High-Yield Superovulation in Adult Mice by Anti-Inhibin Serum Treatment Combined with Estrous Cycle Synchronization1

Abstract

Producing many mature oocytes is of great importance for assisted reproductive technologies. In mice, superovulation by consecutive injections of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) has been the gold standard for oocyte collection. However, the yield of mature oocytes by this regimen can fluctuate according to the stage of the estrous cycle, strain, and age. Therefore, our objective was to develop a high-yield superovulation protocol to collect higher numbers of oocytes from adult female mice of different strains and ages. First, we aimed to synchronize the estrous cycle using C57BL/6 (B6) female mice. Most (93%) were synchronized to metestrus after two daily injections of progesterone. Second, we found that with the injection of anti-inhibin serum (AIS) instead of eCG, the mean number of ovulated oocytes almost doubled (21 vs. 41 per mouse). Third, by combining estrous cycle synchronization with two AIS injections, we obtained 62 oocytes per mouse, about three times that with the eCG-hCG protocol. Importantly, this approach increased the proportion of mice that ovulated >25 oocytes from about 40% (eCG-hCG) to 90%. The same protocol was also effective in other inbred (BALB/cA), outbred (ICR), and hybrid (B6D2F1) strains. In addition, B6 female mice aged over 1 yr ovulated 1.8-fold more oocytes by this protocol. Thus, estrous cycle synchronization followed by AIS-hCG yielded a broadly applicable, highly efficient superovulation. This protocol should promote the effective use of invaluable female mouse strains and decrease the numbers of animals euthanized.

EFCAB9 is a pH-Dependent Ca2+Sensor that Regulates CatSper Channel Activity and Sperm Motility

“…Female mice were caged with heterozygous or homozygous EFCAB9 - mutant or EFCAB9 , CatSperz double mutant males for two months to record pregnancy and litter size when gave births. For IVF, 5-7 weeks B6D2F1 female mice were superovulated by injecting progesterone and anti-inhibin serum (Central Research Co, Ltd) (Hasegawa et al, 2016), and the oocytes were collected after injecting 13 hr from 5 U of human chorionic gonadotrophin (EMD millipore). Prepared epididymal sperm were capacitated at 37 °C for 90 min, and inseminated to oocytes with 2 x 10 5 cells/ml concentration.…”

Development of assisted reproductive technologies in small animal species for their efficient preservation and production

“…Using this method of estrous synchronization followed by the AIS-hCG treatment, we collected 59 normal oocytes on average from each C57BL/6J female mouse (Fig. 2, Tables 3, 4) [40,62]. This corresponds with an approximately 3.5-fold increase in oocyte recovery compared with that following eCG-hCG treatment.…”

“…Results of superovulation with eCG-hCG injections or two daily injections of both P4 and AIS-hCG in inbred (C57BL/6J and BALB/cA), outbred (ICR) and hybrid (B6D2F1) strains[62].…”

Histone H3.3 Hira chaperone complex contributes to zygote formation in mice and humans

“…Female mice were superovulated by administration of 7.5 I.U. pregnant mare serum gonadotrophin (PMSG from Prospec Protein Services, USA) or 80µl ultra-superovulation reagent (CARD HyperOva Cosmo Bio, Japan; (Hasegawa et al, 2016)) and 48 hours later fully-grown oocytes were isolated. For in vitro maturation (IVM), oocytes were cultured in M16 medium (Sigma) at 37°C, 6%CO2 and 5%O2 for 18 to 24 hours.…”