Premature ovarian aging in mice deficient for Gpr3 - PubMed (original) (raw)

Premature ovarian aging in mice deficient for Gpr3

Catherine Ledent et al. Proc Natl Acad Sci U S A. 2005.

Abstract

After becoming competent for resuming meiosis, fully developed mammalian oocytes are maintained arrested in prophase I until ovulation is triggered by the luteotropin surge. Meiotic pause has been shown to depend critically on maintenance of cAMP level in the oocyte and was recently attributed to the constitutive Gs (the heterotrimeric GTP-binding protein that activates adenylyl cyclase) signaling activity of the G protein-coupled receptor GPR3. Here we show that mice deficient for Gpr3 are unexpectedly fertile but display progressive reduction in litter size despite stable age-independent alteration of meiotic pause. Detailed analysis of the phenotype confirms premature resumption of meiosis, in vivo, in about one-third of antral follicles from Gpr3-/- females, independently of their age. In contrast, in aging mice, absence of GPR3 leads to severe reduction of fertility, which manifests by production of an increasing number of nondeveloping early embryos upon spontaneous ovulation and massive amounts of fragmented oocytes after superovulation. Severe worsening of the phenotype in older animals points to an additional role of GPR3 related to protection (or rescue) of oocytes from aging. Gpr3-defective mice may constitute a relevant model of premature ovarian failure due to early oocyte aging.

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Figures

Fig. 1.

Fig. 1.

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).

Fig. 2.

Fig. 2.

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.)

Fig. 3.

Fig. 3.

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.]

Fig. 4.

Fig. 4.

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.

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