RESUMEN
The present study aimed to: (i) identify the exogenous factors that allow in vitro differentiation of mouse spermatogonial stem cells (SSCs) from embryonic stem cells (ESCs); (ii) evaluate the effects of Sertoli cells in SSC enrichment; and (iii) assess the success of transplantation using in vitro differentiated SSCs in a mouse busulfan-treated azoospermia model. A 1-day-old embryoid body (EB) received 5 ng/ml of bone morphogenetic protein 4 (BMP4) for 4 days, 3 µM retinoic acid (RA) in a SIM mouse embryo-derived thioguanine and ouabain resistant (STO) co-culture system for 7 days, and was subsequently co-cultured for 2 days with Sertoli cells in the presence or absence of a leukaemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and RA composition, and in the presence of these factors in simple culture medium. Higher viability, proliferation and germ cell gene expression were seen in the presence of the LIF, bFGF and RA composition, on top of Sertoli cells. Immunocytochemistry results showed higher CDH1 expression in this group. Sertoli co-culture had no effects on SSC proliferation. Eight weeks after transplantation, injected cells were observed at the base of the seminiferous tubules and in the recipient testes. The number of spermatogonia and the mass of the testes were higher in transplanted testes relative to the control group. It seems that transplantation of these cells can be useful in infertility treatment.
Asunto(s)
Azoospermia/cirugía , Células Madre Embrionarias/fisiología , Espermatogénesis/fisiología , Espermatozoides/trasplante , Animales , Diferenciación Celular/fisiología , Modelos Animales de Enfermedad , Masculino , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Células de Sertoli/fisiología , Testículo/cirugíaRESUMEN
One of the treatments to infertility is In Vitro Fertilization (IVF). In the course of IVF, fertilization rate can be improved through stress reduction. Probably one of the causes of low outcome of IVF is changes in uterine glycoconjugates that are first site of contact between blastocyst and uterus, due to stress, e.g., stress of injection. Thus, the study of the injectional stress effects on implantation period is very important to improve the outcome of IVF. Sixteen mature female rats were divided to experimental and control groups. Experimental rats injected with 0.5cm3 distilled water intraperitoneally in diestrus or proestrus and 10 I.U HCG in estrus phase. Control rats injected only with 10 I.U HCG in estrus phase. The experimental and control rats mated with proven fertile male rats, sacrificed at 5.5 day of gestation (time of implantation) and their uterus horns removed. Uterine sections were stained with WGA, DBA, PNA, ConA, SBA and UEA lectins and grading of the intensity of the reaction in apical membrane, Golgi zone and basement membrane of uterine epithelial cells and uterine glands were performed by an arbitrary method. The intensity of the reaction to WGA and DBA in apical membrane and Golgi zone was significantly high in experimental group. It seems that injectional stress can decrease the rate of implantation through alteration in uterine glycoconjugates, e.g. increase in negatively charged glycoconjugates such as sialic acid, which reduce the receptivity of uterus for blastocyst.
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Blastocisto/efectos de los fármacos , Implantación del Embrión/efectos de los fármacos , Endometrio/efectos de los fármacos , Glicoconjugados/análisis , Estrés Fisiológico , Animales , Blastocisto/fisiología , Gonadotropina Coriónica/administración & dosificación , Endometrio/química , Endometrio/fisiología , Ciclo Estral/efectos de los fármacos , Ciclo Estral/fisiología , Femenino , Fertilización In Vitro , Glicoconjugados/biosíntesis , Humanos , Inmunohistoquímica , Inyecciones Intraperitoneales , Masculino , Lectinas de Plantas/química , Ratas , Ratas Sprague-DawleyRESUMEN
Premature ovarian insufficiency (POI) is a complex disorder that can result in varying degrees of infertility. Recently, mesenchymal stem cell (MSC) therapy and its derivatives, such as exosomes, have been introduced as novel strategies for the treatment of POI. This review discusses the features, limitations, and challenges of MSC and exosome therapy in the treatment of POI and provides readers with new insights for comparing and selecting chemical agents, optimizing doses, and other factors involved in study design and treatment strategies. MSC therapy has been shown to improve ovarian function in some animals with POI, but it can also have side effects such as high cost, time-consuming processes, limited lifespan and cell sources, loss of original characteristics during in vitro proliferation, dependence on specific culture environments, potential immune reactions, unknown therapeutic mechanisms, etc. However, exosome therapy is a newer therapy that has not been studied as extensively as MSC therapy, but that it has shown some promise in animal studies. The evidence for the effectiveness of MSC and exosome therapy is still limited, and more research is needed to determine whether these therapies are effective and safe for women with POI. This study presents a new perspective for researchers to advance their research in the fields of cell-based and cell-free therapies.
RESUMEN
Infertility is one of the most prevalent health disorders in reproductive-age males and females. Ficus carica (Fc), an herbal plant, has been used traditionally for the treatment of different diseases such as infertility especially in Iranian folk medicine. This study examined the effects of Fc leaf extract on the proliferation of mice spermatogonial stem cells (SSCs). Phenolic, flavonoid content, major polyphenolic compounds and antioxidant activity of the extract was evaluated respectively by Folin-Ciocateu, aluminum chloride, HPLC and the FRAP and DPPH methods. Testicular cells of neonate mice were extracted and their identity was confirmed using cytokeratin for Sertoli and Oct-4, CDHI and PLZF for SSCs. Effects of Fc (0.0875, 0.175, 0.35, 0.71 and 1.42 mg/ml) was evaluated at third, 7th, 9th and 14th days of culture by colony assay. The expression of the Mvh, GFRα1 and Oct-4 genes and the viability and proliferation of cultured cells was assessed at the end of the culture period. The extract has a rich phenolic and flavonoid content such as Rutin, Psoralen, Bergapten and Caffeoylmalic acid using HPLC analysis. It also had a potent reducing and radical scavenging activity. Morphology of colonies was similar in all groups. Higher viability, proliferation, colony number and diameter of SSCs was seen in the presence of Fc leaf extract in a dose-dependent manner so that higher number and diameter of colonies were observed in two higher doses of 0.71 and 1.42 mg/ml, separately for each time point relative to other groups. The Mvh, Oct-4 and GFRα1 genes expression had no significant differences between groups. It seems that Fc leaf extract not only had no any cytotoxic effects on the viability and proliferation of SSCs but also support their stemness state. So, this culture system can be employed for enrichment of germ stem cells for use in clinical applications.
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Calcium channels play essential roles in sperm motility. A family of sperm-specific cation channels including CatSper1-4 has been identified as voltage-dependent ion channels that act as sperm motility regulators. Methamphetamine is known to cause apoptosis in seminiferous tubules and affect sperm quality. This research was conducted to investigate the effects of methamphetamine on expression of the CatSper family and Mvh genes. Thirty-six adult Wistar rats were divided into four groups of nine rats each: the control and experimental groups 1, 2, and 3. The control group received no solvents or drugs, but experimental groups 1, 2, and 3 were daily given 0.2 mL of a solution by gavage that contained 0.5, 1, and 2 mg of methamphetamine, respectively, for 45 days. The rats were then anesthetized, and one testis removed from each rat was used in a reverse transcription-polymerase chain reaction (RT-PCR). Analysis of variance (ANOVA) and Tukey's posthoc test were used to analyze the data at the P < 0.05 significance level. Treatment with methamphetamine resulted in decreased testis and epididymis weights compared to the control rats. The results showed that the mRNA fold expression level of the CatSper family and Mvh genes decreased significantly in experimental groups compared to that in the control (P < 0.05). Methamphetamine decreased the expression levels of the CatSper and Mvh genes, and thus, it seemed that it can increase the probability of infertility through sperm motility reduction by lowering the expression levels of these genes.
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One of the female causes of infertility is anovulation which is treatable with gonadotropin hormones. These hormones affect the molecular organization of the uterus such as glycoconjugates that are the first site of contact between the blastocyst and the uterus. The objective of this project was to study the alteration of glycoconjugates on the uterine apical, Golgi zone, and basement membrane of epithelial cells and the uterine gland after hyperstimulation with pregnant mare serum gonadotropin (PMSG) (4, 8, 16, 24, and 40 IU), during the implantation period. Injection of PMSG (in experimental groups) and injection of distilled water (in the control group) were followed by HCG administration (10 IU), mating, isolation of positive vaginal plug rats, and killing at 5.5 days of pregnancy. Histochemistry was done on the pregnant uterine horns with the use of WGA, DBA, PNA, ConA, SBA, and UEA lectins. The intensity of the immunohistochemical staining was scored, and quantitative data were generated. 4 IU did not show any significant differences with the control, 8 IU had less effect on the alteration of the Golgi zone, and apical and basement membrane glycoconjugates and 40 IU had the least effects on the alteration of uterine gland glycoconjugates. Also, 24 IU had the most effect on the alteration of uterine glycoconjugates. Understanding of the effects of gonadotropin hormones at the uterine level in implantation time helps to optimize hormonal manipulation for improving the outcome of assisted reproductive procedures. It seems that the optimal dose for superovulation and less alteration in uterine glycoconjugates of rats at implantation time were induced by the administration of 8 IU PMSG.
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Gonadotropina Coriónica/metabolismo , Implantación del Embrión/efectos de los fármacos , Glicoconjugados/metabolismo , Útero/metabolismo , Animales , Blastocisto/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Aparato de Golgi/metabolismo , Caballos , Inmunohistoquímica , Lectinas/química , Embarazo , Preñez , Ratas , Ratas Sprague-DawleyRESUMEN
An in vitro system that supports primordial germ cells (PGCs) survival and proliferation is useful for enhancement of these cells and efficient transplantation in infertility disorders. One approach is cultivation of PGCs under proper conditions that allow self-renewal and proliferation of PGCs. For this purpose, we compared the effects of different concentrations of retinoic acid (RA), and the effect of PGCs co-culture (Co-C) with SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cells on the proliferation of embryonic stem cells (ESCs)-derived PGCs. One-day-old embryoid body (EB) was cultured for 4 days in simple culture system in the presence of 5 ng/ml bone morphogenetic protein-4 (BMP4) (SCB group) for PGC induction. For PGC enrichment, ESCs-derived germ cells were cultured for 7 days in the presence of different doses (0-5 µM) of RA, both in the simple and STO Co-C systems. At the end of the culture period, viability and proliferation rates were assessed and expression of mouse vasa homologue (Mvh), α6 integrin, ß1 integrin, stimulated by retinoic acid 8 (Stra8) and piwi (Drosophila)-like 2 (Piwil2) was evaluated using quantitative PCR. Also, the inductive effects were investigated immunocytochemically with Mvh and cadherin1 (CDH1) on the selected groups. Immunocytochemistry/PCR results showed higher expression of Mvh, the PGC-specific marker, in 3 µM RA concentrations on the top of the STO feeder layer. Meanwhile, assessment of the Stra8 mRNA and CDH1 protein, the specific makers for spermatogonia, showed no significant differences between groups. Based on the results, it seems that in the presence of 3 µM RA on top of the STO feeder layer cells, the majority of the cells transdifferentiated into germ cells were PGCs.
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Proliferación Celular/efectos de los fármacos , Transdiferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Espermatogonias/citología , Tretinoina/farmacología , Proteínas Adaptadoras Transductoras de Señales/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Argonautas/efectos de los fármacos , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Biomarcadores/metabolismo , Proteína Morfogenética Ósea 4/metabolismo , Proteínas Cdh1/efectos de los fármacos , Proteínas Cdh1/metabolismo , Diferenciación Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , ARN Helicasas DEAD-box/efectos de los fármacos , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Células Madre Embrionarias/citología , Inmunohistoquímica , Integrina alfa6beta1/efectos de los fármacos , Integrina alfa6beta1/genética , Integrina alfa6beta1/metabolismo , Masculino , Ratones , Espermatogonias/metabolismo , TranscriptomaRESUMEN
The present study aims to confirm and analyse germ cell-related patterns and specific gene expressions at a very early stage of cell commitment. Following the XY cytogenetic confirmation of the CCE mouse embryonic stem cells (mESCs) line, cells were cultured to form embryoid bodies (EBs). Expression pattern assessment of the mouse vasa homologue (Mvh), Stra8, α6 and ß1 integrin genes in ESC and 1-3-day-old EB showed that all genes except α6 integrin were expressed in the ESC. The mean calibration of Mvh, Stra8 and α6 integrin expression significantly increased upon EB formation compared with the ESCs. During mouse embryogenesis, the signalling of bone morphogenetic protein (BMP) is essential for germ-line formation. To investigate its role in germ-line induction in vitro, mESCs were cultured as 1-day-old EB aggregates with BMP4 for 4 days in STO co-culture systems, in the presence and absence of 5 ng/ml BMP4. At the end of the culture period, colony assay (number and diameter) was performed and the viability percentage and proliferation rate was determined. There were no significant statistical differences in the abovementioned criteria between these two groups. Moreover, the expression of Mvh, α6 and ß1 integrins, Stra8 and Piwil2 genes was evaluated in co-culture groups. The molecular results of co-culture groups showed higher-but insignificant-Piwil2 and significant α6 integrin expressions in BMP4 treated co-culture systems. These results confirmed that the EB system and the presence of BMP4 in a STO co-culture system improve the differentiation of ESCs to germ cell.
RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Date palm (Phoenix dactylifera L.) pollen (DPP) is widely used as a folk remedy for male infertility treatment, and has well known medicinal effects. AIM OF THE STUDY: This study aimed to determine the in vitro effects of DPP on the efficiency of neonate mouse spermatogonial stem cells (SSCs) proliferation. MATERIAL AND METHODS: Sertoli and SSCs were isolated from 6 to 10-days-old mouse testes, and their identity was confirmed using immunocytochemistry against cytokeratin for sertoli cells and PLZF, Oct-4 and CDH-1 for SSCs. Isolated testicular cells were cultured in the absence or presence of 0.06, 0.25 and 0.62mg/ml concentrations of DPP aqueous extract for 2 weeks. The number and diameter of SSC colonies were assessed during third, 7th, 9th and 14th day of culture, and the expression of the Mvh, GFRα-1 and Oct-4 was evaluated using quantitative PCR at the end of the culture period. The significance of the data was analyzed using ANOVA and paired samples t-test and Tukey and Bonferroni test as post hoc tests at the level of p≤0.05. RESULTS: Pattern assay of colony formation showed that SSCs numbers increased in the present of 0.62mg/ml concentration of DPP extract with higher slop relative to other groups (P <0.05). Colony diameters had no significant difference between groups in 3th, 7th, 9th and 14th days after culture. The Mvh and Oct-4 genes expression had no significant difference between groups, while GFRα1 expression was increased significantly in cells treated with 0.06mg/ml concentration relative to other groups (P<0.05). CONCLUSION: It seems that co-culture of SSCs with sertoli sells in the presence of low doses of DPP can increase SSCs proliferation and keep their stemness state, while higher concentrations can differentiate the treated cells.
Asunto(s)
Phoeniceae , Extractos Vegetales/farmacología , Polen/química , Células de Sertoli/efectos de los fármacos , Espermatogonias/citología , Células Madre/efectos de los fármacos , Animales , Animales Recién Nacidos , Proteínas Cdh1/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , ARN Helicasas DEAD-box/genética , Expresión Génica/efectos de los fármacos , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Masculino , Ratones , Factor 3 de Transcripción de Unión a Octámeros/genética , Células de Sertoli/citología , Células de Sertoli/metabolismo , Células Madre/citología , Células Madre/metabolismoRESUMEN
Formaldehyde (FA) is the leading cause of cellular injury and oxidative damage in testis that is one of the main infertility causes. There has been an increasing evidence of herbal remedies use in male infertility treatment. This assay examines the role of Ficus carica (Fc) leaf extracts in sperm parameters and testis of mice intoxicated with FA. Twenty-five adult male mice were randomly divided into control; sham; FA-treated (10 mg/kg twice per day); Fc-treated (200 mg/kg); and FA + Fc-treated groups. Cauda epididymal spermatozoa were analyzed for viability, count, and motility. Testes were weighed and gonadosomatic index (GSI) was calculated. Also, histoarchitecture of seminiferous tubules was assessed in the Haematoxylin and Eosin stained paraffin sections. The findings showed that FA significantly decreased GSI and increased percentage of immotile sperm compared with control group. Disorganized and vacuolated seminiferous epithelium, spermatogenic arrest, and lumen filled with immature germ cells were also observed in the testes. However, Fc leaf extracts improved sperm count, nonprogressive motility of spermatozoa, and GSI in FA-treated testes. Moreover, seminiferous tubule with spermatogenic arrest was rarely seen, indicating that Fc has the positive effects on testis and epididymal sperm parameters exposed with FA.
RESUMEN
There is a fast growing tendency in the use of herbal remedies in developing countries. One of the traditional medicines used for male infertility treatment is date palm (Phoenix dactylifera) pollen (DPP). Isolated spermatogonial stem cells and sertoli cells using enzymatic digestion were grown in Dulbecco's modified Eagle's medium supplemented with 4% foetal bovine serum in the absence or presence of 0.06, 0.25 and 0.62 mg/mL concentrations of aqueous extract of DPP for 2 weeks. The assessment of mean number of the whole cells and the living cells showed that there were no significant differences between the mean viability percentage and proliferation rate between control and experimental groups (P>0.05). As there are no cytotoxicity effects of DPP in our cultural system, this system can be utilised for the enrichment or differentiation of these cells in clinical applications, cell replacement therapy, tissue regeneration and tissue engineering applications.
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Phoeniceae/química , Extractos Vegetales/farmacología , Polen/química , Células de Sertoli/efectos de los fármacos , Espermatogonias/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Masculino , Ratones , Extractos Vegetales/química , Testículo/citologíaRESUMEN
Presence of specific growth factors and feeder layers are thought to be important for in vitro embryonic stem cell (ESCs) differentiation. In this study, the effect of bone morphogenetic protein 4 (BMP4) and mouse embryonic fibroblasts (MEFs) co-culture system on germ cell differentiation from mouse ESCs was evaluated. One-day-old embryoid body was cultured for 4 d in simple culture systems or on top of the MEFs, both in the presence or absence of BMP4. Data showed significant higher viability percent and proliferation rate in simple culture media compared to co-culture systems. Analysis of gene expression indicated that the germ cell-specific genes (VASA and Stra8) were expressed in a significant higher ratio in BMP4-treated cells in simple culture system. Also, the results of immunocytochemistry in simple culture systems showed that the mean percentage of immunostaining cells of VASA, the primordial germ cell (PGC) marker, was increased significantly in BMP4-treated cells compared with BMP4-free group. Meanwhile, CDH1, the late premiotic germ cell marker, showed no significant difference between these two groups. The results suggest that BMP4 is an efficient inducer in PGC derivation from mouse ESC. However, the employment of MEFs as feeder has no apparent effect on PGC derivation.