• Reproductive and Developmental Biology
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• v.29 no.4
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• pp.241-245
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• 2005

Porcine oviduct epithelial cells (POEC) are widely used in co-culture experiments to improve early embryonic development, in vitro fertilization in embryo transfer programs for domestic animals and in vitro maturation of immature germ cells. POEC were mechanically isolated and cultured in tissue culture medium 199. Cells grew continuously, and confluent monolayers were formed after 7 days. After forming confluent monolayer of epithelial cells, supernatant was collected as the condition medium for maturing round spermatids in vitro. Round spermatids were also separated mechanically and cultured in the POEC condition medium. In this study we observed that $20\%$ of round spermatid cultured were matured into elongating spermatid after 24 h, and about $10\%$ of round spermatid cultured showed complete elongation (elongated spermatid) within $24\~48$ h of in vitro culture. No further development was observed within $50\~72$ h and transformed cells lost their viability after 72 h. These preliminary findings suggest that the condition medium from POEC may be possible to overcome the round spermatid block by improving the milieu of culture system.

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.395-403
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• 1998

In this study we determined fertilization processes and developmental ability of porcine oocytes following injection of round spermatid in the presence and absence of artificial activation. Electrical stimulation at 2 h before spermatid injection significantly increased the incidence of normal fertilization as compared to those following injection without stimulation or with stimulation immediately after injection. The incidences of two pronuclear formation and apposition were not different in oocytes between following intracytoplasmic spermatid and spermatid nucleus injection. Indirect immunocyto chemistry and laser scanning confocal microscopy study revealed that microtubuels were organized from the oocyte cortex following round spermatid injection, and this seemed to move both male and female pronuclei into the oocyte center. At 6 days following injection blastocoele formation was seen in the eggs following round spermatid (25%) and round spermatid nucleus injection (27%). However, none of oocytes developed to the blastocyst stage at 6 days following sham injection. The average cell numbers of blastocysts at 8 days following injection of spermatid and spermatid nucleus were 87 to 99. These results suggested that either round spermatid or it's nudeus can be used to produce viable embryos by injection into unfertilized oocytes in the pig.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.327-331
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• 1996

Normal fertilization and pregnancy by round spermatid was achieved from nonobstructive azoospermia patient to be believed untreatable. Therefore, it is suggested that application of round spermatid in human ART program seems to be new treatment of male infertility. Also, it will be needed further research for evaluated fertilization mechanism by round spermatid injection.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.193-201
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• 1999

In this study we determined fertilization processes and developmental ability of porcine oocytes following injection of round spermatid in the presence of artificial activation. Electrical stimulation at 3 h before spermatid injection significantly increased the incidence of normal fertilization as compared to those following injection without stimulation or with stimulation immediately after injection. The incidences of two pronuclear formation and apposition were not different in oocytes between following intracytoplasmic spermatid and spermatid nucleus injection. Indirect immunocytochemistry and laser scanning confocal microscopy study revealed that micro tubules were organized from the oocyte cortex following round spermatid injection, and this seemed to move both male and female pronuclei into the oocyte center. Paternal mitochondria which are introduced with spermatid have been observed up to 4-cell. Our study indicated that either round spermatid or it's nucleus can be used to produce viable bovine embryos by injection into unfertilized oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.233-237
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• 1998

We demonstrated that the normal pregnancy and delivery by round spermatid injection (ROSI) into oocytes was achieved from nonobstructive azoospermia patient. In this case, the normal fertilization rate was 50%. All of the two pronuclear stage embryos cleaved and were transferred to the patient's uterus. A singleton pregnancy was achieved and resulted in the birth of normal female infant. This resuJt show that intracytoplasraic injection of round spermatid seems to be new treatment of nonobstmctive azoospermia male infertility. Further research is needed to evaluate the required culture conditions to induce progression of the round spermatid into a more elongated stage.

• Korean Journal of Animal Reproduction
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• v.23 no.4
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• pp.359-364
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• 1999

Recent advances in intracytoplasmic sperm (ICSI) and round spermatid injection (ROSI) would provide exciting opportunities not only for the male infertility but also for studying gamete physiology during fertilization and early development. Furthermore, intracytoplasmic sperm injection could be used to produce transgenic animal (Perry et al., 1999). However, it is not clear in the fertilization processes in mammalian oocytes following intracytoplasmic injection of spermatozoon, isolated sperm head or round spermatid. (omitted)

• Applied Microscopy
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• v.26 no.1
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• pp.33-45
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• 1996

The spermatogenesis of Korean slug, Incilaria fruhstorferi are observed by electron microscope. The results are as follows: The spermatogenesis of Korean slug, Incilaria fruhstorferi, is processed through the five stages; Spermatogonia, primary spermatocyte, secondary spematocyte, spermatid and spermatozoon. The spermiogenesis, the differentiation of the spermatid, is also processed through the five stages. In stage 1, the numerous and round mitochomdria in the cytoplasm are moved around the nucleus of spermatid. In stage 2, the nucleus of spermatid transformed into the oval shape, and the oval nucleus is surrounded by many rough endoplasmic reticulum. In stage 3, the oval nucleus of spermatid is changed to be curved as an arrow, and then two centrioles appeared behind nucleus. The centriole is sucked into the cytoplasm. and almost all the chromatins are changed into heterochromatins. In stage 4, the nucleus of spermatid are transformed into the oval shape, when the lamella plate chromatin of spermatid form in the nucleoplasm. In stage 5, the oval nucleus is then transformed into the stream-line shape when the lamella plate chromatin of spematid gradually concentrated in the nucleus, and long axoneme ($65{\mu}m$ in length) form from the distal centriole. Two long mitochondria in the middle piece and the main piece of spermatozoon array spirally along a long axoneme, and the mitochondria matrix is gradually filled with electron-dense glycogen particles ($0.1{\mu}m$ in size). The axoneme of spermatozoon consists of typical 9+2 microtubular pattern.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2003.10a
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• pp.133-133
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• 2003

• Korean Journal of Animal Reproduction
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• v.19 no.3
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• pp.171-179
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• 1995

This study was carried out to investigate the fertilizing ability of round spematids isolated from seminiferous tubules. A round spermatid was introduced into the perivitelline space of a mature oocyte using Leitz micromanipulators and then subjected to electrofusion. Electrofusion was induced by applying a single DC pulse of 90V with a duration of 60$\mu$sec using Model 611 Square Wave Stimulator(Phipps and Bird, U.S.A) in 0.3 M sucrose fusion medium containing 0.05mM CaCl2 and 0.1mM MgSO4, Oocyte pre-activation was conducted by exposure to a single DC(80V, 80$\mu$sec) pulse in electrofusion medium at 1 hour before electrofusion. The incidence of fusion with pre-activated oocytes(23.8%, 57/239) was higher than that with nonactivated oocytes(6.7%, 3/45). The most of electro-stimulated mouse oocytes cleaved regardless of the success or failure of fusion. Karyotyping of embryos that developed into blastocysts after exposure to the fusion pulse were performe. We found that blastocysts from the fused oocytes were diploid whereas blastocysts from the unfused oocytes were haploid. About 11.7 and 11.5% of fused and unfused oocytes were developmental potentials of fused and unfused oocytes. Therefore, these results suggest that the mouse mture oocyte can be fertilized by fusion with a round spermtid and subsequently developed normally.

• Development and Reproduction
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• v.7 no.2
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• pp.89-93
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• 2003

The present study observed the ultrastructure of testis of bluespotted mud hopper(Boleophthalmus pectinirostris), and sperrnatogenesis was discussed also. The testis was surrounded by a thin adventitia, inside which spermatocyst composed the parenchyma of testis. Each lobule was enwrapped by many spermatocysts, which were filled with different kinds of spermatogenic cell clusters at the same developmental stage. In the lobule lumen there are large numbers of spermatozoa The thin adventitia(outer wall) of testis was composed of outer epithelium, and the underlying layers, such as collagen fiber layer, and myoid tissue. The myoid tissue elongated into the inside of testis, became the main componentof interstitium between spermatocyst where sperrnatogenesis occurred. In addition interstitial cells containing dense homogeneous nucleus and abundant mitochondria were observed. Spermatogonia contained round nucleus with diffuse chromatin and nucleolus, and dense nuclear bodies surround by mitochondria in cytoplasm. The synaptonemal . complex was observed in primary spermatocytes clearly. Early spermatid presented larger round nucleus composed of granular chromatin, which was located in the center of cytoplasm. The nucleus of mid-spermatid composed of finely granular chromatin lied on one side of spermatid, and abundant mitochondria had migrated another side. A nuclear fossa appeared in the site near mitochondria in late-spermatid, and the centriole was formed in nuclear fossa.