Forced Suppression of let-7a-5p in Mouse Blastocysts Improves Implantation Rate.

Embryo implantation requires appropriate communication between the blastocyst and endometrium. Recurrent implantation failure is an essential component of assisted reproductive technology. Also, miRNA-mediated gene expression impacts the implantation process, and the downregulation of some miRs, such as mmu-let-7a, improves this process. In the present study, we evaluated the effect of let-7a forced suppression on the mouse implantation rate. In total, 100 adult female mice and 10 adult male mice were included (Strain CD-1). We analysed the expression of let-7a and its potential mRNAs targets (Igf1, Il1a, Itgb3 and Tgfb1) in control, sham and antagomir-treated blastocysts using quantitative reverse transcription PCR (qRT-PCR). The control and treated blastocysts were transferred to the 20 pseudopregnant mice so that the effect of let-7a suppression on the rate of implantation could be determined. The expression level of let-7a in the treatment group was significantly downregulated (P=0.001) In contrast, no significant expression changes were observed for let-7a or mRNAs targets when the sham and control groups were compared (P>0.05). In comparison to the controls, the antagomir-treated group exhibited significantly upregulated expression levels of Igf1 (0.0167), Itgb3 (0.045) and Tgfb1 (0.0115). Additionally, the implantation rate was significantly higher in the treatment group (78%) than the control group (61%) (P=0.0098). We found that forced suppression of mmu-let-7a-5p through successful transfection of Anti-miR leads to upregulation of downstream genes, Igf1, Itgb3 and Tgfb1, which directly involved in the trophoblast-endometrium attachment and improve the implantation rate.

Morphological and ultrastructural studies of human spermatogonial stem cells from patients with maturation arrest.

Destruction of spermatogonial stem cells (SSCs) along the chemotherapy and radiotherapy is one of the side effects of cancer treatments that lead to infertility. In vitro propagation of hSSCs is necessary to obtain an adequate number of cells for successful transplantation. In this study, hSSCs were isolated from testis biopsies of the patients with maturation arrest and proliferated in DMEM in the presence of LIF and bFGF for 5 weeks. The various types of human spermatogonia were identified in culture system and compared with testis tissue using morphological criteria at the ultrastructural level. The results showed that although many various types of spermatogonia were identified, but no remarkable difference was observed between spermatogonial cells in culture system and testis tissue. Electron and light microscopic studies of hSSC colonies did not show differentiated SSCs in the culture system. The results also showed that probably the suitable time for transplanting of SSCs in recipient testis is 2-3 weeks after culture. Because apoptosis which may affect the development of germ cells has not started in colony cells at this time and the population of apoptotic cells are low.

Derivation of motor neuron-like cells from neonatal mouse testis in a simple culture condition.

Embryonic stem cell (ESC) therapy is an exciting way to treat neurodegenerative disease and central nervous system injury. However, many ethical and immunological problems surround the use of embryonic stem cells. Finding an alternative source of stem cells is therefore pertinent. In this study, spermatogonia stem cells (SSCs) were used to generate mature motor neurons. SSCs were extracted from neonatal testes and cultured in DMED/F12 medium for 3 weeks. Characterisation of SSC-derived ESC-like cells was confirmed by RT-qPCR, immunostaining, alkaline phosphatase activity and their ability to form embryoid bodies (EBs). The EBs were induced by retinoic acid and Sonic hedgehog and trypsinised to obtain single induced cells. The single cells were cultured in neural medium for 18 days. Characterisation of neural precursors and motor neuron-like cells was confirmed by RT-qPCR and immunocytochemical analysis at the 7th day (early stage) and 18th day (late stage), respectively, of culturing. The neural precursors were found to be positive for nestin and Sox2, and a small fraction of cells expressed ß-tubulin III. Upon further differentiation, multipolar neurons were detected that expressed ß-tubulin III and MAP2 markers. Moreover, the expression levels of Olig2 and PAX6 were significantly lower, while HB9, Isl1 and Isl2 expression levels were higher at the late stage when compared to the early stage. These results show that SSCs have the potential to differentiate to motor neuron-like cells and express markers specific for mature motor neurons. However, the functional ability of these cells remains to be evaluated in future studies.

Effect of extracellular matrix on bovine spermatogonial stem cells and gene expression of niche factors regulating their development in vitro.

Extracellular matrix (ECM) could influence cells function through providing structural and functional networks facilitating the cellular interactions. The present study was conducted to evaluate the effect of culture on ECM versus plastic on bovine spermatogonial stem cells (SSCs) and growth factors regulating their development. Following isolation, bovine testicular cells were cultured on ECM-coated or uncoated (control) plates for 12 days. The colonization of SSCs was assessed by inverted microscope and the gene expression was evaluated using quantitative real-time PCR. The colonization rate was greater in ECM than the control group (P<0.05). The expression of markers of undifferentiated spermatogonia increased in response to conventional culture (P<0.05). Conversely, the expression of ckit as a marker for differentiated spermatogonia was reduced following culture in the control and ECM groups (P<0.05), but this decrease was less in ECM group (P<0.05). Accordingly, while cells cultured on uncoated plates had greater expression of markers of undifferentiated spermatogonia (P<0.05), cells cultured on ECM-coated plates showed higher expression of ckit (P<0.05). Moreover, culture on ECM resulted in higher expression of kit ligand (Kitlg; P<0.05), whereas culture on plastic led to greater expression of glial cell line-derived neurotrophic factor (Gdnf; P<0.05). In conclusion, the present study revealed that the permissive effect of ECM on bovine SSCs differentiation in vitro, which was probably mediated through upregulation of KITLG expression. Moreover, the results imply that GDNF might contribute to germ cells self-renewal during conventional culture.

Xenotransplantation assessment: morphometric study of human spermatogonial stem cells in recipient mouse testes.

The purpose of this study was (i) To establish in vitro propagation of human spermatogonial stem cells (hSSCs) from small testicular biopsies to obtain a high number of cells; (ii) to evaluate the presence of functional hSSCs in culture system by RT-PCR using DAZL, α6-Integrin, ß1-Integrin genes; and (iii) to evaluate the effects of cell concentration on successful xenotransplantation of hSSCs in mice testis. Donor hSSCs were obtained from men with maturation arrest of spermatogenesis duration 1 year ago. These cells were propagated in DMEM containing 1 ng ml(-1) bFGF (basic fibroblast grow factor) and 1500 U ml LIF (leucaemia inhibitory factor) for 5 weeks. Different concentrations of hSSCs transplanted into seminiferous tubules of busulfan-treated immunodeficient mice and analysed up to 8 weeks after transplantation. The results showed that expression of DAZL and α6-Integrin mRNA was increased as well as the colony formation of SSCs in vtro culture during 5 weeks. Proliferation occurred about 4 weeks after transplantation, but meiotic differentiation was not observed in recipient testis after 8 weeks. The difference in donor cells concentration had effect on homing spermatogenesis in recipient testis. Homologous transplantation of proliferated SSCs to seminiferous tubules of that patient individually may allow successful differentiation of transplanted cells.

Oligoprogenitor cells derived from spermatogonia stem cells improve remyelination in demyelination model.

Embryonic stem (ES) like cells-derived testis represents a possible alternative to replace of neurons and glia. Here, we differentiated spermatogonia cells to oligoprogenitor (OP) like cells and transplanted them to demyelination model and assess their recovery potential in a demyelinated corpus callosum model in rats. ES like cells were differentiated to OP like cells using appropriate inducers and were transplanted in situ to demyelinated corpus callosum. Cell integration as well as demyelination extension and myelination intensity changes were evaluated using histologic studies and immunocytochemistry after 2 and 4 weeks post transplantation. Investigation of Nestin, NF68, Olig2, and NG2 by immunocytochemical technique indicated the differentiation of ES like cells to neuroprogenitor and oligodendrocyte like cells in each induction stage. Histologic findings showed a significant decrease in demyelination extension and a significant increase in remyelination intensity in cell transplanted groups. Also on the base of PLP expression, differentiation of transplanted cells was confirmed to myelinogenic cells using immunocytochemistry technique. We conclude that ES like cells derived from spermatogonia cells can be differentiated to OP like cells that can form myelin after transplantation into the demyelination model in rat, this represents recovery potential of spermatogonia cells which opens new window for cell therapeutic approaches using spermatogonial stem cells.

Evaluation of the effects of cryopreservation on viability, proliferation and colony formation of human spermatogonial stem cells in vitro culture.

Proliferation of spermatogonial stem cells (SSCs) in vitro system is very important. It can enhance SSCs numbers for success of transplantation and treatment of infertility in cancer patients. In this study, testicular cells that obtained from azoospermia patients (n=8) by enzymatic digestion were cryopreserved at the beginning and after 2 weeks of culture. Then, frozen-thawed SSCs were co-cultured on fresh Sertoli cells (experimental group 1), and frozen-thawed Sertoli cells (experimental group 2) for another 3 weeks. In control group, fresh SSCs were co-cultured on fresh Sertoli cells. Viability rate after enzymatic digestion was 93.4%±5.0. Frozen-thawed testicular cells after 2 weeks of culture had a significantly (P<0.05) higher percentage of living cells compared to frozen-thawed testicular cells at the beginning of culture (59.2±7.05 and 46.3±8.40 respectively). The number of colonies in the experimental group 1 was significantly higher than experimental group 2 (19.6±2.8 and 8.33±1.5, respectively, P<0.05). The diameter of the colonies in the experimental group 1 was significantly higher than control and experimental group 2 (P<0.05) after 3 weeks of culture (269.7±52.1, 204.34±24.1 and 112.52±23.5 µm, respectively). Cryopreservation technique will raise the possibility of banking SSCs for men who have a cancer-related illness and waiting for radiotherapy and/or chemotherapy.

Impaired reproductive function of male rats infected with Toxoplasma gondii.

Toxoplasmosis is one of the classical conditions known to have an adverse effect on female reproductive functions, but a few investigations into male reproductive parameters have been performed. This work was carried out to study the effects of Toxoplasma gondii on reproductive function in male rats. Male rats were infected with the RH strain of T. gondii tachyzoites, and following every 10 days from 10 to 70 postinfection (PI), the percentage of body weight to testis weight ratio as well as epididymal sperm parameters (number, motility, viability, and morphology rates), serum testosterone (ST), intratesticular testosterone (ITT), serum lactate dehydrogenase (SLDH), intratesticular lactate dehydrogenase and fructose in seminal vesicles and coagulating glands were measured. The results of the study showed sperm motility, viability and concentration rates were significantly decreased temporary after infection up to 70 days. Sperm abnormality was also increased during these days. In addition, temporary alteration in ST, ITT, SLDH, intratesticular LDH and fructose in seminal vesicle and coagulating gland was observed PI. These findings suggest that toxoplasmosis can cause impermanent impairment on the reproductive parameters of male rats.

Comparison of colony formation in adult mouse spermatogonial stem cells developed in Sertoli and STO coculture systems.

This study aimed to compare the in vitro effects of coculture with Sertoli and SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) feeder layer cells on the efficiency of adult mouse spermatogonial stem cells (SSCs) colony formation. Sertoli and SSCs were isolated from testes, and their identity was confirmed using immunocytochemistry against Oct4, CDH1, PLZF and C-kit for SSCs and vimentin for Sertoli cells. SSCs were cultured in a simple culture system (control group) and on top of the Sertoli and STO feeder layers for 2 weeks. The number and diameter of colonies were evaluated during third, 7th, 10th and 14th day of culture, and the expression of the Oct-4, α6 and ß1 integrins was assessed using quantitative RT-PCR. Significant differences were observed between the three groups, separately for each time (P < 0.05), with higher mean in number and diameter for Sertoli cells (P < 0.05). The results of RT-PCR showed higher gene expression of ß1 integrin in Sertoli group, but no significant differences were observed in Oct-4 and α6 integrin gene expression among the three groups. Based the on the optimal effect of Seroli cells on the colony formation of SSCs, it is suggested to use these cells for better colonisation of SSCs.

Effects of basic fibroblast growth factor and leukaemia inhibitory factor on proliferation and short-term culture of human spermatogonial stem cells.

In this study, isolated spermatogonial stem cells (SSCs) and Sertoli cells using enzymatic digestion from patients with maturation arrest of spermatogenesis were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum in three different groups: (1) SSCs cultured without Sertoli cells (2) SSCs co-cultured with Sertoli cells (as control group), (3) SSCs co-cultured with Sertoli cells and adding different concentrations of basic fibroblast growth factor (0.1, 1, 10 ng ml(-1)) and human leukaemia inhibitory factor (1000, 1200, 1500 unit ml(-1)) as experimental groups. The assessment of colonies every 10 days during 5-week cultures showed that in the first group, the average number and diameter of the colonies were significantly lower than in the other groups (P < 0.05). The largest number of colonies was observed in control condition (32.29 ± 9.15) in day 30. The largest diameter of colonies was formed in combination dosages of 1 ng ml(-1) basic fibroblast growth factor (bFGF) + 1500 unit ml(-1) leukaemia inhibitory factor (LIF) (302.93 ± 37.68) and 10 ng ml(-1) bFGF and 1200 unit ml(-1) LIF (262.87 ± 35.54) in day 30 respectively. Isolated SSCs were positive for spermatogonial cell markers such as Oct4, Stra8, Piwil2 and Vasa but negative for Nanog. Transplantation technique indicated that hSSCs have good efficiency for colonisation of mouse seminiferous tubules after proliferation in culture system.

Germ cell apoptosis induced by experimental cryptorchidism is mediated by molecular pathways in mouse testis.

The aim of the study was to characterise the alterations in expression of some apoptosis regulators in unilaterally and bilaterally heat-treated mouse testes at different time intervals to 42 days after surgery. Cryptorchidism was induced in immature mice by returning the testis to the abdominal cavity via a surgical procedure. Transcript levels of Bax, Bcl-2 proper, p53 and survivin mRNA and protein were determined in normal and cryptorchid testes using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. RT-PCR data verified the elevation of p53 expression and decrease of Bax and Bcl-2 proper mRNA in the cryptorchid testis in a time-dependent manner. The expression of survivin 140 and 40 variants strongly decreased in the bilateral groups compared with unilateral and control groups. These changes were significantly different in the bilateral groups in comparison with the unilateral groups. Immunohistochemistry data showed that the intensity of p53 and Bax expression mainly increased in the remainder cells in the cryptorchid testis and the rates of Bcl-2 proper and survivin expression decreased mainly in the bilateral groups. These observations suggest that multiple molecular pathways participate in the germ cell apoptosis induced by cryptorchidism.

Differential gene expression and alternative splicing of survivin following mouse sciatic nerve injury.

STUDY DESIGN: In vivo studies using an axotomy model in adult male Naval Medical Research Institute mice. OBJECTIVE: Survivin, a unique member of the inhibitor of the apoptosis (IAP) protein family, is expressed during embryonal development, but is undetectable in terminally differentiated cells and tissues. Owing to the vital role of survivin in cellular proliferation and apoptotic cell death, and also to the necessity of treatment of the nervous system injuries, we have monitored survivin gene expression as well as its alternative splicing changes at different time points within injured mouse sciatic nerves. SETTING: Department of Genetics, School of Basic Sciences, Tarbiat Modares University, Tehran, Iran. METHODS: The sciatic nerves of adult male Naval Medical Research Institute mice were transected and the expression of survivin was examined in the distal and proximal parts of the dissected nerves as well as in the corresponding segments within the spinal cord of the animals, using semi-quantitative RT-PCR and immunohistochemistry. RESULTS: Survivin is not expressed in undamaged sciatic nerves, but, after sciatic nerve injury, it is gradually upregulated in proximal and distal parts of the dissected nerve (P<0.05). Survivin140 is the main variant expressed after injury, accompanied by a low expression of survivin40 (P<0.05). There was no expression of the survivin121 variant after injury. CONCLUSIONS: Survivin is differentially expressed and spliced in damaged nerve and spinal cord. Future works on the manipulation of expression and/or the splicing of survivin could decipher the potential effects of survivin variants on the regeneration of nerve and/or spinal cord injuries.

Effect of growth hormone on testicular dysfunction induced by methotrexate in rats.

Methotrexate (MTX) is a chemotherapeutic agent causing defective oogenesis and spermatogenesis. This study was performed to assess the role of human growth hormone (GH) on testis recovery after treatment with MTX. Forty male Wistar rats were selected and randomly divided into four groups (n = 10): control (vehicle), GH group (0.3 mg kg(-1) GH for 28 days, IP), MTX group (MTX 1 mg kg(-1) week(-1) for 4 weeks, IP) and GH/MTX group (0.3 mg kg(-1) GH for 28 day plus 1 mg kg(-1) week(-1) MTX for 4 weeks, IP). On days 14 and 28, five rats from each group were killed, testes of rats of all groups were removed, spermatozoa were collected from epididymis and then prepared for analysis. MTX caused significant increase in interstitial tissue and capsular thickness and decrease of testicular and body weight (P < 0.05). Moreover, it caused significant decline in seminiferous tubule diameter and epithelium thickness (P < 0.05). There was no obvious change in morphometrical parameters between MTX/GH and control groups. In MTX group, sperm parameters decreased significantly (P < 0.05). Administration of GH plus MTX reduced the effects of MTX on sperm parameters and testosterone concentration. These results suggested that GH had a protective effect on almost all destructive effects caused by MTX in rat testes and thus improved sperm parameters.

Efficiency of adult mouse spermatogonial stem cell colony formation under several culture conditions.

The aim of this study was to compare the in vitro effects of glial cell line-derived neurotrophic factor, stem cell factor, granulocyte macrophage-colony stimulating factor, and co-culture with Sertoli cells on the efficiency of adult mouse spermatogonial stem cells colony formation. For these purpose, both Sertoli and spermatogonial cells were isolated from adult mouse testes. The identity of the cells was confirmed through analysis of alkaline phosphatase activity, immunocytochemistry against OCT-4, c-kit, and vimentin, and also by transplantation of these cells in the recipient testes. The isolated spermatogonial cells were treated either with various concentrations of the above mentioned factors or co-cultured with Sertoli cells for 3 wk. The spermatogonial cells of the resulting colonies were transplanted via rete testis into the mouse testes, which were irradiated with 14 Gy. The results indicated that glial cell line-derived neurotrophic factor is the most appropriate factor for in vitro colonization of adult mice spermatogonial cells compared with other cytokines and growth factors. A short-term co-culture with Sertoli cells showed a significant increase in the number and diameter of the colonies compared with the treated growth factors and the control group. We have also demonstrated that mouse spermatogonial stem cells in the colonies after co-culturing with Sertoli cells could induce spermatogenesis in the recipient testes after transplantation.

CatSper genes expression, semen characteristics and histology of testes in the contusive spinal cord-injured mice model.

STUDY DESIGN: Experimental study. OBJECTIVE: A recently characterized CatSper genes, encodes for unique Ca(2+) channels in the testes, where they play essential roles in sperm motility. The aim of this research is to evaluate potential changes in the expression of CatSper genes, sperm parameters and testis histology following spinal cord injury (SCI). SETTING: Department of Anatomical Sciences, Tarbiat Modares University, Tehran, Iran. METHODS: A total of 75 adult NMRI mice were divided into three groups (25 in each group) of SCI, sham and control. Following laminectomy, SCI group was subjected to injury at the ninth thoracic vertebra. The epididymal sperm parameters were studied at day 1 and at weeks 1, 2, 4 and 6 after injury. One testis was removed for morphological study and the other testis were used for molecular study. Reverse transcription-PCR was performed at different time in all the groups. RESULTS: Our results revealed that SCI affects spermatogenesis as well as sperm quality and quantity. In gene expression analysis, there was a significant downregulation of CatSpers 1 and 2 at 4 and 6 weeks following contusion. There were no differences between the semen parameters and CatSper genes expression at the different time points in the sham and control groups. CONCLUSIONS: Our data indicated that the SCI mouse model causes a significant reduction in all sperm parameters, along with deleterious effects on spermatogenesis as well as the expression of CatSpers 1 and 2.

In vitro maturation of fresh and frozen-thawed mouse round spermatids.

Both initiation and maintenance of spermatogenesis are hormonally regulated by follicle stimulating hormone (rFSH) and testosterone. Co-culture systems also have important roles in the maintenance of spermatogenic cells. In this study, the effects of FSH and testosterone, co-culture system with Vero cells and co-culture supplemented with the hormones for maturation of frozen-thawed spermatids were determined. Testicular cells were suspended from the testis of National Medical Research Institute (NMRI) male mice and divided into two parts. The first aliquot of suspension was allocated for using as fresh and the rest was quickly cryopreserved. The frozen specimens were thawed and washed using Dulbecco modified Eagle's minimum essential medium (DMEM) medium. The fresh specimens were cultured in four groups: control (cultured on DMEM with 10% FBS), hormone (cultured on a medium supplemented with rFSH and testosterone), co-culture (cultured on Vero cells) and co-culture + hormone (cultured on Vero cells combined with rFSH and testosterone). The frozen-thawed specimens were cultured accordingly. The number of spermatids was recorded daily and the survival rates of each group were evaluated using Trypan blue test. The results showed that the number of the elongating spermatids was increased during the first day of the culture of fresh hormone, co-culture and co-culture + hormone groups. Viability rates of all kinds of the spermatid reduced during the 96 h of culturing. Our findings showed that the addition of hormone could support cell viability better than the co-culture. They also confirmed that the fresh round spermatid cells can progress into elongating and elongated spermatid only within the first 2 days of the culture in hormone, co-culture and co-culture + hormone groups. In the frozen-thawed specimens no extra significant increase in the number of cells was observed.