Koji Niwa

Activation, Pronuclear Formation, and Development In Vitro of Pig Oocytes Following Intracytoplasmic Injection of Freeze-Dried Spermatozoa

Abstract The fertilization of pig oocytes following intracytoplasmic injection of freeze-dried spermatozoa was evaluated. Activation and male pronuclear (MPN) formation were better in oocytes injected with isolated freeze-dried sperm heads than whole freeze-dried spermatozoa, but cleaved embryos were generally difficult to develop to the morula or blastocyst stage. When spermatozoa were freeze-dried for 24 h, oocyte activation and MPN formation in activated oocytes after sperm head injection were inhibited. Embryo development to the blastocyst stage was only obtained after injecting sperm heads isolated from spermatozoa freeze-dried for 4 h and stored at 4°C. The proportion of embryos that developed to the blastocyst stage was not increased by the treatment of injected oocytes with Ca ionophore (5–10 μM). Increasing the sperm storage time did not affect oocyte activation or MPN formation, but blastocyst development was observed only after 1 mo of storage. These results demonstrate that pig oocytes can be fertilized with appropriately freeze-dried spermatozoa and that the fertilized oocytes can develop to the blastocyst stage.

Vitrification of rat embryos at various developmental stages

The effect of developmental stage on the survival of cryopreserved rat embryos was examined. Wistar rat embryos at various developmental stages were vitrified by a 1-step method with EFS40, an ethylene glycol-based solution, or by a 2-step method with EFS20 and EFS40. After warming, the survival of the embryos was assessed by their morphology, their ability to develop to blastocysts (or expanded blastocysts for blastocysts) in culture, or their ability to develop to term after transfer. Most (91-100%) of the embryos recovered after vitrification were morphologically normal in all developmental stages. However, the developmental ability of 1-cell embryos was quite low; exposing them to EFS40 for just 0.5 min decreased the in vitro survival rate from 76 to 9%. The survival rates of 2-cell embryos and blastocysts, both in vitro and in vivo, were significantly higher with a 2-step vitrification process than with a 1-step vitrification process. Very high in vitro survival rates (94-100%) were obtained in 4- to 8-cell embryos and morulae in the 1-step method. Although survival rates in vivo of 4-cell (40%) and 8-cell (4%) embryos vitrified by the 1-step method were comparatively low, the values were similar to those obtained in non-vitrified fresh embryos. When morulae vitrified by the 1-step method were transferred to recipients, the in vivo survival rate (61%) was high, and not significantly different from that of fresh embryos (70%). These results show that rat embryos at the 2-cell to blastocyst stages can be vitrified with EFS40, and that the morula stage is the most feasible stage for embryo cryopreservation in this species.

Development of rat oocytes following intracytoplasmic injection of sperm heads isolated from testicular and epididymal spermatozoa

The possibility of obtaining normal development of rat oocytes following intracytoplasmic injection of rat sperm heads, obtained by sonicating spermatozoa from testes and epididymides, was evaluated. Irrespective of the source of spermatozoa, sperm heads were successfully injected into approximately 45% of oocytes used; after 9-12h of culture, approximately 55% of injected oocytes still had normal morphology. Of the oocytes injected with testicular sperm heads 45% were activated, with a female pronucleus and a second polar body, but significantly more oocytes (approximately 68%) injected with caput and cauda epididymal sperm heads were activated. Male pronuclear formation was observed in 67-84% of the activated oocytes, with no difference in the proportions among the different sources of sperm heads. When zygotes showing two pronuclei and a second polar body at 10h after injection were cultured in conditions that support development of 1-cell embryos produced in vivo, no embryos derived from testicular sperm heads developed to blastocysts after 120 h of culture. Development of embryos derived from cauda sperm heads was significantly higher at all points of assessment, while embryos from caput sperm showed an intermediate degree of development, compared with embryos from testicular spermatozoa. However, similar proportions (2-4%) of 1-cell embryos derived from all three groups of sperm heads developed into normal offspring after transfer to foster mothers; of the limited number of offspring tested, all were fertile. These results demonstrate that sperm heads from all sources tested are similar in their ability to contribute to full development of normal, fertile offspring.

Penetration in vitro of zona-free pig oocytes by homologous and heterologous spermatozoa

We examined the penetrability of pig, rat and bull spermatozoa into zona-free pig oocytes. Frozen-thawed boar spermatozoa penetrated into both zona-intact and zona-free oocytes with similar efficacy in a modified Tris-buffered medium (mTBM) supplemented with BSA and caffeine, but not in medium without caffeine. Rat epididymal spermatozoa did not readily penetrate into zona-free pig oocytes in mTBM with BSA. However, when a modified Krebs-Ringer bicarbonate solution was used, penetration rate varied with sperm concentrations at insemination: 79% of the oocytes were penetrated at 1.0 x 10(6) cells/ml, but very few at 0.1 x 10(6) and 10.0 x 10(6) cells/ml. In all oocytes penetrated, no activation was observed and the sperm nucleus was fully decondensed but did not transform into a male pronucleus. Frozen-thawed bull spermatozoa were also found to penetrate into zona-free pig oocytes in mTBM with BSA, caffeine and heparin: higher penetration rates were obtained with 1.0 x 106 and 10.0 x 10(6) spermatozoa/ml compared with 0.1 x 10(6) spermatozoa/ml. The penetration rate with 1.0 x 10(6) spermatozoa/ml was stable in five different bulls. All oocytes penetrated were activated and male pronuclear formation was observed in 57-79% of the penetrated oocytes. These results suggest that capacitation or the acrosome reaction is required for boar, rat, and possibly, bull spermatozoa to penetrate into zona-free pig oocytes. Bull spermatozoa can easily induce activation of pig oocytes and form male pronuclei, but rat spermatozoa cannot do so, indicating species differences in the ability of spermatozoa to activate pig oocytes and to transform to male pronuclei in the ooplasm.

In Vivo Development of Vitrified Rat Embryos: Effects of Timing and Sites of Transfer to Recipient Females

Abstract In cryopreserved rat embryos, survival rates obtained in vitro are not always consistent with the rates obtained in vivo. To determine the optimal conditions for in vivo development to term, rat embryos at the 4-cell, 8-cell, and morula stages were vitrified in EFS40 by a one-step method and transferred into oviducts or uterine horns of recipients at various times during pseudopregnancy. Vitrified and fresh 4-cell embryos only developed after transfer into oviducts of asynchronous recipients on Days −1 to −2 of synchrony (i.e., at a point in pseudopregnancy 1–2 days earlier than the embryos). Approximately half the vitrified embryos transferred into oviducts on Day −1 developed to term, but only a minority of embryos, whether vitrified (10%–34%) or fresh (24%–33%), transferred at later times did so, suggesting that this may not be the most suitable stage for cryopreservation. Very few 8-cell embryos, either vitrified or fresh, developed when transferred into oviducts on Day 0 to −0.5. However, when transferred into uterine horns, high proportions of vitrified 8-cell embryos (∼63%) developed to term in reasonably synchronous recipients (Day 0 to −0.5) but not in more asynchronous ones (6%; Day −1). A majority of vitrified morulae also developed to term (52%–68%) in a wider range of recipients (Days 0 to −1), the greatest success occurring in recipients on Day −0.5. Similar proportions of vitrified and fresh 4-cell embryos, 8-cell embryos, and morulae developed to term when appropriate synchronization existed between embryo and recipient. Thus, vitrification of preimplantation-stage rat embryos does not appear to impair their developmental potential in vivo.

Develop to Term Rat Oocytes Injected with Heat-Dried Sperm Heads

This study investigated the development of rat oocytes in vitro and in vivo following intracytoplasmic injection of heads from spermatozoa heat-dried at 50°C for 8 h and stored at 4°C in different gas phases. Sperm membrane and chromosome are damaged by the process of heat-drying. Oocyte activation and cleavage of oocytes were worse in oocytes injected with spermatozoa heat-dried and stored for 1 week than unheated, fresh spermatozoa, but in heat-dried spermatozoa, there were no differences in these abilities of oocytes between the samples stored in nitrogen gas and in air. The oocytes injected with heat-dried spermatozoa stored for 1 week could develop to the morula and blastocyst stages without difference between the samples stored in nitrogen gas and in air after artificial stimulation. Cleavage of oocytes and development of cleaved embryos were higher when heat-dried spermatozoa were stored for 3 and 6 months in nitrogen gas than in air. However, the ability of injected oocytes to develop to the morula and blastocyst stages was not inhibited even when heat-dried spermatozoa stored in both atmosphere conditions for as long as 6 months were used. When 2-cell embryos derived from oocytes injected with heads from spermatozoa heat-dried and stored for 1 week and 1 month were transferred, each 1 of 4 recipients was conceived, and the conceived recipients delivered 1 live young each. These results demonstrate that rat oocytes can be fertilized with heat-dried spermatozoa and that the fertilized oocytes can develop to term.