siRNAs targetting sphingomyelin phosphodiesterase 1 protect mouse oocytes from apoptosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the potential of siRNAs targeting sphingomyelin phosphodiesterase 1 (SMPD1) in protecting the oocytes from apoptosis, and explore new approaches to female fertility preservation.</p><p><b>METHODS</b>Chemically synthesized siRNA targeting SMPD1 were introduced into mouse oocytes retrieved by hyperstimulation, and the cell apoptosis was analyzed by comic assay 48 and 72 h later.</p><p><b>RESULTS</b>In the oocytes without any siRNA injection, oocyte DNA damage occurred after 24 h, and large amount of DNA fragments migrated from the cells 48 h later. In oocytes injected with siRNA003, DNA migration decreased significantly as compared with the control and the other two groups injected with siRNA001 and siRNA002 (P<0.01).</p><p><b>CONCLUSION</b>siRNA targeting SMPD1 may protect the oocytes from apoptosis, and has the potential for use in future female fertility preservation.</p>

Mesenchymal stem cells do not differentiate into "quasi-sperm" / 中华男科学杂志

<p><b>OBJECTIVE</b>To determine whether the surviving mesenchymal stem cells (MSCs) in the testis after transplantation can differentiate into quasi-sperm.</p><p><b>METHODS</b>(1) Making an animal model with sterilized testes. Forty 4-week old white male BASB/C mice were used to establish an animal model with sterilized testes and divided randomly into an experimental and a control group. (2) Cell preparation. The MSCs from 10 gray male 129-mice were isolated, cultured and purified by Percoll density gradient centrifugation combined with the adherent method. When the MSCs grew to an adequate number, they were made into a cell suspension with NS at a concentration of 1 million cells/ml. (3) Xenogeneic transplantation of the MSCs into the testis. The MSC suspension was blindly injected into the testes of the mice in the experimental group and NS into the testes of the controls. (4) Post-transplantation observation. Forty white female BASB/C mice were adopted, each put into a box with a male mouse from the experimental group or the control group, and then observed for pregnancy.</p><p><b>RESULTS</b>In the experimental group, 8 cases of pregnancy (40%) were observed at 31-46 d (38.5 d on average), the offspring all white. In the control group, only 1 case of pregnancy (5%) was seen at 45 d, the offspring all white, too. It was suggested that the MSCs of the 129-mice failed to differentiate into functional quasi-sperm and pass their genes to their offspring, as would expectedly have been presented by a mixture of black and white. The pregnancy rates of the two groups were significantly different (P < 0.05), which indicated that MSCs could promote the healing of the testis damage.</p><p><b>CONCLUSION</b>MSCs cannot differentiate into quasi-sperm after heterogeneity transplantation into the testis, but can promote the healing of the testis damage.</p>

Making of the animal model with sterilized testes / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the methods of making an animal model with sterilized testes.</p><p><b>METHODS</b>(1) X-ray local irradiation. Seventy 8-10-week-old male mice were equally divided into 6 experiment groups and a control group. The testes of the mice in the 6 experiment groups were irradiated sequentially by 1000, 1200, 1400, 1600, 1800 and 2000 cGy X-ray for 10 minutes, while those in the control group remained untreated. And then the pregnancy test was performed. (2) Cyclophosphamide injection. Forty 4-5-week-old male mice were divided into 3 experiment groups and a control group, the former treated with different doses of Cyclophosphamide via ip and the latter Natiichloridi Saline (N.S.) via i.p., followed by the pregnancy test. (3) Diphereline injection. Twenty 8-10-week-old male mice were equally divided into an experiment group and a control group, the former treated with Diphereline via ip and the latter N.S. via i.p., followed by the pregnancy test. (4) Identification by such pathologic examinations as TUNE1. technology, HE staining and immunohistochemical staining.</p><p><b>RESULTS</b>(1) X-ray local irradiation. The male mice of Group 1 and 2 made their female partners pregnant respectively 10 and 15 days after the X-ray irradiation, but not those of Group 3 and 4 in our 3-month observation, and those of Group 5 and 6 died respectively 2 and 5 days after the X-ray irradiation. By comparison, the controls got their female partners pregnant within 3 days after placed together. (2) Cyclophosphamide injection. The male mice of Group 1 gained weight about 7 g and achieved pregnancy 9-14 days after drug termination, those of Group 2 gained around 4 g but failed to effect pregnancy, and those in Group 3 lost weight and died respective at 3, 4 and 5 weeks during the medication, while the controls all got their female partners pregnant within 3 days after put together. (3) Diphereline injection. The 10 male mice of the experiment group effected pregnancy 3 weeks after drug termination, while the 10 controls achieved the same result with 3 days after placed together. (4) Pathologic identification: TUNEL technology showed that apoptotic cells were occasionally seen (0.71 +/- 0.12)% in the testis tissue of the control group and remarkably increased (10.36 +/- 1.48)% in the model group, with significant difference between the two groups (P < 0.05). HE staining revealed normal testis tissues and convoluted seminiferous tubules with large numbers of germ cells in the control group, but atrophied convoluted seminiferous tubules and estranged cell linkage with only Ledig's cells but no germ cells in the model group. Immunohistochemical staining showed that the positive expression rates of CD29, Hsp90alpha and CD117 were respectively (50.30 +/- 5.2)%, (41.6 +/- 3.5)% and (73.6 +/- 3.7)% in the control group, as compared with (1.3 +/- 0.2)%, 0% and (1.6 +/- 0.3)% in the model group, with significant difference (P < 0.01). The positive expression rate of p53 was (19.7 +/- 0.8)% in the control group, significantly different from that of the model group, which was (39.4 +/- 2.9)% (P < 0.01).</p><p><b>CONCLUSION</b>The animal model with sterilized testes can be made either by X-ray local irradiation of the testis or by Cyclophosphamide injection via i.p..</p>

Isolation and culture of mouse marrow mesenchymal stem cells (MSCs) and biological characteristics of MSCs cultured in conditions for spermatogonia in vitro / 中华男科学杂志

<p><b>OBJECTIVE</b>To isolate, culture and purify mouse bone marrow mesenchymal stem cells (MSCs) and observe the main biological characteristics of MSCs cultured in conditions for spermatogonia in vitro.</p><p><b>METHODS</b>The tibias and femurs were dissected from 5 - 6-week old mice and the marrow in the tibias and femurs was flushed out with medium. MSCs were isolated, cultured, purified in vitro by Percoll density gradient centrifugation combined with adherent method and identified by dynamic observation of stem cell characteristics by transmission electron microscope, HE staining, and immunohistochemical detection of cell markers. The quantities of such cytokines as IL-6, IL-8, G-CSF and SCF in culture liquid with MSCs were measured by ELISA, and compared with those of the control group. MSCs of the third generation were divided into two groups to be induced and cultured. MSCs of the control group were cultured with basal medium, while those of the experimental group with conditional medium. The results were analysed by microscopic observation, HE staining and immunohistochemical methods.</p><p><b>RESULTS</b>Pure MSCs were obtained. The cultured cells, with stem cell characteristics, shuttle-shaped at HE staining, immature under the transmission electron microscope and CD44 and CD90 positive by immunohistochemical detection, could be identified as MSCs. Compared with the control group, the quantities of IL-6, IL-8, G-CSF and SCF in the experimental group increased significantly (P < 0.05). The shapes of MSCs changed and immunohistochemical staining for CD27, CD119 and Oct-4 was positive in the experimental group, but both were just the opposite in the control group.</p><p><b>CONCLUSION</b>Pure MSCs can be obtained by Percoll density gradient centrifugation combined with adherent method and identified by dynamically observing stem cell characteristics, HE staining, observation under the transmission electron microscope and immunohistochemical detection of cell markers. MSCs can secrete cytokines such as IL-6, IL-8, G-CSF, SCF, and so on. MSCs cultured in conditions for spermatogonia may show some biological characteristics of spermatogonia.</p>

Transplantation of mouse bone marrow mesenchymal stem cells into the xenogeneic testis / 中华男科学杂志

<p><b>OBJECTIVE</b>To study transplantation of mouse bone marrow mesenchymal stem cells (MSCs) into the xenogeneic testis.</p><p><b>METHODS</b>(1) The tibias and femurs were dissected from 5-6-week-old mice. The marrow in the tibias and femurs was flushed out with medium. MSCs were isolated, cultured and purified in vitro by Percoll density gradient centrifugation combined with adherent method. (2) MSCs of the third generation were adopted and marked with Hoechest33342 for observation, and then made into cell-suspending fluid. (3) The marked MSCs were transplanted into the testis of the xenogeneic mouse by testis net injection. The biopsies of the testis tissues were carried out at different time and made into frost slices at three sites for observation.</p><p><b>RESULTS</b>(1) A lot of purified MSCs were obtained at the third generation. (2) The nucleoli of the marked MSCs showed light-yellow under the fluoroscope. (3) Xenogeneic transplantation of mouse bone marrow MSCs by testis net injection was successful, without immunoreaction. On the 1 st day after transplantation, MSCs only concentratively distributed in the medial slices, the nucleoli being light-yellow; On the 1 st and 3 rd day, MSCs dispersively distributed in the medial slices; On the 6th, 9th and 12th day, MSCs presented in all the slices of the three sites, some ranging tubally; On the 15th and 18th day, the fluorescence of MSCs weakened; On the 21 st day, the fluorescence of MSCs disappeared.</p><p><b>CONCLUSION</b>Transplantation of mouse bone marrow MSCs into the xenogeneic testis by testis net injection is effective and feasible, without immunoreaction. MSCs can survive after transplantation.</p>