The biology and methodology of assisted reproduction in deer mice (Peromyscus maniculatus)

Although laboratory-reared species of the genus Peromyscus—including deer mice—are used as model animals in a wide range of research, routine manipulation of Peromyscus embryogenesis and reproduction has been lagging. The objective of the present study was to optimize conditions for oocyte and/or embryo retrieval and for in vitro culturing. On average, 6.4 oocytes per mouse were recovered when two doses of 15 IU of pregnant mare serum gonadotropin (PMSG) were given 24 h apart, followed by 15 IU of hCG 48 h later. Following this hormone priming, females mated overnight with a fertile male yielded an average of 9.1 two-cell stage embryos. Although two-cell stage embryos developed to 8-cell stage in Potassium Simplex Optimized Medium (KSOM; Millipore-Chemicon, Billerica, MA, USA) in vitro, but not further, embryos recovered at the 8- to 16-cell stages developed into fully expanded blastocysts when cultured in M16 media in vitro. These blastocysts had full potential to develop into late stage fetuses and possibly into live pups. As a result of the present work, all stages of Peromyscus preimplantation development are now obtainable in numbers sufficient for molecular or other analyses. These advances provide the opportunity for routine studies involving embryo transfer (e.g., chimeras, transgenics), and preservation of genetic lines by cryopreservation.     

Importance of culture conditions during the morula-to-blastocyst period on capacity of inner cell-mass cells of bovine blastocysts for establishment of self-renewing pluripotent cells

The hypothesis was tested that the pluripotency of the inner cell mass (ICM) of the bovine embryo is enhanced by the glycogen synthase kinase-3β inhibitor CHIR99021 and the MAPK1 and MAPK3 inhibitor PD032591. Treatment with the two inhibitors from Days 6 to 8 after insemination increased blastocyst steady state concentrations of mRNA for NANOG (P < 0.05) and SOX2 (P = 0.055) and tended to decrease (P = 0.09) expression of GATA6. To evaluate pluripotency, the inner cell mass was isolated by immunosurgery at Day 8, seeded on a feeder layer of bovine embryonic fibroblasts, and cultured in the presence of the inhibitors. Ten of 52 (19%) ICM from control embryos had primary outgrowth formation vs. 23 of 50 (46%) of the ICM from embryos cultured with inhibitors (P < 0.01). For ICM outgrowths from embryos cultured without inhibitors, colonies either did not persist through Passage 2 or became differentiated. In contrast, for the inhibitor group, four colonies survived beyond Passage 2, and one line persisted for 19 passages. This cell line possessed alkaline phosphatase activity, expressed several genes characteristically expressed in pluripotent cells, and differentiated into embryoid bodies when cultured in the absence of the signal transduction inhibitors and the feeder layer. Propagation of the cells was difficult due to slow growth and inefficiency in survival through each passage. In conclusion, exposure to inhibitors during the morula-blastocyst transition facilitated formation of self-renewing pluripotent cell lines from bovine blastocysts.     

子宫内膜接受性的建立和分子调控

胚泡着床是一个复杂的生理过程,依赖于胚泡发育和子宫内膜获得接受能力的同步进行.着床只发生在具有接受性的子宫内膜,而子宫内膜只在很短的时间内具有接受性.被称为"着床窗口".子宫内膜接受性的建立涉及子宫腔上皮的形态学改变,以及甾类激素和许多细胞因子复杂的调控作用.本文综述了子宫内膜接受性的建立及其分子调控.     

Characteristics of cells derived from the girdle region of the pre-implantation blastocyst of the donkey

Summary The establishment of a monolayer culture of cells derived from the girdle region of a 34-day-old donkey conceptus is described. These cells have had over 100 repeated passages in culture. Low levels of pregnant mares' serum gonadotrophin (PMSG, eCG) could be detected in the cells by indirect immunofluorescence using some monoclonal anti-eCG antibodies, but the cells did not secrete eCG as measured by radioimmunoassay or inhibition of haemagglutination. There was marked nuclear polymorphism with binucleate and occasional multinucleate cells. The cells were strongly reactive with wheatgerm agglutinin and concanavalin A suggesting the synthesis of many glycosylated products. Some cells were reactive with antisera to prekeratin, others with antisera to vimentin. The cells also contained actin (showing peculiar intercellular communications), -actinin and tubulin. They were able to metabolize certain steroid precursors, but there was no definitive evidence for the presence of aromatase or ⁵-3-hydroxysteroid dehydrogenase in these cells. This cell line appears to resemble trophectodermal girdle epithelium at a stage of development prior to the onset of eCG production, and may be useful in studies on the control of expression of this substance.Dr. S. Kellie is now at the Imperial Cancer Research Fund Laboratories, Lincoln Inn's Fields, London     

Morphometric estimation of stromal edema during delayed implantation in the rat

Summary Morphometric methods were used at the light microscopic level to investigate the appearance of edema in the endometrial stroma of rats during estradiol-induced implantation after an experimental delay. Comparisons between blastocyst-free and blastocyst-containing sites were made at 8, 12, 16 and 24 h after injection of estradiol (h.a.e.). The development of stromal edema during implantation was found to be diphasic. First, during the initial 8–12 h.a.e., a generalized edema developed all along the uterine horns. Later, from 16 h.a.e. onwards, a local edema was present around the blastocysts. The Pontamine Blue Reaction (PBR) became visible between 20 and 24 h.a.e. The results indicate that the blastocyst is recognized by the stroma considerably before the PBR. The appearance of a local edema around the blastocysts before the PBR might be related to a slow increase in vascular permeability and/or to the increased stromal cell synthetic activity that is known to precede the PBR during early implantation.     

Significant improvement of pig cloning efficiency by treatment with LBH589 after somatic cell nuclear transfer

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) associates with epigenetic aberrancy, including the abnormal acetylation of histones. Altering the epigenetic status by histone deacetylase inhibitors (HDACi) enhances the developmental potential of SCNT embryos. In the current study, we examined the effects of LBH589 (panobinostat), a novel broad-spectrum HDACi, on the nuclear reprogramming and development of pig SCNT embryos in vitro. In experiment 1, we compared the in vitro developmental competence of nuclear transfer embryos treated with different concentrations of LBH589. Embryos treated with 50 nM LBH589 for 24 hours showed a significant increase in the rate of blastocyst formation compared with the control or embryos treated with 5 or 500 nM LBH589 (32.4% vs. 11.8%, 12.1%, and 10.0%, respectively, P < 0.05). In experiment 2, we examined the in vitro developmental competence of nuclear transfer embryos treated with 50 nM LBH589 for various intervals after activation and 6-dimethylaminopurine. Embryos treated for 24 hours had higher rates of blastocyst formation than the other groups. In experiment 3, when the acetylation of H4K12 was examined in SCNT embryos treated for 6 hours with 50 nM LBH589 by immunohistochemistry, the staining intensities of these proteins in LBH589-treated SCNT embryos were significantly higher than in the control. In experiment 4, LBH589-treated nuclear transfer and control embryos were transferred into surrogate mothers, resulting in three (100%) and two (66.7%) pregnancies, respectively. In conclusion, LBH589 enhances the nuclear reprogramming and developmental potential of SCNT embryos by altering the epigenetic status and expression, and increasing blastocyst quality.     

Nuclear maturation and embryo development of porcine oocytes vitrified by cryotop: Effect of different stages of in vitro maturation

The present study was designed to evaluate the viability, meiotic competence and subsequent development of porcine oocytes vitrified using the cryotop method at different stages of in vitro maturation (IVM). Cumulus–oocyte complexes (COCs) were cultured in IVM medium supplemented with 1 mM dibutyryl cAMP (dbcAMP) for 22 h and then for an additional 22 h without dbcAMP in the medium. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), anaphase I/telophase I (AI/TI) and metaphase II (MII) were found to occur predominantly at 0–22, 26, 32, 38 and 44 h of IVM, respectively. Oocytes were exposed to cryoprotectant (CPA) or vitrified after different durations of IVM (0, 22, 26, 32, 38 and 44 h). After CPA exposure and vitrification, surviving oocytes that were treated before completion of the 44 h maturation period were placed back into IVM medium for the remaining maturation period, and matured oocytes were incubated for 2 h. CPA treatment did not affect the viability of oocytes matured for 26, 32, 38 or 44 h, but significantly decreased survival rate of oocytes matured for 0 or 22 h. CPA treatment had no effect on the ability of surviving oocytes to develop to the MII stage regardless of the stage during IVM; however, blastocyst formation following PA was severely lower (P < 0.05) than that in the control. At 2 h post-warming, the survival rates of oocytes vitrified at 26, 32, 38 and 44 h of IVM were similar but were higher (P < 0.05) than those of oocytes vitrified at 0 or 22 h of IVM. The MII rates of surviving oocytes vitrified at 0 and 38 h of IVM did not differ from the control and were higher (P < 0.05) than those of oocytes vitrified at 22, 26 or 32 h of IVM. After parthenogenetic activation (PA), both cleavage and blastocyst rates of vitrified oocytes matured for 22, 26, 32, 38 and 44 h did not differ, but all were lower (P < 0.05) than those matured 0 h. In conclusion, our data indicate that survival, nuclear maturation and subsequent development of porcine oocytes may be affected by their stage of maturation at the time of vitrification; a higher percentage of blastocyst formation can be obtained from GV oocytes vitrified before the onset of maturation.     

小鼠囊胚的细胞凋亡:体内发育和体外培养的比较

小鼠胚体外培养到囊胚期的成功率很高,但质量是否能及体内发育的囊胚还不太清楚。细胞的数量和凋亡程度是胚胎质量鉴定的重要指标。本文采用TUNEL法分别对2-、8-细胞和桑椹胚培育成的鼠囊胚及体内发育而成的鼠囊胚细胞凋亡情况进行了检验。结果表明90%以上的2-、8-细胞及桑椹胚经过72h、48h和24h的培养发育到囊胚期。由桑椹胚发育成的与体内发育成的囊胚细胞凋亡指数没有显著差异,但由2-、8-细胞胚培育成的囊胚细胞凋亡指数显著高于体内发育成的囊胚。由此可见,体外长时间培养会增加胚胎的细胞凋亡程率。为培养出高质量的囊胚,胚胎培养条件还需进一步改善。     

Individual blastomeres of 16- and 32-cell mouse embryos are able to develop into foetuses and mice

Cell and developmental studies have clarified how, by the time of implantation, the mouse embryo forms three primary cell lineages: epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE). However, it still remains unknown when cells allocated to these three lineages become determined in their developmental fate. To address this question, we studied the developmental potential of single blastomeres derived from 16- and 32-cell stage embryos and supported by carrier, tetraploid blastomeres. We were able to generate singletons, identical twins, triplets, and quadruplets from individual inner and outer cells of 16-cell embryos and, sporadically, foetuses from single cells of 32-cell embryos. The use of embryos constitutively expressing GFP as the donors of single diploid blastomeres enabled us to identify their cell progeny in the constructed 2n↔4n blastocysts. We showed that the descendants of donor blastomeres were able to locate themselves in all three first cell lineages, i.e., epiblast, primitive endoderm, and trophectoderm. In addition, the application of Cdx2 and Gata4 markers for trophectoderm and primitive endoderm, respectively, showed that the expression of these two genes in the descendants of donor blastomeres was either down- or up-regulated, depending on the cell lineage they happened to occupy. Thus, our results demonstrate that up to the early blastocysts stage, the destiny of at least some blastomeres, although they have begun to express markers of different lineage, is still labile.     

Trichostatin A-treated eight-cell bovine embryos had increased histone acetylation and gene expression, with increased cell numbers at the blastocyst stage

The objective was to determine the effects of trichostatin A (TSA), a potent histone deacetylase inhibitor, on eight-cell bovine embryos. That treatment increased histone acetylation was confirmed by immunostaining with anti-AcH4K5 and AcH4K8 antibodies. Embryos treated with TSA (100 nM) for various intervals (4, 8, and 12 h) developed to the blastocyst stage as frequently as untreated embryos (average development rate, 49.5%). Treatment with TSA for 12 h increased (P < 0.05) the numbers of inner cell mass (ICM) cells and total cells (TC), as well as the ICM/TC ratio in the blastocyst, but the number of cells in the trophectoderm decreased (P < 0.05). Treated embryos had increased relative abundance (RA) of OCT3/4 and E-CADHERIN mRNA relative to controls at the morula stage (P < 0.05), however, the RA of CDX2 mRNA was unchanged. In conclusion, TSA-treated eight-cell stage embryos had increased histone acetylation and gene expression, which increased ICM and TC numbers and the ICM/TC ratio, but significantly decreased the number of cells in the trophectoderm of resulting blastocysts.