Mouse Oviduct Research Articles

Expression of progesterone receptors A and B in the mouse ovary during the estrous cycle.

Progesterone plays a central role in the regulation of ovarian function. The progesterone receptor (PR) has been shown to be essential for ovulation because mice lacking PR fail to ovulate and are infertile. PR is expressed as two isoforms, PRA and PRB, which have been shown to have different functional activities. In this study, we investigated the cellular distribution of PRA and PRB in the ovaries and oviducts of cycling mice using immunohistochemistry with isoform-specific monoclonal antibodies. In the ovary, on the evening of proestrus before ovulation, both the granulosa and theca cells of the preovulatory follicles expressed both PR isoforms. PRA and PRB staining was also observed in the theca cells of preantral and antral follicles, whereas only PRB was observed in the granulosa cells of primary, preantral, and antral follicles and in the corpus luteum. In the oviduct, PRA was the predominant isoform observed, expressed in both the epithelial and stromal cells, whereas PRB was only detected in the epithelial cells. The differences in PRA and PRB localization in the ovary and oviduct may reflect diverse functions for PRA and PRB in reproductive tissues and may have important implications in understanding the mechanisms of progesterone action.

The Embryotrophic Activity of Oviductal Cell-derived Complement C3b and iC3b, a Novel Function of Complement Protein in Reproduction

The oviduct-derived embryotrophic factor, ETF-3, enhances the development of trophectoderm and the hatching process of treated embryos. Monoclonal anti-ETF-3 antibody that abolishes the embryotrophic activity of ETF-3 recognized a 115-kDa protein from the conditioned medium of immortalized human oviductal cells. Mass spectrometry analysis showed that the protein was complement C3. Western blot analysis using an antibody against C3 confirmed the cross-reactivities between anti-C3 antibody with ETF-3 and anti-ETF-3 antibody with C3 and its derivatives, C3b and iC3b. Both derivatives, but not C3, were embryotrophic. iC3b was most efficient in enhancing the development of blastocysts with larger size and higher hatching rate, consistent with the previous reported embryotrophic activity of ETF-3. Embryos treated with iC3b contained iC3b immunoreactivity. The oviductal epithelium produced C3 as evidenced by the presence of C3 immunoreactivity and mRNA in the human oviduct and cultured oviductal cells. Cyclical changes in the expression of C3 immunoreactivity and mRNA were also found in the mouse oviduct with the highest expression at the estrus stage. Molecules involving in the conversion of C3b to iC3b and binding of iC3b were present in the human oviduct (factor I) and mouse preimplantation embryo (Crry and CR3), respectively. In conclusion, the present data showed that the oviduct produced C3/C3b, which was converted to iC3b to stimulate embryo development.

Sperm-induced modification of the oviductal gene expression profile after natural insemination in mice.

In mammals, the physiological interaction between spermatozoa and oviductal epithelia involves intimate and specific contact between the two cell types. Spermatozoa may undergo stringent selection processes within the female reproductive tract before they meet and fertilize oocytes. The physiological basis of the sperm selection process is largely unknown. Here we tested the hypothesis that the oviduct has a recognition system for spermatozoa that can detect the arrival of spermatozoa in the oviduct after insemination, resulting in alterations of the oviductal transcriptome. We initially performed a global screening of the oviductal transcriptome in mice 1) at the time of estrus (mating) and 2) 6 h after mating. Transcriptional alterations in the oviduct after mating were attributed to the presence of spermatozoa in the oviduct after mating and also to changes in the hormonal environment as female mice underwent the transition from estrus to diestrus. To distinguish these possibilities, female mice were then mated with T145H mutant mice, which because of spermatogenic arrest, produce seminal plasma but no spermatozoa. Focusing on two molecules that in the first experiment were upregulated after mating, it was found that adrenomedullin and prostaglandin endoperoxidase synthase 2 transcripts were upregulated in the oviducts of mice only after mating with fertile males; those mated with T145H infertile males showed significantly less response. These results indicate that it is the arrival of spermatozoa in the oviduct that activates one or more signal transduction pathways and leads to changes in the oviductal transcriptome profiles.

279.Infertility in mice with null mutation of the Egr-1 transcription factor

Female infertility has been reported in two lines of mice with mutation of the Egr-1 gene. One underlying cause of this defect is deficient LH production by pituitary gonadotropes. However, Egr-1 is also acutely regulated by both FSH and LH in ovarian granulosa cells (1). A role for this transcription factor in regulating gonadotrophin responsive target genes and ovarian function is hypothesised. Indeed the LH-receptor is a proposed target of Egr-1 regulation, but this has not been investigated in detail in vivo and is difficult to reconcile with the pattern of Egr-1 expression. In this study, the role of Egr-1 within the ovarian follicle was investigated using exogenous gonadotropin replacement in Egr-1–/– mice . Adult Egr-1–/– female mice superovulated by sequential PMSG and hCG stimulation and mated with proven male breeders failed to produced offspring while 90% of heterozygous females got pregnant and produced litters (7.4 � 2.9 pups per litter) within 22 days of stimulation. Recovery of oocytes from oviducts of immature superovulated mice revealed a reduced ovulation rate in null females (6.3 � 3.8 oocytes) compared to their heterozygous (18.0 � 6.5) and WT (17.8 � 6.8) littermates. Gross morphology and histology of exogenously stimulated ovaries were indistinguishable from their heterozygous or WT counterparts. Surprisingly, no alteration was detectable in the mRNA expression of previously reported direct Egr-1 responsive genes, namely LH-receptor and membrane prostaglandin E synthase (mPGES). Nor were mRNA for two critical ovulatory genes with putative Egr-1 response elements, ADAMTS-1 or versican V1 altered. Temporal and spatial expression of genes involved in ovarian steroidogenesis, P450scc and Cyp17 and LH-receptor, were indistinguishable from normal littermates during exogenously controled follicular development. Combined observations of acute Egr-1 induction by gonadotropins, reduced ovulation and complete infertility suggest an important role for Egr-1 in ovarian function. However, genes identified as targets of Egr-1 regulation in other studies proved to be Egr-1 independent in this model. (1) Russell et al. (2003) Mol. Endo. 17, 520.

Establishment of transgenic mice carrying gene encoding human zinc finger protein 191.

Human zinc finger protein 191 (ZNF191) was cloned and characterized as a Kruppel-like transcription factor, which might be relevant to many diseases such as liver cancer, neuropsychiatric and cardiovascular diseases. Although progress has been made recently, the biological function of ZNF191 remains largely unidentified. The aim of this study was to establish a ZNF 191 transgenic mouse model, which would promote the functional study of ZNF191. Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. The offsprings were identified by PCR and Southern blot analysis. ZNF 191 gene expression was analyzed by RT-PCR. Transgenic founder mice were used to establish transgenic mouse lineages. The first generation (F1) and the second generation (F2) mice were identified by PCR analysis. Ten-week transgenic mice were used for pathological examination. Four mice were identified as carrying copies of ZNF191 gene. The results of RT-PCR showed that ZNF 191 gene was expressed in the liver, testis and brain in one of the transgenic mouse lineages. Genetic analysis of transgenic mice demonstrated that ZNF 191 gene was integrated into the chromosome at a single site and could be transmitted stably. Pathological analysis showed that the expression of ZNF 191 did not cause obvious pathological changes in multiple tissues of transgenic mice. ZNF 191 transgenic mouse model would facilitate the investigation of biological functions of ZNF191 in vivo.

Recombinant phospholipase Czeta has high Ca2+ sensitivity and induces Ca2+ oscillations in mouse eggs.

Sperm-specific phospholipase Czeta (PLCzeta) is known to induce intracellular Ca(2+) oscillations and subsequent early embryonic development when expressed in mouse eggs by injection of RNA encoding PLCzeta (Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K., and Lai, F. A. (2002) Development 129, 3533-3544). The present study addressed characteristics of purified mouse PLCzeta protein that was synthesized using the baculovirus/Sf9 cell expression system. Microinjection of recombinant PLCzeta protein into mouse eggs induced serial Ca(2+) spikes quite similar to those produced by the injection of sperm extract, probably because of repetitive Ca(2+) release from the endoplasmic reticulum caused by continuously produced inositol 1,4,5-trisphosphate. Recombinant PLCdelta1 also induced Ca(2+) oscillations, but a 20-fold higher concentration was required compared with PLCzeta. In the enzymatic assay of phosphatidylinositol 4,5-bisphosphate hydrolyzing activity in vitro at various calcium ion concentrations ([Ca(2+)]), PLCzeta exhibited a significant activity at [Ca(2+)] as low as 10 nm and had 70% maximal activity at 100 nm [Ca(2+)] that is usually the basal intracellular calcium ion concentration level of cells. On the other hand, the activity of PLCdelta1 increased at a [Ca(2+)] between 1 and 30 microm. EC(50) was 52 nm for PLCzeta and 5.7 microm for PLCdelta1. Thus, PLCzeta has an approximately 100-fold higher Ca(2+) sensitivity than PLCdelta1. The ability of purified PLCzeta protein to induce Ca(2+) oscillations qualifies PLCzeta as a proper candidate of the mammalian egg-activating sperm factor. Furthermore, such a high Ca(2+) sensitivity of PLC activity as PLCzeta that can be active in cells at the resting state is thought to be an appropriate characteristic of the sperm factor, which is introduced into the ooplasm upon sperm-egg fusion, triggers Ca(2+) release first, and maintains Ca(2+) oscillations.

Expression of Fas and Fas ligand protein and mRNA in mouse oocytes and embryos.

During mammalian embryonic development, abnormal eggs and embryos are eliminated by apoptosis; however, the precise apoptotic pathways remain as yet unidentified. In the present study, expression of Fas and Fas ligand - the proximal members of the death receptor pathway, was evaluated in mouse preimplantation embryos by immunofluorescence and in situ hybridization techniques. Ovulated oocytes were collected from oviducts of cyclic mice on the day of oestrus (day 0), and one-cell, two-cell embryos and eight-cell morulae were collected from oviducts of mated animals on days 1, 2 and 3 of pregnancy, respectively. Blastocysts were flushed from the uterine horns on day 4. Expression of Fas and Fas L mRNAs and proteins was absent from embryos at days 0, 1 and 2. A marked increase in Fas and Fas L mRNA and protein expression was detected in all morphologically normal embryos on day 3 and day 4. In addition, embryos on days 3 and 4 were positive for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining; however, absence of caspase 8 and 3 and intense localization of proliferating cell nuclear antigen confirmed the proliferative status of these embryos. Furthermore, TUNEL staining was absent in postimplantation embryonic sections obtained on day 6. The results of the present studies thus indicate an equilibrium between proliferation and apoptosis in the preimplantation embryo.

Metaphase II nuclei generated by germinal vesicle transfer in mouse oocytes support embryonic development to term.

Cytoplasmic defects are thought to cause aneuploidies in oocytes and embryos and oocyte 'reconstruction' by germinal vesicle (GV) transfer may circumvent such defects. In mice 'reconstructed' oocytes undergo meiosis and fertilize normally, but early embryonic development is compromised if their ooplasm matured in vitro. This study employs sequential MII spindle and/or pronucleus (PN) transfer to assess the embryonic potential of MII nuclei that form following GV transfer. Mouse embryos generated by these procedures were transferred to the oviducts of pseudopregnant mice to monitor pregnancy outcome. Following GV transfer, the resultant metaphase II (MII) nuclei were activated either in situ or transferred and activated in ooplasts from in-vivo matured oocytes. When exchanged with the female PN of a fertilized zygote, only the PNs that developed in in-vivo matured ooplasts generated live offspring. Viable offspring also resulted when MII nuclei were transferred to in-vivo matured ooplasts and fertilized by insemination with sperm or by artificial activation and male PN transfer. Significantly, the offspring displayed normal fertility as adults. This report of live births following GV transfer in mice illustrates the importance of the maturational history of the ooplasm at PN formation for normal embryonic and fetal development.

The role of leptin during the development of mouse preimplantation embryos

Leptin is known to regulate diverse reproductive functions, and recent studies have implicated involvement of leptin in the early mouse embryo development. The aim of the present study was to investigate the expression of leptin and its functional receptor (OB-Rb) in mouse oocyte and preimplantation embryo, and to examine whether leptin influenced the early embryo development. Leptin mRNA was detected in blastocyst and hatched blastocyst, and OB-Rb mRNA was detected in oocytes, 1-cell, 2-cell, morula, blastocyst and hatched blastocyst. As for the origin of leptin, leptin mRNA was identified in both the oviduct and uterus of the pregnant mouse. Furthermore, in the pregnant mouse, the levels of leptin in uterine fluid were higher than those in the non-pregnant mouse. Supplementation of culture medium with leptin promotes the development of preimplantation embryos from 2-cell stage to the blastocysts, fully expanded blastocysts and hatched blastocysts. Leptin significantly increased the total cell number of blastocysts, and the effect was preferentially observed in the trophectoderm. These findings raise the possibility that leptin regulates the development of mouse preimplantation embryo through a prarcrine pathway.

Expression of messenger RNA for prostaglandin E receptor subtypes EP4/EP2 and cyclooxygenase isozymes in mouse periovulatory follicles and oviducts during superovulation.

Prostaglandin (PG) E(2) is synthesized from arachidonic acid by cyclooxygenase (COX) and acts as a regulator in ovulation and fertilization reactions. We present the temporal and regional expression patterns of mRNAs for the two Gs-coupled PGE receptors, EP2 and EP4, and for COX-1 and COX-2 in mouse periovulatory follicles and oviducts during superovulation. Analysis using reverse transcription polymerase chain reaction revealed that the mouse ovaries express a significant amount of EP4 mRNA in addition to EP2 mRNA during superovulation. In situ hybridization results revealed that the signals for EP4 mRNA were localized mostly to oocytes in the preantral follicles. Three hours after hCG injection, the signals for EP4 and EP2 mRNA were present in both granulosa and cumulus cells. However, 9 h after hCG injection, just before ovulation, the signals for EP4 mRNA were still detectable in both cell types, whereas those for EP2 mRNA were found only in cumulus cells. COX-2 mRNA expression was present in both granulosa and cumulus cells at 3 h but was present only in cumulus cells at 9 h. COX-1 mRNA expression was not found in granulosa cells at 3 h but was found in these cells at 9 h. In the oviduct, the expression of EP4 and COX-1 mRNA was localized to epithelial cells, whereas expression of EP2 mRNA was localized to the smooth muscle layer. The tightly regulated expression of both EP2 and EP4 in the preovulatory follicles may reflect the essential role of PGE(2) in the ovulation process.

Characterization of newly established clonal oviductal cell lines and differential hormonal reculation of gene expression

Oviductal functions have been studied mainly in primary epithelial cell culture and organ culture. However, secretory cells and ciliated cells coexist in the epithelium, and the small size of the oviduct limits the sources of both epithelial and stromal cells. To circumvent the limits, we attempted to establish clonal cell lines from an oviduct of a p53-deficient mouse. An oviduct was enzymatically digested and cultured in medium containing 10% fetal calf serum supplemented with estradiol-17beta. Morphologically distinct clones (10 epithelial and 4 fibroblastic clones) were established, and all clones expressed estrogen receptor alpha and progesterone receptor. Expression of a mouse oviduct-specific glycoprotein gene as a marker of secretory cells was limited in one clone and was stimulated by estrogens and suppressed by progesterone. Expression of helix factor hepatocyte nuclear factor/forkhead homologue-4 gene as a marker of ciliated cells was limited in two clones and was suppressed by estrogens. The two genes were never coexpressed in any clones. The results strongly suggest that the oviductal epithelium consists of two functionally determined populations. To our knowledge, this is the first establishment of functional clonal cell lines of the oviduct and makes it possible to study independently two oviductal functions, secretion and ciliogenesis.

Establishment of transgenic mice carrying the gene of human nuclear receptor NR5A2 (hB1F).

Human hepatitis B virus enhancer II B1 binding factor (hB1F) was cloned and characterized as a novel member of the Ftz-F1 (NR5A) nuclear receptor subfamily. Although progresses have recently been made, its biological function remains largely unidentified. The aim of this study was to establish an hB1F transgenic mouse model to promote the functional study of hB1F. Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudopregnant female mice. The offsprings were identified by PCR and Southern blot analysis. Transgene expression was analyzed with RT-PCR and Western blot analysis. Transgenic founder mice were used to establish transgenic mouse lineages. The F1 and F2 mice were identified by PCR analysis. Seven mice were identified as carrying copies of transgene. RT-PCR and Western blotting results showed that the transgene was expressed in heart, liver, lung, kidney and stomach in one of the transgenic mouse lineages. Genetic analysis of the transgenic mice demonstrated that the transgene was integrated into the chromosome at a single site, and was transmitted stably. In this study we established an hB1F transgenic mouse model, which will facilitate the investigation of the biological function of hB1F in vivo.

Experimental evaluation of cross-contamination between cryotubes containing mouse 2-cell embryos and murine pathogens in liquid nitrogen tanks.

It has been suspected that embryos stored in liquid nitrogen tanks may become contaminated with murine pathogens, if some pathogens had been introduced to the tanks accidentally. To examine this, we stored tubes containing embryos with tubes containing mouse hepatitis virus (MHV) or Pasteurella pneumotropica in liquid nitrogen tanks and examined whether progeny mice derived from the embryos were contaminated with the pathogens or not. After storing for 6 months or 1 year the frozen embryos were thawed and implanted into the oviducts of pseudopregnant female mice, and the mice were bred in vinyl isolators. We could not detect serum antibodies to MHV and isolate Pasteurella pneumotropica in the progeny mice, suggesting that cross-contamination between tubes in a liquid nitrogen tank scarcely occurs.

Early Developing Embryos Affect the Gene Expression Patterns in the Mouse Oviduct

Fertilization and development of mouse embryos occur in the ampullae of oviduct. We hypothesize that fetal-maternal communication exists in the preimplantation period, allowing optimal development of embryos. It is known that embryotrophic factors from oviduct affect the development of embryos. Although embryos affect their own transport in the oviduct, the mechanism of action is unknown. As a step toward understanding the action of embryos on oviductal physiology, we adopted suppression subtractive hybridization (SSH) to compare the gene expression in the mouse oviduct containing early embryos with that of oviduct containing oocytes. Ten to twelve 1-cell mouse embryos were transferred to one oviduct of a foster mother and similar number of oocytes were transferred to the contralateral oviduct. The animals were sacrificed after 48 h and their oviducts were excised for mRNA study. Using SSH, we screened out 250 putative positive clones from the subtracted embryo-containing oviduct library and 97 of them were screened positive by reverse dot-blot analysis. DNA sequence analysis identified genes that shared high homology with sequences in GenBank/EMBL database with unknown functions. Overall, 13 of the 90 high-quality sequences (14%) were homologous to 6 different genes previously described. Reverse Northern analysis confirmed that the expression of these genes were higher in the embryo-containing oviduct than in the oocyte-containing oviduct. About 12% of these clones (11/90) were novel. This article is the first to report identification of genes in the oviduct that are upregulated in the presence of embryos during the preimplantation period.

Essential role of ZP molecules in tubal transport of embryos in mice.

Our understandings of the molecular and cellular mechanisms underlying tubal transport of embryos are poor. This study describes the essential role of the molecules on the zona pellucida (ZP) in the tubal transport of mouse embryos. The bovine and porcine embryos that were interspecifically transferred to the mouse oviduct were selectively retained in the oviduct and rarely transported to the uterus. Antiserum ZP3-9 against synthetic peptides that are specific for mouse ZP3, significantly interfered with tubal transport of the treated embryos. The treatment of mouse embryos with antiserum ZP2-20 against the synthetic peptides, deduced from the sequences that are conserved in the structure of ZP2 from mouse and human, also inhibited their tubal transport. Among various proteolytic and glycosidic enzymes, treatments with trypsin and beta-glucosidase prior to transfer to the oviduct, significantly interfered with the tubal transport of the enzyme-treated mouse embryos. We hypothesize that species-specific epitopes on the ZP may be recognized by the oviductal cilia and/or the epithelial cells of ducts for tubal transport.

Differential expression of mucins in mouse oviduct and uterus.

Objective: The membrane-bound and gel-forming mucins of epithelial cells are expressed in a tissue-specific manner. The lumenal surface of the reproductive tract is covered by several kinds of mucins, which play important roles in gamete and embryo transport, implantation, and protection against infections. Mouse blastocysts can attach and implant to the receptive phase uterus. However, implantation in the mouse oviduct has not been observed at any phase. As a first in the evaluation of the specific function of mucins in the implantation process, a systemic analysis of mucin expression in the mouse oviduct and uterus was performed.

Expression and progestational control of OVGP1 messenger RNA in the mouse uterus and oviduct.

Objective: To evaluate the physiologic changes of OVGP1 (oviductin, oviduct-specific glycoprotein, MUC9) messenger RNA (mRNA) in each stage of the estrous cycle and during the early pregnancy period, and to analyze the effect of progesterone (P) on the expression of OVGP1 in the mouse uterus and oviduct.

Localization and distribution of platelet activating factor receptors in the mouse ovary and oviduct during the estrous cycle and early pregnancy.

This study determined platelet activating factor (PAF) binding to ovaries and oviduct tissues to identify the location of PAF tissue interaction. Mouse ovaries and oviducts taken during the estrous cycle and days 1, 4 and 7 post-conception (pc) were analyzed by [3H]PAF binding using frozen sections and autoradiography. For the outer epithelium of estrous and metestrous ovaries there was no significant difference in PAF binding (P>0.05); however, both stages were significantly different from proestrous (P<0.001). Ovarian stroma PAF binding was significantly higher (P<0.001) at estrous than metestrous, with no PAF binding at proestrous. Binding of PAF to estrous ovarian follicles was significantly greater (P<0.001) than at proestrous and metestrous. Estrous oviduct binding of PAF was significantly increased for stroma, inner epithelium and lumen (P<0.001 in all cases). At days 1 and 7 pc, all ovarian tissues had the greatest PAF binding with day 4 pc failing to bind PAF except for a significant decrease in corpora luteal binding (P<0.001). Oviduct binding of PAF was greatest at day 1 pc. Ovarian and oviduct PAF receptor expression corresponds to peak embryo PAF synthesis and establishes the basis for PAF PAF receptor mediated early pregnancy signaling system.

Suppression Subtractive Hybridization Identifies Genes Expressed in Oviduct during Mouse Preimplantation Period

Fertilization and development of mouse embryos occur in the ampullae of oviduct. Various growth factors and embryotrophic factors produced by the oviductal cells have been demonstrated to enhance embryo development in vitro. As a step towards understanding the genetic changes of mouse oviduct during mouse embryos preimplantation period, we adopted suppression subtractive hybridization (SSH) to establish four subtracted cDNA libraries to identify (1) oviduct-expressing genes, and (2) genes that may support embryo development in vivo. Using this method, we isolated 82, 88, 99, and 109 clones from four mouse libraries prepared from 0 (day 0), 24 (day 1), 48 (day 2), and 72 h (day 3) post-human chorionic gonadotropin (hCG) treated mice. Reverse dot-blot analysis confirmed that 25 (day 0), 24 (day 1), 40 (day 2), and 29 (day 3) clones were highly expressed in mouse oviduct when compared to other tissues. DNA sequence analysis identified genes encoding mouse oviduct-specific glycoprotein (MOGP), actin-binding protein 280, and several viral genes. Northern analysis confirmed that the genes were mainly expressed in oviduct, with some viral genes also expressed in uterus. About 9% of these oviduct expressing clones (11/118) were novel. We further demonstrated that one of the novel clones ODEG0-17 was expressed in the oviduct during early embryo preimplantation period and rarely in other tissues by RT-PCR. Our results show that SSH is a powerful method applicable to identifying tissue-specific transcripts on fertilization and development.

Effect of flanking matrix attachment regions on the expression of microinjected transgenes during preimplantation development of mouse embryos.

The efficiency of transgenic animal production would increase if microinjected embryos with a successfully integrated transgene could be identified prior to transfer. It is possible to detect microinjected DNA in embryos. However, no reliable system is able to distinguish between transgenes merely present as extrachromosomal DNA and those that have been integrated into chromatin. The experiments reported here were designed to determine if the inclusion of matrix attachment regions (MARs) would enhance the efficiency of transgenic embryos identification using a selection scheme based on the expression of green fluorescent protein (GFP). Pronuclei of mouse embryos were microinjected with GFP reporter gene under the control of three different promoters and flanked or not by three different MAR elements. Transgene expression profiles were followed by direct visualization of GFP in cultured microinjected embryos. Embryos at different developmental stages were classified according to their GFP expression and groups with the same expression pattern were transferred into oviducts of pseudopregnant female mice. Fetuses were collected between days 12-15, and their genomic DNA was purified and analyzed to detect transgene integration. We did not find any statistically significant difference between the percentage of transgenic fetuses produced from GFP-positive or GFP-negative embryos transferred at 4-cell, morula, or blastocyst stage. However, when MAR elements were included in the construct, we found that GFP-positive embryos transferred at the 2-cell stage produced a significantly higher percentage of transgenic fetuses than GFP-negative embryos, but MAR sequences did not completely eliminate false positives.