Developmental ability of oocytes retrieved from Meishan neonatal ovarian tissue grafted into nude mice

Ovarian xenografting makes it possible to obtain oocytes with fertilization ability from immature pigs of Western breeds. In this study, we applied these methods to the Meishan, an indigenous Chinese pig breed, and investigated the developmental competence of oocytes grown in their neonatal tissue after grafting into nude mice. First, mice harboring neonatal ovarian tissue were infused with follicle stimulating hormone (FSH) (62.5 U/ml) for 13 days starting at 10, 30, and 60 days after vaginal opening (D10‐, D30‐, and D60‐FSH groups, respectively). Development of antral follicles and their oocytes was most enhanced in the D60‐FSH group. For the next step, we examined the in vitro maturation ability of the oocytes recovered from host mice after infusion with FSH at a dose of 62.5 U/ml or 125 U/ml (FSH‐62.5 or ‐125 group) for 13 days starting at 60 days after vaginal opening. Many more oocytes with maturation ability were obtained from the FSH‐125 group. The FSH‐125 mature oocytes were fertilized in vitro, as shown by formation of male and female pronuclei, but did not reach the blastocyst stage. These results indicate that Meishan neonatal ovaries are able to produce oocytes with fertilization ability after being grafted into nude mice.     

Embryo development of porcine oocytes after injection with miniature pig sperm and their extracts

This study examined embryo development of porcine oocytes after microinjection of sperm extracts (SE) in porcine intracytoplasmic sperm injection (ICSI). SE was prepared from miniature pig sperm by a nonionic surfactant, and various concentrations (0.02, 0.04 and 0.08 mg/mL) of SE were injected into the matured oocytes with a first polar body. In the pronuclear stage, the rate of oocytes with two pronuclei and a second polar body (21.4%) in the sperm and SE (0.04 mg/mL) injection group was significantly higher (P < 0.05) compared to other groups. The rate of 2–4‐cell stage in sperm and SE (0.04 mg/mL) injection group was 38.1%, and it was significantly higher than that in the sperm injection group (22.9%). The rate of blastocyst stage in sperm and SE (0.04 mg/mL) injection group was 21.4%, the value was significantly higher than those in SE (0.08 mg/mL) injection group (0%), sperm injection group (5.7%), and sperm and SE (0.08 mg/mL) injection group (2.6%). These results suggest that SE induces activation of porcine oocytes and their further embryonic development, and that SE is effective for porcine ICSI.     

奶山羊睾丸组织冷冻保存及复苏后精原干细胞的分离培养

以关中奶山羊为材料,比较了2种玻璃化冷冻法和1种慢速冷冻法对睾丸组织保存的效率,复苏后发现3种冻存方法均有较高的成活率(超过50%),其中玻璃化Ⅱ组达65%以上,培养及检测后均有精原干细胞存活,在体外培养可以形成胚胎干细胞样克隆,其表达精原干细胞和多能性干细胞的相关特异性标记:碱性磷酸酶(AP)、CD49f、CD133、C-Myc、Oct4和Klf4阳性,证明睾丸组织冷冻可以提供一种有效的奶山羊精原干细胞的保存方法。试验中所使用的低温保存方法简便易行,效果良好,表明这些方法可为癌症治疗等造成的无精症及少精症等不育症患者的医治以及一些珍稀濒危物种和优良畜禽的生殖细胞保存提供一种简捷有效的途径。     

Removal of sperm tail using trypsin and pre-activation of oocyte facilitates intracytoplasmic sperm injection in mice and rats

We examined various methods to enhance the accessibility of intracytoplasmic sperm injection (ICSI) technology to more users by making the technique easier, more efficient, and practical. First, the methods for artificially removing the mouse sperm tail were evaluated. Trypsin treatment was found to efficiently remove the sperm tails. The resultant sperm cells had a lower oocyte activation capacity; however, the use of activated oocytes resulted in the same fecundity as that of fresh, untreated sperm. Pre-activated oocytes were more resistant to physical damage, showed higher survival rates, and required less time per injection. Testing this method in rats yielded similar results, although the oocyte activation method was different. Remarkably, this method resulted in higher birth rates of rat progeny than with conventional methods of rat ICSI. Our method thereby streamlines mouse and rat ICSI, making it more accessible to laboratories across many disciplines.     

Successful blastocyst production by intracytoplasmic injection of sperm after in vitro maturation of follicular oocytes obtained from immature female squirrel monkeys (Saimiri boliviensis)

Advanced reproductive technologies are being applied for the propagation of squirrel monkeys, to ensure their preservation as a genetic resource and the effective use of their gametes in the future. In the present study, oocytes and spermatozoa were collected from live squirrel monkeys, following which piezo intracytoplasmic sperm injection (ICSI) was performed using these gametes. Follicular development was induced by administering equine chorionic gonadotropin (eCG) containing inhibin antiserum to an immature squirrel monkey female. The unilateral ovary was excised after the administration of human chorionic gonadotropin (hCG), to induce ovulation, following which the larger developed follicular oocytes were collected. Follicular oocytes were prepared for ICSI using sperm from the epididymal tail of a unilateral testis extracted from a mature male. The embryos were continuously incubated in CMRL 1066 medium supplemented with 10% (v/v) fetal bovine serum. Embryo culture was performed with cumulus cells. Two experiments of ICSI carried out with three females resulted in 14 mature oocytes from the 49 cumulus-oocyte complexes collected and five embryos, three of which developed into blastocysts. These blastocysts were vitrified, thawed, and transferred to recipient monkeys, but no pregnancies resulted. In conclusion, the present study is the first to successfully produce ICSI-derived blastocysts from MII oocytes obtained by means of hormone administration (a combination of eCG+inhibin antiserum and hCG) and in vitro maturation in immature squirrel monkeys.