonial stem cells (SSCs) are at the base of male fertility. This study investigated the effects of low intensity ultrasound stimulation (LIUS) and low intensity pulsed ultrasound stimulation (LIUPS) on the expression of germ cell-specific and pluripotency genes in SSCs in vitro. MATERIALS AND METHODS: In this experimental study, isolated SSCs from neonatal male mice were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS). In addition, to confirm identification of SSCs, PLZF protein was detected positively in SSCs derived colonies. SSCs were stimulated by LIUS and LIUPS for 5 days, followed by assessment of expression of integrin-α6 (Itga6) and ß1 (Itgß1), as two germ cell-specific genes, and Oct- 4, as a pluripotency gene, on day 21st by quantitive reverse transcriptase-polymerase chain reaction (qRT-PCR). To investigate the proliferation rate and colonization of SSCs in different groups, counting whole number of the cells and colonies as well as analysis of the respective diameters were performed on days 7th, 14th and 21th. Data was analyzed by ANOVA test. RESULTS: LIUS and LIUPS treatment of mouse SSCs increased expression of Itga6 and Itgß1 genes in the experimental groups, compared to the control group (P<0.05), whereas there was no significant difference between the groups, regarding the expression of Oct-4 gene. These treatments maintained survival rate, while they increased proliferation rate and colonization of SSCs during the first week of culture. However, within the second week, proliferation rate and colonization were decreased in the experimental groups. CONCLUSIONS: These results suggested that LIUS and LIUPS treatment had good effect on SSCs proliferation and colonization, based on the gene-specific marker expression during 21 days culture in vitro.