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Study hypothesisWe hypothesized that the mitochondria of granulosa cells (GC) and/or oocytes might be abnormal in a mouse model of fragile X premutation (FXPM).Study findingMice heterozygous and homozygous for the FXPM have increased death (atresia) of large ovarian follicles, fewer corpora lutea with a gene dosage effect manifesting in decreased litter size(s). Furthermore, granulosa cells (GC) and oocytes of FXPM mice have decreased mitochondrial content, structurally abnormal mitochondria, and reduced expression of critical mitochondrial genes. Because this mouse allele produces the mutant Fragile X mental retardation 1 (Fmr1) transcript and reduced levels of wild-type (WT) Fmr1 protein (FMRP), but does not produce a Repeat Associated Non-ATG Translation (RAN)-translation product, our data lend support to the idea that Fmr1 mRNA with large numbers of CGG-repeats is intrinsically deleterious in the ovary.What is known alreadyMitochondrial dysfunction has been detected in somatic cells of human and mouse FX PM carriers and mitochondria are essential for oogenesis and ovarian follicle development, FX-associated primary ovarian insufficiency (FXPOI) is seen in women with FXPM alleles. These alleles have 55-200 CGG repeats in the 5' UTR of an X-linked gene known as FMR1. The molecular basis of the pathology seen in this disorder is unclear but is thought to involve either some deleterious consequence of overexpression of RNA with long CGG-repeat tracts or of the generation of a repeat-associated non-AUG translation (RAN translation) product that is toxic.Study design, samples/materials, methodsAnalysis of ovarian function in a knock-in FXPM mouse model carrying 130 CGG repeats was performed as follows on WT, PM/+, and PM/PM genotypes. Histomorphometric assessment of follicle and corpora lutea numbers in ovaries from 8-month-old mice was executed, along with litter size analysis. Mitochondrial DNA copy number was quantified in oocytes and GC using quantitative PCR, and cumulus granulosa mitochondrial content was measured by flow cytometric analysis after staining of cells with Mitotracker dye. Transmission electron micrographs were prepared of GC within small growing follicles and mitochondrial architecture was compared. Quantitative RT-PCR analysis of key genes involved in mitochondrial structure and recycling was performed.Main results and the role of chanceA defect was found in follicle survival at the large antral stage in PM/+ and PM/PM mice. Litter size was significantly decreased in PM/PM mice, and corpora lutea were significantly reduced in mice of both mutant genotypes. Mitochondrial DNA copy number was significantly decreased in GC and metaphase II eggs in mutants. Flow cytometric analysis revealed that PM/+ and PM/PM animals lack the cumulus GC that harbor the greatest mitochondrial content as found in wild-type animals. Electron microscopic evaluation of GC of small growing follicles revealed mitochondrial structural abnormalities, including disorganized and vacuolar cristae. Finally, aberrant mitochondrial gene expression was detected. Mitofusin 2 (Mfn2) and Optic atrophy 1 (Opa1), genes involved in mitochondrial fusion and structure, respectively, were significantly decreased in whole ovaries of both mutant genotypes. Mitochondrial fission factor 1 (Mff1) was significantly decreased in PM/+ and PM/PM GC and eggs compared with wild-type controls.Limitations, reasons for cautionData from the mouse model used for these studies should be viewed with some caution when considering parallels to the human FXPOI condition.Wider implications of the findingsOur data lend support to the idea that Fmr1 mRNA with large numbers of CGG-repeats is intrinsically deleterious in the ovary. FXPM disease states, including FXPOI, may share mitochondrial dysfunction as a common underlying mechanism.Large scale dataNot applicable.Study funding and competing interestsStudies were supported by NIH R21 071873 (J.J./G.H), The Albert McKern Fund for Perinatal Research (J.J.), NIH Intramural Funds (K.U.), and a TUBITAK Research Fellowship Award (B.U.). No conflict(s) of interest or competing interest(s) are noted.

Study hypothesisWe hypothesized that the mitochondria of granulosa cells (GC) and/or oocytes might be abnormal in a mouse model of fragile X premutation (FXPM).Study findingMice heterozygous and homozygous for the FXPM have increased death (atresia) of large ovarian follicles, fewer corpora lutea with a gene dosage effect manifesting in decreased litter size(s). Furthermore, granulosa cells (GC) and oocytes of FXPM mice have decreased mitochondrial content, structurally abnormal mitochondria, and reduced expression of critical mitochondrial genes. Because this mouse allele produces the mutant Fragile X mental retardation 1 (Fmr1) transcript and reduced levels of wild-type (WT) Fmr1 protein (FMRP), but does not produce a Repeat Associated Non-ATG Translation (RAN)-translation product, our data lend support to the idea that Fmr1 mRNA with large numbers of CGG-repeats is intrinsically deleterious in the ovary.What is known alreadyMitochondrial dysfunction has been detected in somatic cells of human and mouse FX PM carriers and mitochondria are essential for oogenesis and ovarian follicle development, FX-associated primary ovarian insufficiency (FXPOI) is seen in women with FXPM alleles. These alleles have 55-200 CGG repeats in the 5' UTR of an X-linked gene known as FMR1. The molecular basis of the pathology seen in this disorder is unclear but is thought to involve either some deleterious consequence of overexpression of RNA with long CGG-repeat tracts or of the generation of a repeat-associated non-AUG translation (RAN translation) product that is toxic.Study design, samples/materials, methodsAnalysis of ovarian function in a knock-in FXPM mouse model carrying 130 CGG repeats was performed as follows on WT, PM/+, and PM/PM genotypes. Histomorphometric assessment of follicle and corpora lutea numbers in ovaries from 8-month-old mice was executed, along with litter size analysis. Mitochondrial DNA copy number was quantified in oocytes and GC using quantitative PCR, and cumulus granulosa mitochondrial content was measured by flow cytometric analysis after staining of cells with Mitotracker dye. Transmission electron micrographs were prepared of GC within small growing follicles and mitochondrial architecture was compared. Quantitative RT-PCR analysis of key genes involved in mitochondrial structure and recycling was performed.Main results and the role of chanceA defect was found in follicle survival at the large antral stage in PM/+ and PM/PM mice. Litter size was significantly decreased in PM/PM mice, and corpora lutea were significantly reduced in mice of both mutant genotypes. Mitochondrial DNA copy number was significantly decreased in GC and metaphase II eggs in mutants. Flow cytometric analysis revealed that PM/+ and PM/PM animals lack the cumulus GC that harbor the greatest mitochondrial content as found in wild-type animals. Electron microscopic evaluation of GC of small growing follicles revealed mitochondrial structural abnormalities, including disorganized and vacuolar cristae. Finally, aberrant mitochondrial gene expression was detected. Mitofusin 2 (Mfn2) and Optic atrophy 1 (Opa1), genes involved in mitochondrial fusion and structure, respectively, were significantly decreased in whole ovaries of both mutant genotypes. Mitochondrial fission factor 1 (Mff1) was significantly decreased in PM/+ and PM/PM GC and eggs compared with wild-type controls.Limitations, reasons for cautionData from the mouse model used for these studies should be viewed with some caution when considering parallels to the human FXPOI condition.Wider implications of the findingsOur data lend support to the idea that Fmr1 mRNA with large numbers of CGG-repeats is intrinsically deleterious in the ovary. FXPM disease states, including FXPOI, may share mitochondrial dysfunction as a common underlying mechanism.Large scale dataNot applicable.Study funding and competing interestsStudies were supported by NIH R21 071873 (J.J./G.H), The Albert McKern Fund for Perinatal Research (J.J.), NIH Intramural Funds (K.U.), and a TUBITAK Research Fellowship Award (B.U.). No conflict(s) of interest or competing interest(s) are noted.

Although successful embryo development is dependent upon genetic and epigenetic contributions from both the male and female, the male potential to adversely affect embryo development has been scarcely studied. It is unclear whether the sperm variation among different males would affect the outcome of oocyte evaluation by embryo development following fertilization. In the present study, variation in the developmental potential of mouse embryos was first compared between in vitro fertilization with epididymal spermatozoa from different males and Sr(2+) parthenogenetic activation using oocytes of different qualities, and then the effect of male on fertilization and embryo development was examined using randomly chosen oocytes and spermatozoa from cauda epididymidis, vas deferens or electro-ejaculates. Rates of fertilization and blastocyst formation were significantly higher with spermatozoa from cauda epididymidis or vas deferens than with ejaculated spermatozoa. Rates of embryonic development differed significantly between different males, but not between different ejaculates of the same male. Analysis of standard errors of means and coefficients of variance indicated that as long as multiple males were involved, the variation in oocyte fertilization/activation and blastocyst formation was always higher after fertilization than after Sr(2+) parthenogenetic activation whether spermatozoa were collected from epididymidis, vas deferens or ejaculates and regardless of oocyte qualities. It is concluded that (1) epididymal mouse spermatozoa fertilize more oocytes than ejaculated spermatozoa under identical experimental conditions; (2) like farm animals, the mice also show a remarkable male effect on the developmental potential of in vitro produced embryos although they are supposed to be less genetically diverse; (3) parthenogenetic activation is recommended for assessment of oocyte quality to exclude the effect of male.

Control of oocyte aging in vitro is important for both human-assisted reproduction and animal embryo technologies because fertilization or artificial activation of aged oocytes results in abnormal development. Interactions between somatic and germ cells are also an important issue in current biological research. The role of cumulus cells (CCs) in maturation, ovulation, and fertilization of oocytes has been extensively studied, yet little is known about their role in oocyte aging. Although our previous study has shown that CCs accelerate the aging progression of mouse oocytes, the mechanism by which CCs accelerate oocyte aging is unknown. In this study, cumulus-denuded mouse oocytes (DOs) were co-cultured with cumulus-oocyte complexes (COCs) or CC monolayer or cultured in medium conditioned with these cells and changes in the susceptibility to activating stimuli and in MPF activity of oocytes were evaluated after different aging treatments. The results showed that culture with or in medium conditioned with COCs or CC monolayer promoted activation of DOs, indicating that a soluble factor is responsible for the aging-promoting effect. The in vivo and in vitro-matured DOs did not differ in responsiveness to the aging-promoting factor (APF). Heat shock did not accelerate oocyte aging unless in the presence of CCs. The production of APF was not affected by the age or maturation system of COCs, but increased with their density and duration of culture. The results strongly suggest that CCs accelerated oocyte aging by secreting a soluble APF into the medium. Further analysis showed that the APF was heat labile but stable to freezing, it had a threshold effective concentration and can be depleted by DOs., (Copyright 2007 Wiley-Liss, Inc.)

We studied the effects of the amount of liposome and plasmid, exposure time of cells to the liposome-plasmid complexes, number of cell passages and cell types on GFP gene transfection of mouse somatic cells. The maximal GFP transgene expression (30.7%) was achieved when mouse fetal fibroblast cells (MFFC) at 70%-90% confluence of passage 3 were exposed for 6 h to the complexes of 4 microg liposome (LipofectAMINE) and 0.3 microg plasmid (pEGFP-N1). Under these conditions, we compared the effect of the number (from primary to 15) of passages on the transfection efficiency of MFFC. The transfection efficiency of MFFC was 10.0%, 28.9% and 7.2% at the primary, 3rd and 15th passage, respectively, which indicated that the transfection efficiency decreased with passaging. When MFFC, mouse oviductal epithelial cells (MOEC) and mouse granulosa cells (MGC) were transfected at passage 3, the transfection efficiency was 27.8%, 13.7% and 14.2%, respectively, under the described transfection conditions. When the cell cycle stages of different cell types at transfection were examined, it was found that 17.2% of MFFC, 8.7% of MOEC and 9.9% of MGC were at M phases of the cell cycle. Examination of the cell cycle stages of MFFC at different passages showed that MFFC at the third passage had the highest percentage of M cells and the percentage decreased afterwards. This suggested that the transfection efficiency was correlated with the percentages of cells at M phase, and provided essential data for improvement of the transfection efficiency.

Although ethylene glycol (EG) has been widely used for embryo cryopreservation in domestic animals, few attempts were made to use this molecule to freeze mouse and human embryos. In the few studies that used EG for slow-freezing of mouse and human embryos, complicated protocols for human embryos were used, and the protocols need to be simplified. Besides, freezing mouse morula with EG as a cryoprotectant has not been reported. In this paper, we studied the effects of embryo stages, EG concentration, duration and procedure of equilibration, sucrose supplementation and EG removal after thawing on the development of thawed mouse embryos, using the simple freezing and thawing procedures for bovine embryos. The blastulation and hatching rates (81.92% +/- 2.24% and 68.56% +/- 2.43%, respectively) of the thawed late compact morulae were significantly (P < 0.05) higher than those of embryos frozen-thawed at other stages. When mouse late compact morulae were frozen with different concentrations of EG, the highest rates of blastocyst formation and hatching were obtained with 1.8mol/L EG. The blastulation rate was significantly higher when late morulae were equilibrated in 1.8 mol/L EG for 10 min prior to freezing than when they were equilibrated for 30 min, and the hatching rate of embryos exposed to EG for 10 min was significantly higher than that of embryos exposed for 20 and 30 min. Both rates of blastocyst formation and hatching obtained with two-step equilibration were higher (P < 0.05) than with one-step equilibration in 1.8 mol/L EG. Addition of sucrose to the EG-based solution had no beneficial effects. On the contrary, an increased sucrose level (0.4 mol/L) in the solution impaired the development of the frozen-thawed embryos. In contrast, addition of 0.1 mol/L sucrose to the propylene glycol (PG)-based solution significantly improved the development of the frozen-thawed embryos. Elimination of the cryoprotectant after thawing did not improve the development of the thawed embryos. The cell numbers were less (P < 0.05) in blastocysts developed from the thawed morulae than in the in vivo derived ones. In summary, embryo stage, EG concentration, duration and procedure of equilibration and sucrose supplementation had marked effects on development of the thawed mouse embryos, and a protocol for cryopreservation of mouse embryos is recommended in which the late morulae are frozen in 1.8 mol/L EG using the simple freezing and thawing procedures of bovine embryos after a two-step equilibration and the embryos can be cultured or transferred without EG removal after thawing.