ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of carnitine on human sperm motility and its potential role in the treatment of male infertility diseases.</p><p><b>METHODS</b>We obtained sperm by testis puncture from obstructive azoospermia patients and cultured them in vitro with normal culture solution (the control group) and the solution with L-carnitine at the concentration of 100 and 250 mmol/L, respectively. We observed the changes in sperm motility and morphology before and after the treatment, detected the expressions of the germ-specific genes, Vasa, Dazl, Acr, Prm1 and ATPase 6.0 by RT-PCR, and investigated the relationship between L-carnitine and the genes associated with sperm development and maturation.</p><p><b>RESULTS</b>After 24 -72 hours of treatment, the percentage of motile sperm was significantly higher in the 100 mmol/L L-carnitine group than in the control and 250 mmol/L L-carnitine groups (P < 0.01); the number of forward moving sperm was obviously increased and sperm morphology remained normal in the 100 mmol/L L-carnitine group. RT-PCR showed that L-carnitine increased the expressions of Acr, Prm1, Dazl and ATPase 6. 0 at the concentration of 100 mmol/L, and decreased the expressions of Dazl, Acr and Prm1 at 250 mmol/L.</p><p><b>CONCLUSION</b>L-carnitine at a proper concentration may improve the motility of incubated testicular sperm by upregulating the expressions of some sperm-specific genes, which helps sperm selection for intracytoplasmic sperm injection. However, a higher concentration of L-carnitine may reduce the expressions of these genes, probably due to its increased toxicity.</p>
Subject(s)
Humans , Male , Azoospermia , Genetics , Metabolism , Carnitine , Pharmacology , Cells, Cultured , Gene Expression , Infertility, Male , Reverse Transcriptase Polymerase Chain Reaction , Sperm Count , Spermatozoa , Metabolism , Testis , MetabolismABSTRACT
Retinoic acid plays an important role in maintaining the structure and function in male testis. Recent studies showed that there is a group of genes that can be specially activated by retinoic acid during the development of male reproductive gland. The gene Stra 8 (Stimulated by Retinoic Acid) was one of the gene in this group. In mouse, Stra 8 is restrictively expressed in male germ line cells, and its function is related to the development of sperm. In order to investigate the feature of Stra 8 gene expression,the 1.4 kb (-1407 - +7) promoter region of Stra 8 gene was amplified from mouse genomic DNA. The DNA fragment was then cloned into a promoter less vector to form the construct that contained the 1.4 kb promoter region, and the reporter gene of EGFP that was regulated by 1.4kb Stra 8 promoter. To investigate the specificity of Stra 8 promoter,the vector pStro-EGFP was transfected into undifferentiated mouse stem cells such as ES-129, bone marrow mesenchymal stem cell (mMSC) and spermatogonial stem cell (mSSC). The results showed that the expression of GFP was only observed in the mSSC cells,which indicated that Stra 8 gene was specially regulated in testis tissue. As the gene marker,vector pStra8-EGFP was then transfected to undifferentiated mMSC cells. After being selected by G418 for 2 weeks,the mMSC cells were induced by retinoic acid. After 12 hours induction, some induced cells started to express GFP protein, which was observed under the fluorescence microscope. At the same time, several stem cell specificity biomarkers such as Oct4, and spermatogonial stem cell biomarkers such as CyclinA2 and Stra 8 were detected in the induced cells by RT-PCR method. These results showed that the mMSCs would differentiated to spermatognial stem cells after induced by Retinoic Acid.
Subject(s)
Animals , Male , Mice , Adaptor Proteins, Signal Transducing , Cell Differentiation , Genetics , Cells, Cultured , Gene Expression , Promoter Regions, Genetic , Proteins , Genetics , Spermatogonia , Cell Biology , Metabolism , Stem Cells , Cell Biology , Metabolism , Tretinoin , PharmacologyABSTRACT
Male germ stem cells (mGSCs), which is in testis after sex differentiation, derive from primordial germ cells. In this study, bovine mGSCs were isolated from testis of 20 weeks fetuses. Number of CD9 positive cells of the cells through two-steps adhering plates velocity different was 95.8% by flow cytometer. The carina-type cells clones and the plane-type cells clones appeared in co-cultured system. One cells lines had been successively maintained for 4 passages, and the cells clusters showed AKP positive staining. The cells clusters showed nest-shape in third passage showed SSEA1 and Oct-4 positive staining. These cells can also spontaneously differentiate into c-kit positive staining germ cells, and the cells were directional induced to formaactin positive staining cardiac-like cells cluster and NF positive staining neuron-like cells. The conclusion showed that male germ stem cells from 20 weeks bovine fetuses could be in vitro formed like embryonic stem cells.