A new cryotop vial device system provides an aseptic cryoprotectant-free and centrifuge-free cryopreservation of human spermatozoa (a closed system).

Single sperm cryopreservation is a new method to preserve small numbers of spermatozoa in small droplets. So far, several devices have been introduced for this technique, but more studies are needed for its optimization. The aim of this study was optimizing the previous device for low number of spermatozoa and low volume semen, which led to design of Cryotop Vial device. Normal semen samples from 25 patients were prepared by swim-up method and divided into four groups: Fresh (F), Rapid freezing (R) and ultra-rapid freezing with Cryotop Device (CD) and Cryotop Vial Device (CVD). In R group, the diluted sperm suspension with sperm freezing medium was cooled in the vapor phase and then immersed in liquid nitrogen. Ultra rapid freezing was performed with sucrose in small volume using the Cryotop Device (CD) or Cryotop Vial Device (CVD). Sperm viability, motility, fine morphology, mitochondrial activity and DNA fragmentation were assessed in all samples. All sperm parameters decreased significantly in all the cryo groups compared to the fresh group. The comparison between the cryo groups showed that progressive motility (69.28 ± 6.82 vs. 55.68 ± 9.04, and 54.76 ± 5.34, p < 0.001) and viability (77.36 ± 5.48 vs.68.84 ± 8.51, p < 0.001, and 70.04 ± 7.44, P = 0.002) were significantly higher in the CVD compared to the CD and R groups respectively. DNA fragmentation was also significantly lower in both ultra-rapid freezing groups (CD and CVD) compared to the R group. Fine morphology and mitochondrial activity were not different between the cryo groups. The CVD as a cryoprotectant and centrifuge-free technique preserved sperm motility, viability and DNA integrity after cryopreservation better than other groups.

Changes in the expression of OCT4 in mouse ovary during estrous cycle.

The transcriptional factor OCT4 regulates pluripotency of stem cells and has an important role during oocyte growth. Whereas, its role has remained ambiguous in ovarian tissue during reproductive cycle. Therefore, this study was aimed to investigate the expression patterns of OCT4 in mouse ovaries during the normal estrous cycle. Adult National Medical Research Institute mice were classified as proestrous, estrous, metestrous and diestrous on the basis of vaginal smear cytology. Their ovaries were removed and the protein and gene expression levels of OCT4 were assessed using immunohistochemical staining and real-time quantitative reverse-transcription PCR, respectively. Immunohistochemical staining revealed the expression of OCT4 in the cytoplasm of corpus luteum cells. In the follicles, OCT4 was expressed in the cytoplasm of granulosa cells. Furthermore, the gene expression levels of OCT4 was significantly higher in the proestrous phase than in the other phases of the estrous cycle (p < 0.05). The results indicated that OCT4 gene expression levels are affected by the cyclic pattern of the estrous cycle.