The Chinese hamster gene map. Assignment of four genes (DTS, PGM2, 6PGD, EnoI) to chromosome 2

Mouse morulae and blastocysts express cell surface antigens that fortuitously cross-react with antisera to human chorionic gonadotropin (hCG). In the present study, the cell surface and cytoplasmic expression of these antigens was followed in mouse unfertilized oocytes, different stages of preimplantation embryos and in early post-implantation embryos cultured from blastocysts. In addition to their known stage-dependent cell surface expression on morulae and blastocysts, these antigens (1) were already present in the cytoplasm of mature unfertilized oocytes and pre-morula stages of embryos; (2) remained expressed as cell surface antigens on cells of the inner cell mass (ICM), but not on the surface of trophectodermal cells with further blastocyst development although (3) they persisted as cytoplasmic antigens in trophectodermal cells. In addition, these antigens were also detectable by antiserum to the alpha subunit of hCG.     

Aberrant cannabinoid signaling impairs oviductal transport of embryos

Ectopic pregnancy is a major reproductive health issue. Although other underlying causes remain largely unknown, one cause of ectopic pregnancy is embryo retention in the fallopian tube. Here we show that genetic or pharmacologic silencing of cannabinoid receptor CB1 causes retention of a large number of embryos in the mouse oviduct, eventually leading to pregnancy failure. This is reversed by isoproterenol, a beta-adrenergic receptor agonist. Impaired oviductal embryo transport is also observed in wild-type mice treated with methanandamide. Collectively, the results suggest that aberrant cannabinoid signaling impedes coordinated oviductal smooth muscle contraction and relaxation crucial to normal oviductal embryo transport. Colocalization of CB1 and beta2-adrenergic receptors in the oviduct muscularis implies that a basal endocannabinoid tone in collaboration with adrenergic receptors coordinates oviductal motility for normal journey of embryos into the uterus. Besides uncovering a new regulatory mechanism, this study could be clinically relevant to ectopic pregnancy.     

Identification of the facilitative glucose transporter 12 gene Glut12 in mouse preimplantation embryos

In this study we report the cloning and characterisation of the mouse Glut12 gene and examine for the first time its expression pattern in the earliest stages of development. Mouse Glut12 (mGlut12) was cloned from preimplantation embryos by 5'RACE RT-PCR using primers designed from an EST clone corresponding to a human GLUT12 antigenic sequence after positive immunoreactivity was observed in mouse two-cell embryos by western immunoblotting. The mGlut12 gene contains an open reading frame of 1869 base pairs, potentially encoding a polypeptide of 622 amino acids. The predicted mGLUT12 protein bears all the hallmarks of the SLC2A family of hexose transporters and shares an 83% sequence homology to human GLUT12. Consistent with its human homolog mGlut12 mRNA is found highly expressed in skeletal and cardiac muscle and fat. Additionally, it was also found in the uterus and during early embryogenesis. During early development in the mouse, Glut12 expression is clearly apparent in ovulated oocytes and two-cell embryos but declines in day 3 morulae. With the exception of some Glut12 expression apparent in blastocysts, Glut12 mRNA remains at low to undetectable levels until E11.     

The influence of insulin on the in vitro development of mouse and bovine embryos

To further investigate the role of insulin during preimplantation embryo development, we compared the effects of insulin on the development of mouse and bovine preimplantation embryos and on cell proliferation during culture in vitro in simplex media. The influence of insulin on the development of mouse zygotes was determined during cultivation in mSOF medium, alone or supplemented with glucose. Similarly, the effects of insulin on the bovine preimplantation embryo development were studied in mSOF medium. The addition of insulin into mSOF medium enhanced significantly the number of cells per mouse blastocyst. Moreover, when mSOF medium was supplemented with insulin and 0.2 mmol x l(-1) glucose, the percentage of hatched blastocysts and the mean cell number of mouse blastocysts were significantly higher. Insulin had no significant effect on the development of bovine embryos, produced by in vitro fertilization of in vitro matured oocytes. Neither the rates of developing embryos nor the mean number of cells in blastocysts were different in comparison with control embryos. Our results suggest that the in vitro development of mouse embryos could be enhanced by the addition of insulin to the culture medium and is further improved by the addition of glucose. In contrast to this our results indicate that insulin has no detectable beneficial effect on the preimplantation development of bovine embryos in mSOF medium.     

Is there any delta 5-3 beta hydroxysteroid dehydrogenase activity in preimplantation embryo of rhesus monkey?

This is the first report on the histochemical assessment of delta 5-3 beta hydroxysteroid dehydrogenase activity in all the preimplantation embryonic stages in the rhesus monkey (Macaca mulatta). An apparent stage dependent increase in enzyme activity was obtained, however, distinctively a high degree of non-specificity in enzyme reaction was noted primarily in morulae and blastocysts. Such marked non-specificity in the histochemical enzyme reaction for delta 5-3 beta hydroxysteroid dehydrogenase activity was not found in mouse blastocysts. High amounts of endogenous steroids present within rhesus embryos, or the participation of non-specific dehydrogenases could account for the observed non-specificity. Furthermore, the present report documents the pattern and degree of association (r = 0.9; P less than 0.01) between developmental stage and gestational age of preimplantation rhesus embryos, and thus provides a normal in situ cell cleavage rate of preimplantation embryo in the rhesus monkey.     

Expression of leptin ligand and receptor and effect of exogenous leptin supplement on in vitro development of porcine embryos

The present study investigated the expression of ligand and receptor for leptin, and the effect of leptin supplementation on preimplantation development of porcine in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. The IVF embryos were produced using frozen boar semen and SCNT embryos were obtained by nuclear transfer of fetal fibroblasts into enucleated oocytes. The protein expression of leptin ligand and receptor was investigated in in vitro matured oocytes, 2-, 4- and 8-cell embryos, morulae and blastocysts derived from IVF and SCNT using immunofluorescence. Both the ligand and receptor were detected in in vitro matured oocytes and all stage of IVF and SCNT embryos. The IVF and SCNT embryos were cultured in modified North Carolina State University (mNCSU)-23 medium supplemented with various concentrations (0, 1, 10, 100 or 1000 ng/mL) of leptin. The rates of cleavage at day 2 and blastocyst formation at day 7, and cell number of blastocysts were monitored as experimental parameters. In SCNT embryos, supplementing with 1000 ng/mL leptin significantly (P<0.05) increased the rate of blastocysts formation (20.2% versus 12.9%) and total cell number (54.6+/-17.4 versus 45.1+/-15.2) compared to the control group. In IVF embryos, leptin supplementation did not affect preimplantation embryo development and cell number in blastocysts. In conclusion, the present study demonstrated the expression of leptin ligand and receptor and the embryotropic effect of leptin in SCNT embryos.     

Molecular cloning and expression of the cDNA for a novel alpha 1-adrenergic receptor subtype

A novel alpha 1-adrenergic receptor subtype has been cloned from a bovine brain cDNA library. The deduced amino acid sequence is that of a 466-residue polypeptide. The structure is similar to that of the other adrenergic receptors as well as the larger family of G protein-coupled receptors that have a presumed seven-membrane-spanning domain topography. The greatest sequence identity of this receptor protein is with the previously cloned hamster alpha 1B-adrenergic receptor being approximately 72% within the presumed membrane-spanning domains. Localization on different human chromosomes provides evidence that the bovine cDNA is distinct from the hamster alpha 1B-adrenergic receptor. The bovine cDNA clone expressed in COS7 cells revealed 10-fold higher affinity for the alpha 1-adrenergic antagonists WB4101 and phentolamine and the agonist oxymetazoline as compared with the alpha 1B receptor, results similar to pharmacologic binding properties described for the alpha 1A receptor. Despite these similarities in pharmacological profiles, the bovine alpha 1-adrenergic receptor is sensitive to inhibition by the alkylating agent chloroethylclonidine unlike the alpha 1A-adrenergic receptor subtype. In addition, a lack of expression in tissues where the alpha 1A subtype exists suggests that this receptor may actually represent a novel alpha 1-adrenergic receptor subtype not previously appreciated by pharmacological criteria.     

Serotonin localization and its functional significance during mouse preimplantation embryo development

Serotonin is a neurotransmitter functioning also as a hormone and growth factor. To further investigate the biological role of serotonin during embryo development, we analysed serotonin localization as well as the expression of specific serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos. The functional significance of serotonin during the preimplantation period was examined by studying the effects of serotonin on mouse embryo development. Embryo exposure to serotonin (1 microM) highly significantly reduced the mean cell number, whereas lower concentrations of serotonin (0.1 microM and 0.01 microM) had no significant effects on embryo cell numbers. In all serotonin-treated groups a significant increase in the number of embryos with apoptotic and secondary necrotic nuclei was observed. Expression of serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos was confirmed by in situ hybridization showing a clearly distinct punctate signal. Immunocytochemistry results revealed the localization of serotonin in oocytes and embryos to the blastocyst stage as diffuse punctate cytoplasmic labelling. It appears that endogenous and/or exogenous serotonin in preimplantation embryos could be involved in complex autocrine/paracrine regulations of embryo development and embryo-maternal interactions.     

TGFalpha and EGFR in ovine preimplantation embryos and effects on development

The present study aimed to assess location and relative amounts of transforming growth factor alpha (TGFalpha) and its receptor (EGFR) in ovine oocytes and preimplantation embryos by using immunohistochemical technique that was graded on a relative scale of 0-3, with 0 representing absence of staining, and 3 exhibiting prominent staining, and to evaluate the effects of TGFalpha/EGF on in vitro development of preimplantation embryos by adding different concentrations of EGF and TGFalpha to culture medium. The results showed that EGFR was abundant in cell plasma membranes in immature and mature oocytes, cumulus cells of immature cumulus-oocyte complexes (COC), fertilized oocytes and at different stages of embryo development. However, the relative amounts in inner cell mass (ICM) (1+) was less than that in trophectoderm (TE) cells (2+) at the blastocysts stage. The staining pattern for TGFalpha was a similar to EGFR. However, the staining for TGFalpha slightly increased in the fertilized oocytes (1-2+) as compared to immature and mature oocytes (1+). TGFalpha was mainly detected in the cytoplasm close to the membrane in both ICM and trophectoderm (TE) cells. The developmental rate of 8-cell stage embryos cultured with 5 ng/ml TGFalpha was increased as compared to other treatments (P<0.05). There was no significant difference in the rate of development of blastocysts cultured with 5 ng/ml TGFalpha, 20 ng/ml EGF, 20 ng/ml EGF+5 ng/ml TGFalpha or the control treatment (P>0.05). In addition, there was no significant difference in the number of cells in blastocyst stage as compared with different treatments (P>0.05). However, TGFalpha alone enhanced cell survival rated (P<0.01) and reduced apoptosis. We concluded that TGFalpha can improve development of ovine preimplantation embryos at the 8-cell and blastocyst stages in vitro.