Genetic contributions of the endothelial nitric oxide synthase gene to ovulation and menopause in a mouse model

OBJECTIVE: To investigate the influence of the endothelial nitric oxide synthase gene (Nos3) on ovulatory capacity and reproductive senescence. DESIGN: Prospective, controlled study. SETTING: Academic research institution. SUBJECT(s): Laboratory mice with targeted mutagenesis of Nos3. INTERVENTION(s): Hyperstimulation protocol, oocyte culture, and ovarian histology using wild-type (Nos3(+/+); n = 20), heterozygous (Nos3(+/m); n = 39), and homozygous deficient (Nos3(m/m); n = 11) female mice; observation of reproductive outcomes. MAIN OUTCOME MEASURE(s): Number and survival of oocytes; onset of menarche and menopause. RESULT(s): The mean number of superovulated oocytes (18 +/- 36 vs. 41 +/- 4) and the 48-hour overall survival rate of embryos (65% vs. 81%) were significantly reduced for Nos3(m/m) female mice compared with Nos3(+/+) female mice. Nos3(m/m) females showed a significantly reduced number and size of antral follicles and corpora lutea compared with wild-type controls. Compared with Nos3(+/m) x Nos3(+/m) breedings, Nos3(m/m) x Nos3(m/m) breedings showed a higher female age at first litter (76.2 +/- 10.3 vs. 107.8 +/- 26.6 days), fewer litters (10.5 +/- 3.6 vs. 7. 8 +/- 4.2), and a lower female age at reproductive senescence (400.2 +/- 64.5 vs. 332.1 +/- 27.4 days), respectively. CONCLUSION(s): Our data suggest that Nos3 deficiency is associated with reduced ovulatory capacity and impaired early embryonic viability and that it influences the onset of menarche and menopause.     

具生殖系嵌合能力的C57BL/6J小鼠胚胎干细胞系的建立

目的:建立能实现种系传递的C57BL/6J小鼠胚胎干细胞系。方法:从C57BL/6J小鼠3.5d的囊胚中分离培养内细胞团。经体外适宜培养建系后,将C57BL/6J胚胎干细胞(ES)注入ICR小鼠受体囊胚腔,制备嵌合体小鼠。结果:成功地建立了3个C57BL/6J小鼠胚胎干细胞系,该C57BL/6JES细胞呈集落状生长,正常稳定的核型率>80%,具高水平的磷酸酶活性,并表达ES细胞特殊性细胞表面标记SSEA-1,不表达SSEA-3和SSEA-4;体内外的分化实验也证实mC57ES1具向三胚层组织分化的能力。经显微注入ICR小鼠囊胚腔中,产生4只嵌合体小鼠,经证实其中1只为种系嵌合小鼠。结论:建立了能实现种系传递的C57BL/6J小鼠胚胎干细胞系,该系的ES细胞可用于今后制备转基因动物和基因敲除动物。     

Preimplantation development following in vitro fertilization of mouse oocytes: Effects of timing of superovulation and preincubation in vitro

The early embryonic development of in vitro fertilized oocytes was assessed following superovulation in F₁ hybrid C57BL/6×CBA/Ca mice. Decreasing the time interval between the administration of constant doses of pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) resulted in decreases in the frequency of development to the blastocyst stage but had no significant effect on development to the two-cell stage. Preincubation of postovulatory oocytes in vitro prior to insemination did not compensate for the reduced preovulatory development in vivo but resulted in decreases in the frequency of development to the blastocyst stage. The results indicate that inadequate preovulatory development of superovulated mouse oocytes can adversely affect the preimplantation development of in vitro fertilized embryos in the absence of a visible inhibitory effect on development to the two-cell stage and also that preincubation of postovulatory oocytes in vitro prior to fertilization reduces subsequent developmental capacity.     

Preimplantation genetic diagnosis for polycystic kidney disease

OBJECTIVE: To use preimplantation genetic diagnosis for achieving a polycystic kidney disease (PKD)-free pregnancy for a couple in which the female partner was affected by PKD but whose PKD1 or PKD2 carrier status was not established. DESIGN: Case report. SETTING: The IVF program of Reproductive Genetics Institute, Chicago, Illinois. PATIENT(S): An at-risk couple with the female partner affected by PKD, whose PKD1 or PKD2 carrier status was not established. INTERVENTION(S): Removal of PB1 and PB2 and testing for three closely linked markers to PKD1 (Kg8, D16S664, and SM7) and four closely linked markers to PKD2 (D4S2922, D4S2458, D4S423, and D4S1557) after standard IVF. MAIN OUTCOME MEASURE(S): Deoxyribonucleic acid analysis of PB1 and PB2 indicating whether corresponding oocytes were PKD1 or PKD2 allele free, for the purpose of transferring only embryos resulting from mutation-free oocytes. RESULT(S): Of 11 oocytes tested by PB1 and PB2 DNA analysis, 7 were predicted to contain PKD1 or PKD2, with the remaining 4 free of both mutations. Three embryos resulting from these oocytes were transferred, yielding a twin pregnancy and the birth of two unaffected children. CONCLUSION(S): This is the first preimplantation genetic diagnosis for PKD, which resulted in the birth of healthy twins confirmed to be free of PKD1 and PKD2. Preimplantation genetic diagnosis based on linked marker analysis provides an alternative for avoiding the pregnancy and birth of children with PKD, even in at-risk couples without exact PKD1 or PKD2 carrier information.     

Maternal cell traffic in allogeneic embryos

Blastocysts from CBA and/or C57BL/6 mice were transferred into the uterus of allogeneic, syngeneic or semi-syngeneic pseudopregnant female mice. Observations of embryo status, relative embryo position in the uterus and immunofluorescent staining of embryonic cells for determination of H-2 antigen type shows that cell movement occurs between the maternal host and the fetus and apparently among embryos. Both in-utero maternal cell traffic in the fetus and the rate of fetal resorptions depend on the proximity of allogeneic embryos in utero and the genetic inter-relationship of the surrogate mother to the fetus.     

Immunological relationships between murine surrogate mothers and transplanted embryos, as studied by local GVH reactivity

C57BL/Ks (H-2d) female mice were transplanted with early (stage 2) embryos of the A/J (H-2a) strain. Spleens from mice exhibiting successful pregnancies were tested at days 16 to 19 of gestation in a local graft versus host (LGVH) assay using (C57BL/Ks X A/J)F1 recipients and proved to be significantly more reactive than virgin controls or mice carrying transplanted syngeneic fetuses. This increased reactivity was specific for the transplanted embryo's strain. Other controls included donors with semi-allogeneic (F1) transplanted fetuses and females naturally pregnant by allogeneic males which did not give reactions significantly different from virgin control spleen cells. Para-aortic lymph node cells (PALN) obtained from the same A/J embryo-transplanted females showed a strong T suppressive activity both on their own spleen cell (SC) reaction as well as on the reaction obtained with virgin SC. This suppressive activity also appeared to be embryo-strain specific. Serological tests revealed the presence of mast cell-degranulating (anaphylactic) antibodies but not of hemagglutinating or complement-fixing cytotoxic activities. The A/J offspring obtained after embryo transfer to C57BL/Ks females presented at the age of two months significantly lower LGVH reactivity against the surrogate mother's strain. The differences in the responsiveness of the mice transplanted with allogeneic embryos compared with those with conventional pregnancies are discussed.     

Variation in immunophenotype of lactating mice

Immunological factors have been shown to play a crucial role in mammary remodelling in rodent models of lactation, particularly at the stage of mammary involution. However, the relationship between immunological factors and the ability of normal mammary gland to produce milk, as well as the genetic components contributing to lactation performance remain largely unknown. In this study, we assessed the lactation and immunological phenotypes of 11 inbred mouse strains, namely 129X1/SvJ (129), A/J, AKR, C3H/HeJ (C3H), CBA/CaH (CBA), C57BL/6J (C57), DBA/1J, DBA/2J, FVB/N (FVB), QSi5 and SJL/J (SJL) to identify potential links. Leukocyte analyses showed no direct link between the fraction of splenic leukocytes and lactation performance. However, significant strain differences were discovered in the fraction of CD8+ T lymphocytes (P=0.016) and CD11b+Gr-1 mid-low monocytes (P<0.001). Cytokine profiles in plasma were examined and a subset of plasma cytokines, namely CCL2, CCL3, CCL5, CSF2, CSF3, IL10, IL15, IL1B, IL4, IL5, IL7 and TNF, were fitted to a linear regression model for prediction of lactation performance (R-sq=62%, S=0.309). Significant strain differences in the plasma cytokine levels were also discovered amongst these inbred strains. Analysis of immunological phenotypes showed strong correlations between splenic immune cell subsets and their regulating cytokine levels in plasma. The results demonstrate the extent of genetic variability in the immunological phenotypes of lactating mice, and provide a basis for understanding the role of cytokines in milk production, and identifying potential biomarkers of lactation performance.     

Immunization with a syngeneic regressor tumor causes resorption in allo-pregnant mice

The purpose of our studies is to establish experimental systems in which one can deliberately disrupt the apparent maternal tolerance toward the semiallogeneic fetuses. Bases on the hypothesis that immunization against tumor-associated antigens may lead to a subsequent immune response directed against cross-reacting fetal antigens, we have immunized C57BL/6J female mice with a syngeneic regressor tumor. Mice were subsequently mated to B6D2F1, DBA/2, CBA/J or C57BL/6J males. We show that a high proportion of embryos sired by either B6D2F1 or DBA/2 males undergo resorption whereas those engendered by CBA/J or C57BL/6J males remain fully protected.     

MicroRNA expression in preimplantation mouse embryos from <Emphasis Type="Italic">Ped</Emphasis> gene positive compared to <Emphasis Type="Italic">Ped</Emphasis> gene negative mice

PURPOSE: The mouse preimplantation embryo development (Ped) gene product, Qa-2, influences the rate of preimplantation embryonic development and overall reproductive success. Here we investigated the expression pattern of two microRNAs, miR-125a and miR-125b, known to be involved in development in lower organisms, in preimplantation embryos from the two-cell, four-cell, eight-cell, morula, and blastocyst stages of development from the congenic B6.K1 (Ped negative) and B6.K2 (Ped positive) strains of mice. METHOD: B6.K1 and B6.K2 congenic mice differ only in the absence (B6.K1) or presence (B6.K2) of the genes encoding Qa-2 protein. We analyzed the expression of miR-125a and miR-125b in B6.K1 and B6.K2 preimplantation embryos by using real-time PCR. RESULT: We found no variability in miR-125b expression at any developmental stage in both strains. However, miR-125a expression increased during development in both strains and was ten times higher in Ped negative (B6.K1) embryos than in Ped positive (B6.K2) embryos by the blastocyst stage of development. CONCLUSION: Our results show that the absence of the Ped gene profoundly affects the level of a miRNA (miR-125a) known to regulate early development. The implication is that miR-125a is likely involved in the regulation of timing of early development in mice.     

Neurobehavior effects in four strains of mice offspring exposed prenatally to alprazolam

OBJECTIVE: This study was performed to determine whether prenatal exposure to alprazolam affects offspring behavior in different strains of mice. STUDY DESIGN: Eight to 11 gravid mice of the C3H/He, C57BL/6, A/J, and DBA/2 strains were given either an anxiolytic dose of alprazolam (0.32 mg/kg) or a placebo by gavage on day 18 of an anticipated 19- to 21-day gestation. Neurobehavior tasks were conducted to assess anxiety, learning and memory, and social interaction. Data were analyzed by analysis of variance or a Fisher exact probability test. RESULTS: Anxiety in alprazolam-exposed offspring was reduced in C3H/He (P <.05) and A/J (P <.05) newborn infants by separation vocalization but may be increased in the C3H/He adult strain on the plus maze task. Learning was slower among C57BL/6 mice exposed to alprazolam (P <.01), whereas memory was reduced in exposed A/J and DBA/2 offspring (P <.05). Alprazolam exposure was associated with more aggression among C3H/He and C57BL/6 male offspring (P <.01) and with less group activity by C57BL/6 offspring (P <.05). CONCLUSION: Altered behaviors in several mouse strains after prenatal exposure to alprazolam suggests a vulnerability of GABA-benozdiazepine receptor formation in fetal brain development.     

Differential expression of c-fos in a mouse model of fetal alcohol syndrome

OBJECTIVE: Fetal alcohol syndrome (FAS) results in stillbirth, fetal growth restriction, and mental retardation with injury attributed to oxidative stress. Our objective was to identify signal transduction pathways expressed in a model of FAS and to quantify expression of c-fos, a gene in the stress signal pathway. STUDY DESIGN: Timed, pregnant C57Bl6/J mice were injected on E8 with saline solution or alcohol. RNA was extracted from decidua and embryo 6 and 24 hours later. Microarray analysis was used to screen gene pathways. Differential gene expression was confirmed using real-time polymerase chain reaction with results presented as the ratio of c-fos concentration to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RESULTS: Differential gene expression between alcohol and control was noted for stress signal pathway genes including c-fos. Real-time polymerase chain reaction demonstrated that c-fos messenger RNA expression was greater in the alcohol than control decidua at 6 hours after injection (P<.01). This effect persisted at 24 hours (P<.01). There was no difference in c-fos expression in embryos whose mothers received alcohol versus control after 6 hours (P=.12) or 24 hours (P=.89). CONCLUSION: Alcohol administration during pregnancy results in differential gene expression in the stress signal pathway, particularly in c-fos. C-fos expression in the decidua increases from 6 to 24 hours after alcohol injection, but does not change in the embryo, which may contribute to alcohol-induced damage in FAS.     

Use of knockout transgenic mice in the study of endometriosis: insights from mice lacking beta(2)-microglobulin and interleukin-12p40

OBJECTIVE: To test the possibility of using transgenic knockout mice in the study of endometriosis and to investigate specific immunologic aspects of the disease. DESIGN: Experimental blinded study. SETTING: Academic research center. ANIMAL(S): Thirty-two mice with experimentally induced endometriosis. INTERVENTION(S): Endometriosis was induced in 8 beta(2)-microglobulin-deficient BALB/c mice and 7 wild-type BALB/c controls. Similarly, endometriosis was induced in 8 interleukin-12-deficient C57BL/6 mice and in 9 wild-type C57BL/6 controls. MAIN OUTCOME MEASURE(S): Weight and surface area of endometriotic lesions. RESULT(S): Total weight and surface area of endometriotic lesions was markedly lower in beta(2)-microglobulin-deficient BALB/c mice than in wild-type BALB/c controls. A slight but statistically insignificant increase in total weight and surface area of lesions was observed in interleukin-12-deficient C57BL/6 mice compared to wild-type C57BL/6 controls. CONCLUSION(S): Knockout transgenic mice can be used successfully for the study of endometriosis; however, in these animals, the redundancy of the immunologic cytokine-mediated regulatory mechanisms may lead to compensation from the remaining genome. Results from beta(2)-microglobulin-deficient mice support the critical role of the immune system in the pathogenesis of the disease.     

A polymorphism of the Nos3 gene and age at natural menopause

To investigate the possible influence of a polymorphism of the Nos3 gene on menarche and onset of menopause in humans.Cohort study.Academic research institution.Ninety-one consecutive Caucasian postmenopausal women.Peripheral venous puncture and a patient questionnaire were administered.A tandem repeat polymorphism in intron 4 of Nos3 was analyzed by polymerase chain reaction amplification.The common B allele was identified on 143 of 182 chromosomes (frequency 0.79). The polymorphic A allele was present on 39 chromosomes (frequency 0.21). The genotype frequencies were as follows: 58.2% (B/B), 40.7% (A/B), and 1.1% (A/A). Age at menarche, number of deliveries, number of miscarriages, and onset of menopause did not differ between genotypes. Smoking and increased body mass index were associated with an earlier onset of natural menopause.In contrast to mouse models, in humans Nos3 does not seem to modulate onset and cessation of menses.     

In vitro maturation and fertilization of oocytes isolated from aged mice: A strategy to rescue valuable genetic resources

Purpose This project was to determine whether oocytes isolated from virgin aged mice, up to 18 months old, are competent to undergo cytoplasmic maturation in vitro and undergo fertilization and embryonic development. If so, oocyte maturation in vitro could be used as a strategy to rescue valuable genetic resources.Results Although the number of oocytes recovered from mice was greatly reduced with increasing age, the percentage of oocytes that underwent fertilization, cleavage, and development to the blastocyst stage was essentially unchanged up to 18 months of age. The success of cleavage to the two-cell stage was greater after maturation in vitro (81%) than gonadotropin-induced maturation in vivo (55%). About 20% (20/106) of the embryos derived from oocytes isolated from 18-month-old mice developed to term after embryo transfer.Conclusion Oocytes from virgin aged mice undergo normal cytoplasmic maturation in vitro. Higher percentages of oocytes from aged mice cleave to the two-cell stage after spontaneous maturation in vitro than after gonadotropin-induced maturation in vivo. Therefore, in vitro maturation and fertilization of oocytes could be used to rescue valuable genetic resources that might otherwise be lost because of age-related infertility.     

Differences in immunogenicity indicating polymorphism of sperm antigens from mice of different inbred strains

Polymorphism of mouse sperm was investigated by analysis of immune sera generated in BALB/c female mice against sperm from 6 inbred strains. The immune sera were analyzed by immunofluorescence and Western blot techniques against sperm antigens from the 6 immunizing strains. Immunofluorescence revealed no differences in reactivity patterns or titers. However, several different reaction patterns were detected by Western blot technique which indicated that both the sperm extracts and the antisperm immune sera contained different components. Syngeneic (anti-BALB/c sperm) antisera showed far fewer reactive antibody species than allogeneic immune sera. The anti-BALB/c sera recognized an antigen of 23 kDa in sperm extracts from DBA/2J and C57BL/6 mice, and failed to react with an antigen of the same molecular weight when applied to sperm from A/J and 129/J mice, indicating antigenic differences between sperm from these inbred strains. Anti-C57BL/6 sera contained a unique antibody which reacted with an antigen of 80 kDa in all of the 6 sperm extracts, whereas others antisera did not detect this antigen. These findings indicate antigenic and immunogenic polymorphism in sperm from different inbred strains of mice.     

Impact of genetic background on placental glycogen storage in mice

129 and C57BL/6 are two of the most widely used laboratory mouse strains. While it is well known that genetic modifiers between the two strains can directly influence embryonic and adult phenotypes, less is known regarding morphological differences in placental development. Here we identify differences in the junctional zone, glycogen storage and the maternal-fetal interface between these two strains and provide examples where these differences impact the phenotypic characterisation of placental mutations.     

Effects of simulated microgravity on mammalian fertilization and preimplantation embryonic development in vitro

Objective: To study the effects of simulated microgravity on mammalian fertilization and preimplantation embryonic development in vitro with the use of a horizontal clinostat device.

Design: Controlled animal study.

Setting: Research laboratory at a university medical school.

Animal(s): B6D2F1 (C57BL/6 × DBA/2) and ICR mice between 8 and 10 weeks old.

Intervention(s): The first experiment was performed to investigate whether gravity is required for fertilization in vitro under three conditions: clinostat rotation, rotational control, and stationary control. In the second experiment, one-cell embryos were cultured under each condition and their morphology and viability were assessed at 96 hours.

Main Outcome Measure(s): The fertilized numbers and embryonic numbers at the morula and blastocyst stages were recorded in each condition.

Result(s): In the first experiment, there were no statistically significant differences in the efficiency of achieving normal fertilization in vitro among the conditions. In the second experiment, there was a statistically significant decrease in the number of embryos reaching the morula and blastocyst stages after 96 hours in culture under clinostat rotation.

Conclusion(s): These results suggest that the process of fertilization in vitro is not sensitive to the gravitational vector. However, the possibility exists that the frequency of early embryonic lethality is increased by microgravity.     

Coculture of human oviductal cells maintains mitochondrial function and decreases caspase activity of cleavage-stage mouse embryos

OBJECTIVE: To investigate the mitochondrial function and caspase activity in mouse embryos after human oviductal cell coculture. DESIGN: Experimental laboratory study. SETTING: University gynecology unit. ANIMAL(S): MF-1 (female); BALB/c (male) mice. INTERVENTION(S): Mouse embryos were cocultured with human oviductal cells. MAIN OUTCOME MEASURE(S): Mitochondrial transmembrane potential (Delta psi(m)) and caspase activity. RESULT(S): Compared to embryos after coculture in Chatot-Ziomek-Bavister (CZB) medium supplemented with 0.5 mg/mL of BSA (CZB), Delta psi m of embryos cultured in CZB was significantly lower at the two-cell (CZB, 2.04 +/- 0.412; coculture, 4.34 +/- 0.563) and morula (CZB, 6.06 +/- 0.548; coculture, 7.12 +/- 0.568) stages. Cocultured embryos and in vivo developed embryos had comparable Delta psi m. Caspase activity was not detected in unfragmented cleavage-stage embryos and morula developed in vivo. In vitro cultured morula possessed caspase activity. The activity was significantly reduced in the cocultured morula. CONCLUSION(S): Human oviductal cells maintained the mitochondria function in terms of mitochondrial transmembrane potential and decreased the caspase activity to improve the development of mouse embryo.     

Cryoprotective effects of various saccharides on cryopreserved mouse sperm from various strains

Aim  Cryopreservation of mouse sperm commonly uses raffinose, which is a trisaccharide, plus 3% skim milk. Because of the present lack of knowledge of the effectiveness of any other saccharides, we examined the cryoprotective effects of various saccharides on the viability of mouse sperm from various strains to determine which saccharides are the best cryoprotectants for mouse sperm. Methods  Sperm from the caudae epididymides of mature C57BL/6J mice were frozen with monosaccharides (fructose, glucose, rhamnose, xylose), disaccharides (lactose, maltose, sucrose, trehalose) or trisaccharides (melezitose, raffinose) in a range of concentrations (4–33%). After thawing, the optimal concentration was determined to be the concentration in which there was the highest proportion of motile sperm. In addition, sperm of inbred and hybrid mice were frozen with the saccharides at the optimal concentrations and used for in vitro fertilization. Results  The optimal concentration was 12% for the disaccharides and 18% for the trisaccharides. The fertility of all strains, except C57BL/6J, showed the best cryoprotective effects with maltose, melezitose and raffinose when compared with fresh sperm. Conclusion  Maltose, melezitose and raffinose have the best effects when used as a protectant for cryopreservation of mouse sperm.