Mouse‐rabbit germinal vesicle transfer reveals that factors regulating oocyte meiotic progression are not species‐specific in mammals

Abstract

A series of experiments were designed to evaluate the meiotic competence of mouse oocyte germinal vesicle (GV) in rabbit ooplasm. In experiment 1, an isolated mouse GV was transferred into rabbit GV-stage cytoplast by electrofusion. It was shown that 71.8% and 63.3% of the reconstructed oocytes completed the first meiosis as indicated by the first polar body (PB1) emission when cultured in M199 and M199 + PMSG, respectively. Chromosomal analysis showed that 75% of matured oocytes contained the normal 20 mouse chromosomes. When mouse spermatozoa were microinjected into the cytoplasm of oocytes matured in M199 + PMSG and M199, as many as 59.4% and 48% finished the second meiosis as revealed by the second polar body (PB2) emission and a few fertilized eggs developed to the eight-cell stage. In experiment 2, a mouse GV was transferred into rabbit MII-stage cytoplast. Only 13.0-14.3% of the reconstructed oocytes underwent germinal vesicle breakdown (GVBD) and none proceeded past the MI stage. When two mouse GVs were transferred into an enucleated rabbit oocyte, only 8.7% went through GVBD. In experiment 3, a whole zona-free mouse GV oocyte was fused with a rabbit MII cytoplast. The GVBD rates were increased to 51.2% and 49.4% when cultured in M199 + PMSG and M199, respectively, but none reached the MII stage. In experiment 4, a mouse GV was transferred into a partial cytoplasm-removed rabbit MII oocyte in which the second meiotic apparatus was still present. GVBD occurred in nearly all the reconstructed oocytes when one or two GVs were transferred and two or three metaphase plates were observed in ooplasm after culturing in M199 + PMSG for 8 hr. These data suggest that cytoplasmic factors regulating the progression of the first and the second meioses are not species-specific in mammalian oocytes and that these factors are located in the meiotic apparatus and/or its surrounding cytoplasm at MII stage.

Reconstruction of mammalian oocytes by germinal vesicle transfer: A systematic review

“…Micro-pipettes were made of borosilicate glass (4,(7)(8)(9)(10). Electrocell manipulator and the piezoelectric actuator have been respectively used for electrofusion and intracytoplasmic sperm injection (ICSI) (7,8,(10)(11)(12)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). Modular incubator with 5% CO 2 /5% O 2 / balance N 2 , and 5% CO 2 /21% O 2 / balance N 2 are essential for the process (7-10, 15, 16).…”

“…, or 10 IU of pregnant mare serum gonadotropin (PMSG) (46-48 hr post-injection) (9,12,20,22,23,33). Then cumulus cell-oocyte complexes (COCs) were released using a fine gauge needle or forceps and were collected with medium (HTF) supplemented with 10% fetal calf serum (FCS) (21).…”

“…Some researchers stated that before oocyte stripping, COCs should be released into one of these supplemented media for 1-2 hr (7,9,11,20); HEPES [4-(2-Hydroxyethyl) -1-piperazineethanesulfonic acid]-medium (M2) with dibutyryl cyclic AMP (dbcAMP) (9), medium (M2) with 0.2 mM 3isobutyl-1-methylxanthine (IBMX) as collection media and medium (MEM-a) with 20% fetal bovine serum (FBS) and 0.2 mM IBMX, as culture media (7,11) and medium (HTF) with 10% (v/v) FBS and 0.1 mM IBMX (20). B) Immature oocytes culturing: Immature naked oocytes were cultured in another medium to preparing oocyte for manipulation or removing whole zona pellucida (2,4,6,7,9,10,12,14,18,19,23). It was shown that zona-free GV oocytes can also be used as a GV donor (7,23).…”

“…B) Immature oocytes culturing: Immature naked oocytes were cultured in another medium to preparing oocyte for manipulation or removing whole zona pellucida (2,4,6,7,9,10,12,14,18,19,23). It was shown that zona-free GV oocytes can also be used as a GV donor (7,23). Some research groups have used only one type of supplemented media to culture cumulus-free and zona free GV oocytes 1/2-2 h before and during manipulation (7,32).…”

“…3) medium (M199) with 0.2 mM IBMX, ~2h (10) or medium (Waymouth) with 0.2 mM IBMX, 0.23 μM Pyruvate and 5% FBS until the GVT procedure (12,14) then at least 15 min in medium (M2) with 25 µg/ml CB and 3 mg/ml bovine serum albumin (BSA) (10, 12) and 5 µl droplets of medium (M2) containing 3 mg/ml BSA and 25 µg/ml CB for manipulation (10,12). 4) medium (M199) containing 10% FCS and 20 µg/ml IBMX, 2h (23) then medium (M119) with 0.3% pronase, 3-4 min (23) and IBMX-containing medium for maintaining until injection (23).…”