Serotype Diversity and Antimicrobial Resistance among Salmonella enterica Isolates from Patients at an Equine Referral Hospital.

Although Salmonella enterica can produce life-threatening colitis in horses, certain serotypes are more commonly associated with clinical disease. Our aim was to evaluate the proportional morbidity attributed to different serotypes, as well as the phenotypic and genotypic antimicrobial resistance (AMR) of Salmonella isolates from patients at an equine referral hospital in the southern United States. A total of 255 Salmonella isolates was obtained from clinical samples of patients admitted to the hospital between 2007 and 2015. Phenotypic resistance to 14 antibiotics surveilled by the U.S. National Antimicrobial Resistance Monitoring System was determined using a commercially available panel. Whole-genome sequencing was used to identify serotypes and genotypic AMR. The most common serotypes were Salmonella enterica serotype Newport (18%), Salmonella enterica serotype Anatum (15.2%), and Salmonella enterica serotype Braenderup (11.8%). Most (n = 219) of the isolates were pansusceptible, while 25 were multidrug resistant (≥3 antimicrobial classes). Genes encoding beta-lactam resistance, such as blaCMY-2, blaSHV-12, blaCTX-M-27, and blaTEM-1B, were detected. The qnrB2 and aac(6')-Ib-cr genes were present in isolates with reduced susceptibility to ciprofloxacin. Genes encoding resistance to gentamicin (aph(3')-Ia, aac(6')-IIc), streptomycin (strA and strB), sulfonamides (sul1), trimethoprim (dfrA), phenicols (catA), tetracyclines [tet(A) and tet(E)], and macrolides [ere(A)] were also identified. The main predicted incompatibility plasmid type was I1 (10%). Core genome-based analyses revealed phylogenetic associations between isolates of common serotypes. The presence of AMR Salmonella in equine patients increases the risk of unsuccessful treatment and causes concern for potential zoonotic transmission to attending veterinary personnel, animal caretakers, and horse owners. Understanding the epidemiology of Salmonella in horses admitted to referral hospitals is important for the prevention, control, and treatment of salmonellosis.IMPORTANCE In horses, salmonellosis is a leading cause of life-threatening colitis. At veterinary teaching hospitals, nosocomial outbreaks can increase the risk of zoonotic transmission, lead to restrictions on admissions, impact hospital reputation, and interrupt educational activities. The antimicrobials most often used in horses are included in the 5th revision of the World Health Organization's list of critically important antimicrobials for human medicine. Recent studies have demonstrated a trend of increasing bacterial resistance to drugs commonly used to treat Salmonella infections. In this study, we identify temporal trends in the distribution of Salmonella serotypes and their mechanisms of antimicrobial resistance; furthermore, we are able to determine the likely origin of several temporal clusters of infection by using whole-genome sequencing. These data can be used to focus strategies to better contain the dissemination and enhance the mitigation of Salmonella infections and to provide evidence-based policies and guidelines to steward antimicrobial use in veterinary medicine.

Using the emerging Collaborative Cross to probe the immune system.

The Collaborative Cross (CC) is an emerging panel of recombinant inbred (RI) mouse strains. Each strain is genetically distinct but all descended from the same eight inbred founders. In 66 strains from incipient lines of the CC (pre-CC), as well as the 8 CC founders and some of their F1 offspring, we examined subsets of lymphocytes and antigen-presenting cells. We found significant variation among the founders, with even greater diversity in the pre-CC. Genome-wide association using inferred haplotypes detected highly significant loci controlling B-to-T cell ratio, CD8 T-cell numbers, CD11c and CD23 expression. Comparison of overall strain effects in the CC founders with strain effects at QTL in the pre-CC revealed sharp contrasts in the genetic architecture of two traits with significant loci: variation in CD23 can be explained largely by additive genetics at one locus, whereas variation in B-to-T ratio has a more complex etiology. For CD23, we found a strong QTL whose confidence interval contained the CD23 structural gene Fcer2a. Our data on the pre-CC demonstrate the utility of the CC for studying immunophenotypes and the value of integrating founder, CC and F1 data. The extreme immunophenotypes observed could have pleiotropic effects in other CC experiments.

Characterization of the invasive and inflammatory traits of oral Campylobacter rectus in a murine model of fetoplacental growth restriction and in trophoblast cultures.

Campylobacter species (C. jejuni, C. fetus) are enteric abortifacient bacteria in humans and ungulates. Campylobacter rectus is a periodontal pathogen associated with human fetal exposure and adverse pregnancy outcomes including preterm delivery. Experiments in pregnant mice have demonstrated that C. rectus can translocate from a distant site of infection to the placenta to induce fetal growth restriction and impair placental development. However, placental tissues from human, small-for-gestational age deliveries have not been reported to harbor C. rectus despite evidence of maternal infection and fetal exposure by fetal IgM response. This investigation examined the temporal relationship between the placental translocation of C. rectus and the effects on fetal growth in mice. BALB/c mice were infected at gestational day E7.5 to examine placental translocation of C. rectus by immunohistology. C. rectus significantly decreased fetoplacental weight at E14.5 and at E16.5. C. rectus was detected in 63% of placentas at E14.5, but not at E16.5. In in vitro trophoblast invasion assays, C. rectus was able to effectively invade human trophoblasts (BeWo) but not murine trophoblasts (SM9-1), and showed a trend for more invasiveness than C. jejuni. C. rectus challenge significantly upregulated both mRNA and protein levels of IL-6 and TNFalpha in a dose-dependent manner in human trophoblasts, but did not increase cytokine expression in murine cells, suggesting a correlation between invasion and cytokine activation. In conclusion, the trophoblast-invasive trait of C. rectus that appears limited to human trophoblasts may play a role in facilitating bacterial translocation and placental inflammation during early gestation.

Identification of novel genes in the oral pathogen Campylobacter rectus.

INTRODUCTION: A poorly described bacterium, Campylobacter rectus, has been implicated as an etiological agent of periodontal disease. The aim of this study was to use a comparative genomics approach to identify genes that contribute to the lifestyle of C. rectus as an oral pathogen. METHODS: Suppressive subtractive hybridization was used to identify genes encoded by C. rectus ATCC 33238, but not present in the genome of a related Campylobacter species, Campylobacter jejuni ATCC 11168. RESULTS: Suppressive subtractive hybridization identified 154 unique DNA sequences from the C. rectus genome. Ninety-two of the 154 clones were classified as C. rectus-specific, as they did not show significant sequence homology to genes identified in any strain of C. jejuni (blast E-value >1E-3). blast analysis predicted that the 92 C. rectus-specific gene fragments play a role in a variety of biological processes including signal transduction mechanisms (histidine kinase, response regulators, diguanylate cyclases, chemotaxis receptor) and potentially virulence (S-layer RTX and cysteine desulfhydrase). Further analysis of the C. rectus-specific clones showed that 10 genes had Campylobacter homologues that were only found in species that commonly reside within the oral cavity of humans and 10 other fragments shared homology only with non-campylobacter organisms. CONCLUSIONS: These data provide the first substantial insights into the genomic content of C. rectus, a significant oral pathogen. The genes identified in this study are a valuable resource for initiating new research on the virulence of C. rectus during periodontitis.

Altered trophoblast proliferation is insufficient to account for placental dysfunction in Egfr null embryos.

Homozygosity for the Egfr(tm1Mag) null allele in mice leads to genetic background dependent placental abnormalities and embryonic lethality. Molecular mechanisms or genetic modifiers that differentiate strains with surviving versus non-surviving Egfr nullizygous embryos have yet to be identified. Egfr transcripts in wildtype placenta were quantified by ribonuclease protection assay (RPA) and the lowest level of Egfr mRNA expression was found to coincide with Egfr(tm1Mag) homozygous lethality. Immunohistochemical analysis of ERBB family receptors, ERBB2, ERBB3, and ERBB4, showed similar expression between Egfr wildtype and null placentas indicating that Egfr null trophoblast do not up-regulate these receptors to compensate for EGFR deficiency. Significantly fewer numbers of bromodeoxyuridine (BrdU) positive trophoblast were observed in Egfr nullizygous placentas and Cdc25a and Myc, genes associated with proliferation, were significantly down-regulated in null placentas. However, strains with both mild and severe placental phenotypes exhibit reduced proliferation suggesting that this defect alone does not account for strain-specific embryonic lethality. Consistent with this hypothesis, intercrosses generating mice null for cell cycle checkpoint genes (Trp53, Rb1, Cdkn1a, Cdkn1b or Cdkn2c) in combination with Egfr deficiency did not increase survival of Egfr nullizygous embryos. Since complete development of the spongiotrophoblast compartment is not required for survival of Egfr nullizygous embryos, reduction of this layer that is commonly observed in Egfr nullizygous placentas likely accounts for the decrease in proliferation.

Masking in waved-2 mice: EGF receptor control of locomotion questioned.

It has been suggested that epidermal growth factors (EGF) are responsible for the inhibition of locomotion by light (i.e., masking) in nocturnal rodents (Kramer et al., 2001). The poor masking response of waved-2 (Egfr(wa2)) mutant mice, with reduced EGF receptor activity, was adduced in support of this idea. In the present work, we studied the responses to light over a large range in illumination levels, in a variety of tests, with pulses of light and with ultradian light-dark cycles in Egfr(wa2) mutant mice. No evidence suggested that normal functioning of epidermal growth factor receptors was required, or even involved, in masking.

The math of making mutant mice.

More than ten large-scale mutagenesis projects are now generating hundreds of novel mouse mutants. Projects employ a wide variety of strategies and screens: targeting as much as the whole genome, part of a chromosome or just single genes. In this commentary, we consider the pros and cons of different tactics. We highlight issues of cost, efficiency and defend the impact of this mutagenesis program in an era of sophisticated conditional knockouts and advanced transgenic lines. Given the significant difficulties of adequately phenotyping and mapping randomly generated mutations that cover the whole genome, we tend to favor regional and gene-targeted screens. Whatever the choice of method, whole genome sequence data combined with detailed transcriptome and proteome surveys promise to significantly improve the efficiency with which series of mutations in a large subset of mammalian genes can be generated and cloned.

Profiling proteins from azoxymethane-induced colon tumors at the molecular level by matrix-assisted laser desorption/ionization mass spectrometry.

New developments in mass spectrometry allow for the profiling of the major proteomic content of fresh tissue sections. Briefly, fresh tissue sections are sampled and blotted onto a polyethylene membrane for protein transfer and then subsequently analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Using this technology, we have compared the protein expression of normal and cancerous mouse colon tissue obtained from the same animal. By difference, several protein signals specific to cancerous tissue were observed. A protein extract obtained from the tumors was fractionated by high-performance liquid chromatography and the individual fractions analyzed by MALDI-MS. The fractions containing the targeted proteins were subjected to trypsin digestion. The resulting tryptic peptides were sequenced by tandem mass spectrometry, and based on the recovered partial amino acid sequences, three of the tumor specific protein markers were identified as calgranulin A (S100A8), calgranulin B (S100A9) and calgizzarin (S100A11).

Comparative genomic sequence analysis and isolation of human and mouse alternative EGFR transcripts encoding truncated receptor isoforms.

This study presents the annotated genomic sequence and exon-intron organization of the human and mouse epidermal growth factor receptor (EGFR) genes located on chromosomes 7p11.2 and 11, respectively. We report that the EGFR gene spans nearly 200 kb and that the full-length 170-kDa EGFR is encoded by 28 exons. In addition, we have identified two human and two mouse alternative EGFR transcripts of 2.4-3.0 kb using both computational and experimental methods. The human 3.0-kb and mouse 2.8-kb EGFR mRNAs are predominantly expressed in placenta and liver, respectively, and both transcripts encode 110-kDa truncated receptor isoforms containing only the extracellular ligand-binding domain. We also have demonstrated that the aberrant 2.8-kb EGFR transcript produced by the human A431 carcinoma cell line is generated by splicing to a recombinant 3'-terminal exon located in EGFR intron 16, which apparently was formed as a result of a chromosomal translocation. Finally, we have shown that the human, mouse, rat, and chicken 1.8- to 3.0-kb alternative EGFR transcripts are generated by distinct splicing mechanisms and that each of these mRNAs contains unique 3' sequences that are not evolutionarily conserved. The presence of truncated receptor isoforms in diverse species suggests that these proteins may have important functional roles in regulating EGFR activity.

Cross-talk between epidermal growth factor receptor and protein kinase C during calcium-induced differentiation of keratinocytes.

The induction of epidermal differentiation by extracellular Ca2+ involves activation of both tyrosine kinase and protein kinase C (PKC) signaling cascades. To determine if the differentiation-dependent activation of tyrosine kinase signaling can influence the PKC pathway, we examined the tyrosine phosphorylation status of PKC isoforms in primary mouse keratinocytes stimulated to terminally differentiate with Ca2+. Elevation of extracellular Ca2+ induced tyrosine phosphorylation of PKC-delta, but not the other keratinocyte PKC isoforms (alpha, epsilon, eta, zeta). We have previously demonstrated that activation of the epidermal growth factor receptor (EGFR) pathway induces PKC-delta tyrosine phosphorylation in basal keratinocytes (Denning M F, Dlugosz A A, Threadgill D W, Magnuson T, Yuspa S H (1996) J Biol Chem 271: 5325-5331). When basal keratinocytes were stimulated to differentiate by Ca2+, the level of cell-associated transforming growth factor-alpha (TGF-alpha) increased 30-fold, while no increase in secreted TGF-alpha was detected. Furthermore, Ca2+-induced tyrosine phosphorylation of PKC-delta and phosphotyrosine-association of the receptor adapter protein Shc was diminished in EGFR -/- keratinocytes, suggesting that EGFR activation may occur during keratinocyte differentiation. Tyrosine phosphorylated PKC-delta was also detected in mouse epidermis, suggesting that this differentiation-associated signaling pathway is physiological. These results establish a requirement for the EGFR in Ca2+-induced tyrosine phosphorylation of PKC-delta, and document the production of cell-associated TGF-alpha in differentiated keratinocytes which may function independent of its usual mitogenic effects.

Role of the angiotensin type 2 receptor gene in congenital anomalies of the kidney and urinary tract, CAKUT, of mice and men.

Angiotensin type 2 receptor gene null mutant mice display congenital anomalies of the kidney and urinary tract (CAKUT). Various features of mouse CAKUT impressively mimic human CAKUT. Studies of the human type 2 receptor (AGTR2) gene in two independent cohorts found that a significant association exists between CAKUT and a nucleotide transition within the lariat branchpoint motif of intron 1, which perturbs AGTR2 mRNA splicing efficiency. AGTR2, therefore, has a significant ontogenic role for the kidney and urinary tract system. Studies revealed that the establishment of CAKUT is preceded by delayed apoptosis of undifferentiated mesenchymal cells surrounding the urinary tract during key ontogenic events, from the ureteral budding to the expansive growth of the kidney and ureter.

Gamma 3 gene-disrupted mice selectively deficient in the dominant IgG subclass made to bacterial polysaccharides undergo normal isotype switching after immunization with polysaccharide-protein conjugate vaccines.

Bacterial polysaccharides (PS) are T-independent type 2 Ags that elicit restricted Ab responses of IgM and IgG3 in mice and IgM and predominantly IgG2 in humans. Immunodeficiency in the dominant IgG subclass made to PS is associated with chronic sinus and pulmonary infections with PS-encapsulated bacteria. To elucidate the biologic role of the dominant IgG subclass in the immune response to PS and to make an animal model of human IgG subclass deficiency, we generated mice with a targeted disruption of the exon encoding the CH1 domain of the gamma 3 heavy-chain constant region gene. Homozygotes had no detectable serum IgG3, and their splenocytes did not produce IgG3 after LPS stimulation. IgG3(-/-) mice immunized with PS from Pseudomonas aeruginosa LPS O-side chain or Streptococcus pneumoniae type 19F capsule did not produce any IgG3 anti-PS Abs, in contrast to wild-type mice in which IgG3 was the major IgG subclass. Immunizing both wild-type and IgG3(-/-) mice with 19F PS-protein conjugate elicited IgG1 Abs. We conclude that IgG3(-/-) mice have a selective deficiency in the dominant murine IgG subclass made to T-independent type 2 Ags and may be a useful animal model of IgG subclass deficiency. In addition, we show that the anti-PS Ab class switching to IgG1 that occurs when mice are immunized with a PS-protein conjugate vaccine does not require sequential Ig expression or an intact, upstream gamma 3 heavy-chain gene.

Mitogenic synthetic polynucleotides suppress the antibody response to a bacterial polysaccharide.

Unmethylated bacterial DNA containing a high frequency of the CpG motif, is mitogenic and induces T-cell independent, murine B-cell proliferation. These stimulatory effects are also induced by synthetic oligonucleotides that contain one or more unmethylated CpG dinucleotides (CpG oligo). Such mitogenicity is not seen with highly methylated vertebrate DNA, which has a lower prevalence of the CpG motif than bacterial DNA. Due to their stimulatory effects, CpG oligo have been proposed for use as vaccine adjuvants. In order to determine if a synthetic CpG oligo that was stimulatory for B-cell proliferation could augment the murine antibody response to protective bacterial polysaccharide epitopes (Pseudomonas aeruginosa LPS-O polysaccharide side chain; high-molecular-weight polysaccharide or high-MW PS), BALB/c mice were injected with mitogenic doses of CpG oligo simultaneously with high-MW PS, and antibody titers were measured by ELISA weekly for 4 weeks. Controls received PBS, a nonstimulatory control oligo plus PS, CpG alone, or PS alone. Despite evidence of B-cell mitogenicity and an increase in total IgM in CpG oligo-treated mice, CpG oligo treatment plus PS significantly decreased the high-MW PS antibody response compared to PS alone. The blunting of the anti-PS antibody response could be eliminated by vaccinating the animals with PS prior to CpG oligo. We conclude that despite in vitro and in vivo evidence of B-cell proliferation, this CpG oligo reduces PS-specific antibody responses in an animal model when given simultaneously with a bacterial polysaccharide. Based on results in this model, oligonucleotides containing stimulatory unmethylated CpG dinucleotides may not be useful adjuvants when given simultaneously with bacterial PS vaccines.

Targeted disruption of the epidermal growth factor receptor impairs growth of squamous papillomas expressing the v-ras(Ha) oncogene but does not block in vitro keratinocyte responses to oncogenic ras.

We have assessed the role of epidermal growth factor receptor (EGFR) signaling in biological responses to the v-ras(Ha) oncogene using primary keratinocytes from Egfr -/- mice and wild-type littermates. On the basis of several criteria, Egfr -/- keratinocytes were unresponsive to either acute or chronic exposure to several EGFR ligands but were stimulated to proliferate in response to several other mitogens. Although conditioned medium from primary keratinocytes transduced with v-ras(Ha) retrovirus (v-ras(Ha) keratinocytes) was a potent mitogen for wild-type but not Egfr -/- keratinocytes, v-ras(Ha) transduction of primary keratinocytes of either genotype resulted in a strong mitogenic response, arguing against an obligatory role for EGFR activation in v-ras(Ha)-mediated stimulation of keratinocyte proliferation. Infection with high-titer v-ras(Ha) retrovirus altered the keratin expression pattern in keratinocytes of both genotypes, suppressing differentiation-specific keratins K1 and K10 while activating aberrant expression of K8 and K18. In wild-type but not Egfr -/- cultures, K1 and K10 were also suppressed following infection at lower retroviral titers, presumably as a result of paracrine EGFR activation on uninfected cells present in these cultures. Squamous papillomas produced by grafting Egfr -/- v-ras(Ha) keratinocytes onto nude mice were only 21% of the size of wild-type v-ras(Ha) tumors, and a striking redistribution of S-phase cells was detected by immunostaining for bromodeoxyuridine. In Egfr -/- v-ras(Ha) papillomas, the fraction of total labeled nuclei detected in suprabasal layers was increased from 19 to 39%. In contrast, the basal layer labeling index of Egfr -/- papillomas was reduced to 34%, compared to 43% in wild-type tumors. Our results indicate that, although autocrine EGFR signaling is not required for keratinocyte responses to oncogenic ras in culture or benign tumor formation in nude mouse grafts, disruption of this pathway impairs growth of v-ras(Ha) papillomas by a mechanism that may involve alterations in keratinocyte cell cycle progression and/or migration in vivo.

Genealogy of the 129 inbred strains: 129/SvJ is a contaminated inbred strain.

The 129 mouse is the most widely used strain in gene targeting experiments. However, numerous substrains exist with demonstrable physiological differences. In this study a set of simple sequence length polymorphisms (SSLPs) was used to determine the relatedness of selected 129 substrains. 129/SvJ was significantly different from the other 129 substrains and is more accurately classified as a recombinant congenic strain (129cX/Sv), being derived from 129/Sv and an unknown strain. This mixed genetic background could complicate gene targeting experiments by reducing homologous recombination efficiency when constructs and ES cells are not derived from the same 129 substrain. Additionally, discrepancies due to different genetic backgrounds may arise when comparing phenotypes of genes targeted in different 129-derived ES cell lines.

Genetically null mice reveal a central role for epidermal growth factor receptor in the differentiation of the hair follicle and normal hair development.

Mice harboring a targeted disruption of the epidermal growth factor receptor (EGFR) allele exhibit a severely disorganized hair follicle phenotype, fuzzy coat, and systemic disease resulting in death before 3 weeks. This skin phenotype was reproduced in whole skin grafts and in grafts of EGFR null hair follicle buds onto nude mice, providing a model to evaluate the natural evolution of skin lacking the EGFR. Hair follicles in grafts of null skin did not progress from anagen to telogen and scanning electron micrografts revealed wavy, flattened hair fibers with cuticular abnormalities. Many of the EGFR null hair follicles in the grafted skin were consumed by an inflammatory reaction resulting in complete hair loss in 67% of the grafts by 10 weeks. Localization of follicular differentiation markers including keratin 6, transglutaminase, and the hair keratins mHa2 and hacl-1 revealed a pattern of premature differentiation within the null hair follicles. In intact EGFR null mice, proliferation in the interfollicular epidermis, but not hair follicles, was greatly decreased in the absence of EGFR. In contrast, grafting of EGFR null skin resulted in a hyperplastic response in the epidermis that did not resolve even after 10 weeks, although the wound-induced hyperplasia in EGFR wild-type grafts had resolved within 3 to 4 weeks. Thus, epithelial expression of the EGFR has complex functions in the skin. It is important in delaying follicular differentiation, may serve to protect the hair follicle from immunological reactions, and modifies both normal and wound-induced epidermal proliferation but seems dispensable for follicular proliferation.

Characterization and mutational studies of equine infectious anemia virus dUTPase.

The macrophage tropic lentivirus, equine infectious anemia virus (EIAV), encodes a dUTPase in the pol gene that is required for efficient replication in macrophages. Two naturally occurring variants of the enzyme were expressed as recombinant proteins in Escherichia coli; metal chelate affinity chromatography was used to purify histidine-tagged recombinant enzymes to greater than 80% homogeneity in a single chromatographic step. Biochemical and enzymatic analyses of these preparations suggest that this method yields dUTPase that is suitable for detailed mutational analysis. Specific activities of preparations ranged from 4 x 10(3) to 5 x 10(4) units/mg. Recombinant EIAV dUTPase was highly specific for dUTP with a Km in the range of 3 to 8 microM. The enzyme was sensitive to inhibition by dUDP with little inhibition by other nucleotides or the reaction products, dUMP and PPi. The subunit organization of recombinant EIAV dUTPase was probed by gel filtration, glycerol gradient centrifugation, and chemical cross-linking, and is a trimer. We have begun mutational analyses by targeting a conserved domain present at the carboxyl terminus of all dUTPases that shares high homology to the phosphate binding loops (P-loops) of a number of ATP- and GTP-binding phosphatases. The P-loop-like motif of dUTPases is glycine rich but lacks the invariant lysine found in authentic P-loops. Deletion of this motif leads to loss of dUTPase activity; a series of point mutations that have been shown to inactivate authentic P-loops also abolish EIAV dUTPase activity.

Differential expression of the full-length and truncated forms of the epidermal growth factor receptor in the preimplantation mouse uterus and blastocyst.

The present investigation examined the differential expression of the full-length (fl) and the truncated (tr) forms of the epidermal growth factor receptor (EGFr) in the preimplantation mouse uterus and blastocyst. Northern blot hybridization using a complementary RNA probe specific to the full-length form (EGFr-fl) detected a 6.5-kb transcript, whereas that of the truncated form (EGFr-tr) detected a 2.7-kb transcript in the preimplantation mouse uterus on days 1 and 4 of pregnancy (day 1 = vaginal plug). In situ hybridization using these probes detected the EGFr-fl transcripts only in the stroma and myometrium, but not in the epithelium, whereas EGFr-tr transcripts were detected in all major uterine cell-types. To confirm the results of in situ hybridization, RT-PCR was performed on RNA isolated from separated uterine cell-types on day 4 of pregnancy using sequence specific primers for the two forms of the receptor. The results concur that the EGFr-tr transcript is expressed in the epithelium, stroma and myometrium, whereas that of the EGFr-fl transcript is not expressed in the epithelium. In the preimplantation blastocyst, RT-PCR detected the EGFr-fl messenger RNA, but not the EGFr-tr messenger RNA. These results suggest that the blastocyst, not the uterine epithelium, is the target for EGF family of growth factors in embryo-uterine interaction during implantation.