Isolation And Identification Of Spermatids From Mouse Testis And Semen Of Male Infertile Patients

The first reports on successful clinical use of round and elongated spermatids for assisted reproduction in 1995 suggest the benefits that the technique of spermatid conception can bring to some patients suffering from otherwise untreatable types of male factor infertility. Men with non-obstructive azoospermia(NOA) can now be treated by using intra-oocyte round spermatid injection(ROSI) or elongated spermatid injection(ELSI). Spermatids can be retrieved from semen or from testis biopsy specimens. An ongoing clinical pregnancy has even been reported following ROSI using frozen-thawed testicular spermatids.However, the rates of fertilization and pregnancy with spermatids have been disappointing. Many problems limiting success rates and binding a wide application of this technique still remain unresolved. Except the incomplete maturation of spermatid nuclear and oocyte activation, idendification of a live spermatid is a pivotal procedure. It is difficult to distinguish round spermatids from other round cells such as spermatocytes, monocytes, polymorphonuclear leukocytes and so on. On the other hand, various methods of isolation of spermatids from testis are available. Most commonly used is the mechanical method in which a small piece of testicular tissue is torn apart using needles and the spermatids are released. While enzymatic digestion of human testicular tissue can result in a mixture of cell types including a whole range of spermatogenic cell types. Although with these methods, spermatids can be isolated manually under the microscope for immediate use, it is notpossible to obtain large populations of purified spermatids. But repeated biopsies for retrieving spermatids can further compromise the already deficient seminiferous tubules of patients. Therefore there is a need to develop a method by which large and purified populations of spermatids can be isolated, not only for the immediate requirement but also for cryoproservation for subsequent treatment cycles.In the first trial, combination of enzymatic digestion was used to prepare suspensions of spermatogenic cells from adult mouse testis, and then a modified discontinuous Percoll gradient centrifugation method (15%,22%,30%,40%,50%,60%) was introduced to isolate spermatids from the cellular suspensions. The content of spermatids in each isolated fraction by Percoll method was determined by morphology (Wright-Giemsa stain) and flow cytometry analysis, and the viability of spermatogenic cells was assessed by using eosin Y exclusion test. As a result, more than 97% of the testicular cells remained their viability after enzymatic digestion. Of the six fractions resulted from Percoll centrifugation, the 22% fraction contained mostly spermatids (mean 86.7%, P<0.05) and cell viability was more than 85.5%. While in the 30% fraction, immature spermatogenic cells were present, and more than 92% of the cells remained their viability. So a lagre populations of relatively purified spermatids can be isolated from mouse testis by enzymatic digestion combined with discontinuous Percoll gradient centrifugation method.Whereafter a total of fifteen mouse oocytes were injected with round spermatids and seventeen with elongated spermatids. By three days after injection, the number of embryos which had cleaved to the 2-cell stage was 0 with round spermatids and mree(17.6%) with elongated spermatids.In the second trial, this modified discontinuous Percoll gradient centrifugation method was introduced to isolate spermatids from the semen of fifteen male infertile patients. Then the effect was identified by Wright-Giemsa stain, flow cytometry analysis, immunocytochemistry and fluorescence in situ hybridization (FISH). Similary, the 22% Percoll fraction contained mostly haploid cells [(91.85 ?5.18)%](P<0.005) and the mean density in this fraction was (1.010 ?0.786) x 105/ml.