Changes in the estrous cycle and number of ovulated and fertilized ova in aging female IVCS mice

IV CS mice (ddN origin) aged 90 days (control) and 180, 210, 240, 270, 300, 330, 360 and 420 days were used for observation of the length of the estrous cycle, and the numbers of ovulated and fertilized ova. The estrous cycle between the ages of 90 and 330 days presented a regular 4-day pattern. Thereafter, regularity of the cycle declined steadily between the ages of 360 and 420 days. All mice over 420 days old exhibited cessation of cyclicity and ultimately persistent diestrus. The average number of ova ovulated in mice aged between 90 and 240 days was consistent (11.8 to 11.4 ova), whereas a steady decline was observed for mice between 270 days old (10.5 ova) and 360 days old (4.8 ova). Ova were not recovered from oviducts of mice aged about 420 days. The average number of fertilized ova (2-cell stage) in mice between 90 and 210 days old showed no significant change (11.5 to 10.3), whereas the number began to decrease in mice aged between 240 days (8.7 fertilized ova) and 330 days (2.5 fertilized ova). No fertilized ova were recovered from the oviducts of mice around 360 days old. These findings demonstrate that the decline of reproductive activity is initially observed in the number of fertilized ova, followed by a decline in the number of ovulations and finally by loss of the estrous cycle.     

SPARC synthesis in pre-implantation and early post-implantation mouse embryos

Summary SPARC (secreted protein acidic and rich in cysteine), also known as osteonectin and BM-40, is a secreted protein associated with a variety of embryonic and adult tissue and cell types, including placenta, parietal and visceral endoderm, certain epithelia (e.g. gut, skin, glandular epithelia), and regions of active chondrogenesis and osteogenesis. Although much is known concerning the tissue distribution of this protein, neither the time and location of its initial appearance nor its functions during embryogenesis have been clearly established. We identified the location of SPARC on two-dimensional protein gels. By using two-dimensional gel analysis of both pre- and post-implantation stage mouse embryos, we find that SPARC is initially synthesized between 3.5 and 4.5 days of embryogenesis. This is the earliest time during development at which synthesis of SPARC has been demonstrated. Inner cell masses isolated from 4.5 day blastocysts synthesize SPARC indicating that either primitive ectoderm, primitive endoderm, or both produce this protein. SPARC synthesis is also detectable in isolated trophoblast vesicles. Thus, SPARC is synthesized not only in placenta, parietal endoderm, and visceral endoderm, but in the precursors of these tissues as well. Examination of 7.5 day embryos reveals that SPARC is synthesized in isolated parietal yolk sac and in whole extraembryonic and embryonic regions. Relative to other proteins, synthesis of SPARC was most prevalent in the parietal yolk sac. The possible implications of SPARC synthesis as early as 4.5 days are discussed.     

Aberrant expression of imprinted genes in post-implantation rat embryos

AimImprinted genes are known regulators of embryo growth. Studies from our laboratory have demonstrated that treatment of adult male rats with tamoxifen increased post-implantation loss at around midgestation. Expression of insulin like growth factor 2 (Igf2), a paternally expressed imprinted gene was down-regulated in the resorbing embryos obtained at embryonic day 13. Hypomethylation of Igf2-H19 imprint control region was observed in the resorbing embryo sires and spermatozoa obtained from tamoxifen-treated rats thereby suggesting that errors in imprint acquisition during spermatogenesis can result in embryo loss. The present study aims at studying the expression of other imprinted genes, besides Igf2 in the embryos sired by tamoxifen-treated males.Main methodsGene expression profiles of resorbing versus normal embryos were assessed by microarrays. Real time quantitative RT-PCR for six imprinted genes and four genes involved in cell cycle was done to validate gene expression data. The affected pathways and functions were identified in the resorbing embryos and effect on cell cycle was confirmed by flow cytometry.Key findingsAberrant expression of a number of imprinted genes was observed in the resorbing embryos when compared to the normal embryos at embryonic days 11 and 13. Down-regulation of Notch signaling, Wnt signaling and cell cycle pathway was observed in the resorbing embryos.SignificanceThe study suggests that exposure of male germ cells to tamoxifen during adulthood results in aberrant expression of imprinted genes and down-regulation of development associated pathways in the F₁ progeny thereby causing embryo loss.     

Levels of alpha-inhibin in aging female mice.

alpha-inhibin was immunocytochemically localized in granulosa cells of different stages of developing follicles, freshly formed corpora lutea, and scattered interstitial cells (pigmented or ceroid cells) in ovaries of 6-, 14-, and 23-25-mo-old C57BL/6NNia mice. Developing follicles exhibited the greatest amount of staining. Quantitation of the stain using an image analysis system indicated the staining intensity within ovarian follicles of 14-mo-old mice was greater than that in 23-25-mo-old mice. The levels of plasma alpha-inhibin and estradiol (E2) decreased with age. The number of follicles present in ovaries of middle-aged mice was comparable to those of 6-mo-old mice, yet plasma levels of FSH were significantly higher than those of 6-mo-old mice. This may be due to an age-related loss in the sensitivity of the hypothalamus and/or pituitary of middle-aged mice to ovarian hormones. In contrast, ovaries of 23-25-mo-old mice contained few antral follicles and consequently produced little alpha-inhibin. There appeared to be little negative feedback regulation of FSH secretion in 23-25-mo-old mice as a result of age-related ovarian impairments. This study supports an earlier hypothesis from our laboratory [Biol Reprod 1985; 32:989-997] that the primary defect(s) limiting age-related reproductive performance in mice appears to reside within the hypothalamo-hypophyseal axis, whereas secondary defects arise from the ovary.     

Lipid mediator production by post-implantation rat embryos in vitro

The production of inflammatory lipid mediators by post-implantation rodent embryos was examined in this study. Explanted day 10 rat embryos, either intact or after homogenization, were cultured for 3 hr in vitro and the resulting culture medium and embryonic tissue were assessed for eicosanoids and platelet-activating factor (PAF). The rank order of cyclooxygenase arachidonate products produced by intact embryos was as follows: 6-keto-PGF1 alpha much greater than congruent to PGF2 alpha congruent to TXB2. No lipoxygenase products of arachidonic acid metabolism were detected by either high performance liquid chromatography or radioimmunoassay. PAF production was detectable in embryonic cultures. Homogenization of rat embryos prior to in vitro culture enhanced eicosanoid and PAF production from 2.1-6.9 fold over intact embryos. These findings demonstrate the extent of lipid metabolism by early post-implantation rat embryos and support the concept that potent lipid mediators of inflammation generated by the conceptus may play a role in both the initiation and maintenance of pregnancy.     

Expression of the IGFBP-2 gene in post-implantation rat embryos.

The insulin-like growth factors (IGFs) stimulate mitogenesis in a variety of cell types both in vitro and in vivo. These effects are mediated by both IGF receptors and a family of IGF binding proteins (IGFBPs), which are found complexed with the IGFs in serum and tissue fluids. Here we compare the sites of expression during early rat embryogenesis of the genes encoding the RGD-containing IGF binding protein IGFBP-2 and IGF-II. At all ages from early post-implantation through mid-gestation, the expression of IGFBP-2 was highly complementary to IGF-II. IGFBP-2 mRNA was detected throughout the epiblast of the egg cylinder as early as e7, when IGF-II expression was restricted to trophectoderm and other extraembryonic cells. As gastrulation proceeded, IGFBP-2 expression ceased as IGF-II expression began in the newly formed embryonic and extra-embryonic mesoderm, but was retained in other epiblast derivatives including the surface ectoderm and neuroectoderm, throughout its rostral-caudal extent. By e10-e11, IGFBP-2 expression in neuroectoderm was restricted to the rostral brain of the primary neural tube and was found in the new population of neuroepithelium formed in the tail bud during secondary neurulation. IGFBP-2 expression remained high in the ventricular layer of the rostral brain into mid-gestation ages but decreased or disappeared as cells entered the mantle layer and began to express the neurofilament-related gene alpha-internexin. IGFBP-2 mRNA was abundant in surface ectoderm, particularly that of the branchial arches, and all ectodermal placodes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific expression in adult mice and post-implantation embryos of a transgene carrying the histone H1° regulatory region

Abstract. Histone H1°, a variant of the H1 group, has been found associated with the repressed state of chromatin and its content is increased in terminally differentiated cells. We have cloned a mouse H1° histone gene and introduced the promoter region, ligated to the β-galactosidase reporter gene, into transgenic mice. By histochemistry we demonstrated a strong expression of the transgene in adult kidney, testis and brain. Intestine, uterus and ovarium were also positive. This expression followed the same pattern as that of the endogenous H1° gene, as demonstrated by in situ hybridization with a non-coding fragment of the mRNA, by Northern analysis, and by immunofluorescence with specific antibodies. In post-implantation embryos, the expression was very low up to day ten p.c. At this time, most of the X-Gal staining was found in the brain, retina and some of the large blood vessels. Hence, expression of the transgene as well as of the endogenous H1° gene is not exclusively linked to a differentiated phenotype or to a reduced cell proliferation capacity.     

Early patterning of cloned mouse embryos contributes to post-implantation development

Several research groups have suggested that the embryonic-abembryonic (Em-Ab) axis in the mouse can be predicted by the first cleavage plane of the early embryo. Currently, it is not known whether this early patterning occurs in cloned embryos produced by nuclear transfer and whether it affects development to term. In this work, the relationship between the first cleavage plane and the Em-Ab axis was determined by the labeling of one blastomere in cloned mouse embryos at the 2-cell stage, followed by ex-vivo tracking until the blastocyst stage. The results demonstrate that approximately half of the cloned blastocysts had an Em-Ab axis perpendicular to the initial cleavage plane of the 2-cell stage. These embryos were classified as "orthogonal" and the remainder as "deviant". Additionally, we report here that cloned embryos were significantly more often orthogonal than their naturally fertilized counterparts and overexpressed Sox2. Orthogonal cloned embryos demonstrated a higher rate of post-implantation embryonic development than deviant embryos, but cloned pups did not all survive. These results reveal that the angular relationship between the Em-Ab axis and the first cleavage plane can influence later development and they support the hypothesis that proper early patterning of mammalian embryos is required after nuclear transfer.     

Dynamic transition of Dnmt3b expression in mouse pre- and early post-implantation embryos

The de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns in mouse development. Dnmt3b is more highly expressed in early developmental stages than Dnmt3a, and is thought to have an important role in the epigenetic gene regulation during early embryogenesis. Previous reports suggest that Dnmt3b is expressed preferentially in the embryonic lineage, but less in the extra-embryonic lineage, in early post-implantation embryos. However, it is unclear when this lineage-specific differential expression is established. Here we demonstrate that Dnmt3b shows a dynamic expression change during pre- and early post-implantation development. Contrary to the expectation, Dnmt3b is preferentially expressed in the trophectoderm rather than the inner cell mass at the mid blastocyst stage. Subsequently, the spatial Dnmt3b expression gradually changes during pre- and early post-implantation development, and finally Dnmt3b expression is settled in the embryonic lineage at the epiblast stage. The findings are consistent with the role for Dnmt3b in cell-lineage specification and the creation of lineage-specific DNA methylation patterns.     

Regulation of cellular adhesion molecule expression in murine oocytes,peri-implantation and post-implantation embryos

Expression of the adhesion molecules, ICAM-1, VCAM-1, NCAM, CD44, CD49d (VLA-4, α chain) ,and CDlla (LFA-1, α chain) on mouse oocytes, and pre- and peri-implantation stage embryos was exam-ined by quantitative indirect immunoliuorescence microscopy. ICAM-1 was most strongly expressed at the oocyte stage, gradually declining almost to undetectable levels by the expanded blastocyst stage. NCAM,also expressed maximally on the oocyte, declined to undetectable levels beyond the morula stage. On the other hand, CD44 declined from highest expression at the oocyte stage to show a second maximum at the compacted 8-cell/morula. This molecule exhibited high expression around contact areas between trophecto-derm and zona pellucida during blastocyst hatching. CD49d was highly expressed in the oocyte, remained significantly expressed throughout and after blastocyst hatching was expressed on the polar trophecto-derm. Like CD44, CD49d declined to undetectable levels at the blastocyst outgrowth stage. Expression of both VCAM-1 and CDlla was undetectable throughout. The diametrical temporal expression pattern of ICAM-1 and NCAM compared to CD44 and CD49d suggest that dynamic changes in expression of adhesion molecules may be important for interaction of the embryo with the maternal cellular environment as well as for continuing development and survival of the early embryo.     

Regulation of cellular adhesion molecule expression in murine oocytes,peri-implantation and post-implantation embryos

Expression of the adhesion molecules, ICAM-1, VCAM-1, NCAM, CD44, CD49d (VLA-4, alpha chain), and CD11a (LFA-1, alpha chain) on mouse oocytes, and pre- and peri-implantation stage embryos was examined by quantitative indirect immunofluorescence microscopy. ICAM-1 was most strongly expressed at the oocyte stage, gradually declining almost to undetectable levels by the expanded blastocyst stage. NCAM, also expressed maximally on the oocyte, declined to undetectable levels beyond the morula stage. On the other hand, CD44 declined from highest expression at the oocyte stage to show a second maximum at the compacted 8-cell/morula. This molecule exhibited high expression around contact areas between trophectoderm and zona pellucida during blastocyst hatching. CD49d was highly expressed in the oocyte, remained significantly expressed throughout and after blastocyst hatching was expressed on the polar trophectoderm. Like CD44, CD49d declined to undetectable levels at the blastocyst outgrowth stage. Expression of both VCAM-1 and CD11a was undetectable throughout. The diametrical temporal expression pattern of ICAM-1 and NCAM compared to CD44 and CD49d suggest that dynamic changes in expression of adhesion molecules may be important for interaction of the embryo with the maternal cellular environment as well as for continuing development and survival of the early embryo.     

Regulation of cellular adhesion molecule expression in murine oocytes,peri-implantation and post-implantation embryos

Expression of the adhesion molecules, ICAM-1, VCAM-1, NCAM, CD44, CD49d (VLA-4, α chain),and CD11a (LFA-1, α chain) on mouse oocytes, and pre- and peri-implantation stage embryos was exam-ined by quantitative indirect immunofluorescence microscopy. ICAM-1 was most strongly expressed at theoocyte stage, gradually declining almost to undetectable levels by the expanded blastocyst stage. NCAM,also expressed maximally on the oocyte, declined to undetectable levels beyond the morula stage. On theother hand, CD44 declined from highest expression at the oocyte stage to show a second maximum at thecompacted 8-cell/morula. This molecule exhibited high expression around contact areas between trophecto-derm and zona pellucida during blastocyst hatching. CD49d was highly expressed in the oocyte, remainedsignificantly expressed throughout and after blastocyst hatching was expressed on the polar trophecto-derm. Like CD44, CD49d declined to undetectable levels at the blastocyst outgrowth stage. Expression ofboth VCAM-1 and CD11a was undetectable throughout. The diametrical temporal expression pattern ofICAM-1 and NCAM compared to CD44 and CD49d suggest that dynamic changes in expression of adhesionmolecules may be important for interaction of the embryo with the maternal cellular environment as wellas for continuing development and survival of the early embryo.     

Analysis of post-implantation mouse embryos after maternal heat stress during meiotic maturation

The effects of heat stress during oocyte maturation were studied in post-implantation mouse embryos. Virgin ICR mice were exposed to 35 +/- 1 degree C and 65 +/- 3% RH for 12.5 h beginning immediately after synchronization of ovulation with PMSG and hCG. Embryos of heat-stressed dams were developmentally heterogeneous and showed significant delays in development with as much as 48 h delayed development. Nearly 6% of these embryos were triploid, and another 2% were hyper-diploid. Development of triploid embryos was delayed more than 24 h. Nine embryos with severe developmental delay had heterogeneous chromosome constitutions. Embryo mortality before and after implantation was higher in heat-stressed dams than in controls.     

Tissue-specific RNA interference in post-implantation mouse embryos using directional electroporation and whole embryo culture

In mammals, embryonic development is more difficult to analyze than in non-mammalian species because this development occurs in utero. Interestingly, whole embryo culture allows the normal development of mouse post-implantation embryos for up to 2 days in vitro. One limitation of this technology has been the difficulty of performing loss-of-gene function studies in this system. RNA interference (RNAi), whereby double-stranded RNA molecules suppress the expression of complementary genes, has rapidly become a widely used tool for gene function analyses. We have combined the technologies of mouse whole embryo culture and RNAi to allow the molecular dissection of developmental processes. Here, we review the manipulation by topical injection followed by directional electroporation of endoribonuclease-prepared siRNA to demonstrate that this technology may be useful to knock down genes in a tissue- and region-specific manner in several organs of the developing mouse embryo.     

Regulation of cellular adhesion molecule expression in murine oocytes, peri-implant ation and post-implantation embryos

Expression of the adhesion molecules, ICAM-1, VCAM-1, NCAM, CD44, CD49d (VLA-4, a chain), and CDlla (LFA-1, a chain) on mouse oocytes, and pre- and peri-implantation stage embryos was examined by quantitative indirect immunofluorescence microscopy. ICAM-1 was most strongly expressed at the oocyte stage, gradually declining almost to undetectable levels by the expanded blastocyst stage. NCAM, also expressed maximally on the oocyte, declined to undetectable levels beyond the morula stage. On the other hand, CD44 declined from highest expression at the oocyte stage to show a second maximum at the compacted 8-cell/morula. This molecule exhibited high expression around contact areas between trophecto-derm and zona pellucida during blastocyst hatching. CD49d was highly expressed in the oocyte, remained significantly expressed throughout and after blastocyst hatching was expressed on the polar trophecto-derm. Like CD44, CD49d declined to undetectable levels at the blastocyst outgrowth stage. Expression of both     

Metformin slows down aging and extends life span of female SHR mice

Studies in mammals have led to the suggestion that hyperglycemia and hyperinsulinemia are important factors both in aging and in the development of cancer. It is possible that the life-prolonging effects of calorie restriction are due to decreasing IGF-1 levels. A search of pharmacological modulators of insulin/IGF-1 signaling pathway (which resemble effects of life span extending mutations or calorie restriction) could be a perspective direction in regulation of longevity. Antidiabetic biguanides are most promising among them. Here we show the the chronic treatment of female outbred SHR mice with metformin (100 mg/kg in drinking water) slightly modified the food consumption but decreased the body weight after the age of 20 months, slowed down the age-related switch-off of estrous function, increased mean life span by 37.8%, mean life span of last 10% survivors by 20.8%, and maximum life span by 2.8 months (+10.3%) in comparison with control mice. On the other side, treatment with metformin failed influence blood estradiol concentration and spontaneous tumor incidence in female SHR mice. Thus, antidiabetic biguanide metformin dramatically extends life span, even without cancer prevention in this model.