Aging Gpr3^–/– mice display accelerated reduction of fertility, and Gpr3 is expressed in mouse oocytes and two-cell embryos. (A) Litter size resulting from Gpr3^+/+ (open columns) and Gpr3^–/– (black columns) incrosses as a function of maternal age. Results show a clear difference between the two genotypes at all ages (P < 0.001), and the difference increases with age. Only animals showing spontaneous ovarian cycles were scored. Results are expressed as mean ± SEM. The number of mothers in each group is indicated at the top of the columns. (B) RT-PCR detection of Gpr3 and β-actin mRNAs in preimplantation Gpr3^+/+ embryos at different stages (1c, one cell; 2c, two cell; M, morula; and Bl, blastocyst). (C) Detection of GPR3 on the wild-type oocyte by immunofluorescence confocal microscopy by using an anti-GPR3 polyclonal antibody (a). Incubation of the oocyte with the second antibody alone yielded only a background signal (b).

Oocytes from superovulated Gpr3^–/– females display age-dependent increase in fragmentation. (A) Percentage of fragmented unfertilized oocytes collected in the ampullae of females at different ages after exogenous hormonal stimulation. Total number of oocytes analyzed is indicated above the columns (open columns, Gpr3^+/+; black columns and Gpr3^–/–, oocytes). Whereas both genotypes produced progressively more fragmented oocytes with age (P < 0.001), at all ages, Gpr3^–/– animals produced much more fragmented oocytes than Gpr3^+/+ (P < 0.001). (B) Nonfertilized Gpr3^–/– oocytes observed in light microscopy by using Nomarski optics at time of harvest in the ampullae of superovulated females. Normally shaped unfertilized oocytes with or without polar body, fragmented oocytes, and oocytes exhibiting cytoplasmic condensation are observed. (Bar, 40 μm.) (C) Histology sections of Gpr3^–/– fragmented oocytes observed in follicles (a and b) and oviducts (c) after stimulation by human chorionic gonadotropin. [Bars, (a–c) 20 μm and (b)10 μm.]

Oocytes from Gpr3^–/– mice display age-independent premature resumption of meiosis I, associated with increase in morphological abnormalities. (A) Antral oocytes from 3.5-week (3.5w)- and 6-month (6m)-old ovaries were collected and scored for presence of a GV, GV breakdown (GVB), or extrusion of first polar body (PB) at collection time (d1) and after 18 h in culture (d2). The proportion of oocytes undergoing parthenogenetic division (2c), lysis (L), or fragmentation (FGT) was also scored. For both ages, a clear decrease in the proportion of GV stage was observed in Gpr3^–/– oocytes (P < 0.001) at collection time (d1); spontaneous resumption of meiosis was impaired after 18 h in culture (d2). (B) Meiotic competence expressed as percentage of GV stage oocytes having extruded the first polar body after 18 h in culture. For A and B, open columns, Gpr3^+/+ and black columns, Gpr3^–/– animals. (C) Representative morphology of oocytes observed in light microscopy by using Nomarski optics. (a) Oocyte with GV intact, (b) oocyte showing GV breakdown, (c) oocyte with first polar body extruded, (d) oocyte with two polar bodies extruded, (e) lysed oocyte, and (f) fragmented oocyte. (Bar, 10 μm.)

Decreased fertility of aged Gpr3^–/– females is accounted for by reduction of zygote—early-embryo transition. (A) Number of zygotes produced by spontaneous ovulation (1c), embryonic preimplantation developmental capacity to the two-cell (2c) and blastocyst stages (Bl), number of implanted embryo (Im) and litter size (NB) are indicated for 6-month-old Gpr3^+/+ (open columns) and Gpr3^–/– females (black columns) mated with males of the same genotype. Results are expressed as the mean ± SEM, with the number of mothers tested indicated at the top of the columns. Transition from 1c to 2c stage was strongly decreased in Gpr3^–/– embryos (P < 0.001), as compared with Gpr3^+/+ (P = 0.400), whereas the 2c-to-blastocyst transition was not significantly different for both genotypes (Gpr3^+/+, P = 0.966; Gpr3^–/–, P = 0.492). Similarly, the decrease observed between the number of implantation sites and litter size was not significantly different in the two genotypes (Gpr3^+/+, P = 0.062; Gpr3^–/–, P = 0.404). (B) The percentage of zygotes displaying progression to the two-cell stage in spontaneously ovulating females was much decreased in Gpr3^–/– animals at all maternal ages tested (P < 0.001), and the decrease was more important at 8.5 months of age. The total number of embryos analyzed is indicated above the columns (open columns, Gpr3^+/+; black columns and Gpr3^–/–). (C) Photomicrographs showing coexistence of normally shaped Gpr3^–/– one-cell (a), two-cell (b) and four-cell embryos (c) with Gpr3^–/– fragmented embryos (arrowheads), after spontaneous ovulation, in 6-month-old females. (D) Laser-scanning confocal microscopy of Gpr3^–/– preimplantation embryos after immunofluorescent staining for α-tubulin (green). DNA was counterstained with ethidium homodimer-2 (red). Normally shaped (a–c) and degenerated Gpr3^–/– embryos (d–f) are illustrated: (a) zygote with male and female pronuclei, each displaying a large nucleolus; only the second polar body is visible; (b) two-cell embryo; and (c) six-cell embryo. (d–f) Fragmented embryos with most of the fragments containing chromatin material.