Proteomic analysis of the early bovine yolk sac fluid and cells from the day 13 ovoid and elongated preimplantation embryos

The bovine blastocyst hatches 8 to 9 days after fertilization, and this is followed by several days of preimplantation development during which the embryo transforms from a spherical over an ovoid to an elongated shape. As the spherical embryo enlarges, the cells of the inner cell mass differentiate into the hypoblast and epiblast, which remain surrounded by the trophectoderm. The formation of the hypoblast epithelium is also accompanied by a change in the fluid within the embryo, i.e., the blastocoel fluid gradually alters to become the primitive yolk sac (YS) fluid. Our previous research describes the protein composition of human and bovine blastocoel fluid, which is surrounded by the trophectoderm and undifferentiated cells of the inner cell mass. In this study, we further examine the changes in the protein composition in both the primitive YS fluid and the embryonic cells during early and slightly later stage cell differentiation in the developing bovine embryo. In vitro–produced Day 6 embryos were transferred into a recipient heifer and after 7 days of further in vivo culture, ovoid and elongated Day 13 embryos were recovered by flushing both uterine horns after slaughter. The primitive YS fluid and cellular components were isolated from 12 ovoid and three elongated embryos and using nano-high-performance liquid chromatography, tandem mass spectrometry, and isobaric tag for relative and absolute quantitation proteomic analysis, a total of 9652 unique proteins were identified. We performed GO term and keyword analyses of differentially expressed proteins in the fluid and the cells of the two embryonic stages, along with a discussion of the biological perspectives of our data with relation to morphogenesis and embryo-maternal communication. Our study thereby provides a considerable contribution to the current knowledge of bovine preimplantation development.     

Characterization of equine and other vertebrate TLR3, TLR7, and TLR8 genes

Toll-like receptors 3, 7, and 8 (TLR3, TLR7, and TLR8) were studied in the genomes of the domestic horse and several other mammals. The messenger RNA sequences and exon/intron structures of these TLR genes were determined. An equine bacterial artificial chromosome clone containing the TLR3 gene was assigned by fluorescent in situ hybridization to the horse chromosomal location ECA27q16–q17 and this map location was confirmed using an equine radiation hybrid panel. Direct sequencing revealed 13 single-nucleotide polymorphisms in the coding regions of the equine TLR 3, 7, and 8 genes. Of these polymorphisms, 12 were not previously reported. The allelic frequency was estimated for each single-nucleotide polymorphism from genotyping data obtained for 154 animals from five horse breeds. Some of these frequencies varied significantly among different horse breeds. Domain architecture predictions for the three equine TLR protein sequences revealed several conserved regions within the variable leucine-rich repeats between the corresponding horse and cattle TLR proteins. A phylogenetic analysis did not indicate that any significant exchanges had occurred between paralogous TLR7 and TLR8 genes in 20 vertebrate species analyzed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of dehydration prior to cryopreservation of large equine embryos

Cryopreservation of equine embryos > 300 μm in diameter results in low survival rates using protocols that work well for smaller equine embryos. These experiments tested the potential benefit of incorporating a dehydration step prior to standard cryopreservation procedures. Forty-six, day 7–8, grade 1, equine embryos 300–1350 μm in diameter were subjected to one of the following treatments: (A) 2 min in 0.6 M galactose, 10 min in 1.5 M glycerol, slow freeze (n = 21); (B) 10 min in 1.5 M glycerol, slow freeze (n = 15); (C) 2 min in 0.6 M galactose, 10 min in 1.5 M glycerol, followed by exposure to thaw solutions, then culture medium (n = 5); (D) transferred directly to culture medium (n = 5). Frozen embryos were thawed and subjected to a three-step cryoprotectant removal. Five embryos from each treatment were evaluated morphologically after 24 and 48 h culture (1 = excellent, 5 = degenerate/dead). All treatments had at least 4/5 embryos with a quality score  3 at these time points except treatment B (2/5 at 24 h, 1/5 at 48 h). Subsequent embryos from treatment A (n = 16) or B (n = 10) were matched in sets of two for size and treatment, thawed, and immediately transferred in pairs to 13 recipients. Only two recipient mares were pregnant; one received two 400 μm embryos from treatment A, and the other one 400 and one 415 μm embryo from treatment B. There was no advantage of incorporating a 2 min dehydration step into the cryopreservation protocol for large equine embryos.     

Infection of embryos following insemination of donor mares with equine arteritis virus infective semen

The objective was to evaluate the potential risks associated with embryo transfer from mares bred with equine arteritis virus (EAV) infective semen. Twenty-six mares were embryo donors, whereas 18 unvaccinated and EAV antibody seronegative mares were embryo recipients. Of the 26 donor mares, 15 were unvaccinated and seronegative for antibodies to EAV and 11 were vaccinated for the first time with a commercially available modified live virus vaccine against EVA before breeding and subsequent embryo transfer. All donor mares were bred with EAV-infective semen from a stallion persistently infected with the virus. Twenty-four embryos were recovered 7 d post-ovulation; all were subjected in sequential order to five washes in embryo flush medium, two trypsin treatments, and five additional washes in embryo flush medium (prior to transfer). Twelve and seven embryos (Grades 1 or 2) were transferred from the non-vaccinated and vaccinated donors, respectively, and pregnancy was established in 3 of 12 and 2 of 7. Perhaps trypsin reduced embryo viability and pregnancy rate. The uterine flush fluid of 11 mares (9 of 15 and 2 of 11 from non-vaccinated and vaccinated donor groups, respectively) was positive for EAV by VI (confirmed by real-time RT-PCR); the wash fluid from the embryos of nine of these mares was negative following 10 washes and two trypsin treatments. However, the embryo wash fluid from two mares was still positive for EAV after all 10 washes and the two trypsin treatments, and one embryo was positive for EAV. Two of 18 recipient mares had seroconverted to EAV 28 d after embryo transfer. Virus was not detected in any fetal tissues or fluids harvested after pregnancies were terminated (60 d). In conclusion, we inferred that the washing protocol of 10 washes and two trypsin treatments did not eliminate EAV from all embryos; due to limitations in experimental design, this requires confirmation. Furthermore, there may be a risk of EAV transmission associated with in vivo embryo transfer from a donor mare inseminated with EAV infective semen.     

2D-DiGE analysis of the human endometrial secretome reveals differences between receptive and nonreceptive states in fertile and infertile women

Endometrial secretions in the uterine cavity contain mediators important for endometrial receptivity and embryo implantation. Unbiased analysis of uterine fluid from a receptive versus nonreceptive time of the menstrual cycle and in fertile and infertile women will provide new insights into uterine receptivity. We hypothesized that proteomic analysis of human uterine lavages would identify proteins important for the establishment of pregnancy in humans. Lavages collected from fertile (n = 7) and infertile (n = 8) women during the midsecretory (MS) phase, and from fertile women during the midproliferative (MP) (n = 7) phase, were assessed using 2D-differential in gel electrophoresis (2D-DiGE) over a pI 4-7 range. Statistical analysis revealed 7 spots that were significantly decreased in the MP compared to the MS phase, while 18 spots showed differential expression between fertile and infertile women. A number of proteins were identified by mass spectrometry, including antithrombin III and alpha-2-macroglobulin, whose production was confirmed in endometrial epithelium. Their staining pattern suggests roles during embryo implantation. Assessment of the human endometrial secretome has identified differences in the protein content of uterine fluid with respect to receptivity and fertility.     

Biochemical changes in the equine capsule following prostaglandin-induced pregnancy failure

The equine embryonic capsule, an acellular covering that envelops the conceptus during the second and third weeks of pregnancy, is composed of mucin-like glycoproteins. Its structure is consistent with a dual role during early pregnancy: protection of the conceptus, and communication between the embryo and the mother. Loss of sialic acid from the capsular glycoproteins at day 16 correlates with the time of “fixation,” or loss of conceptus mobility throughout the uterine horns. This study investigated how the structure of the capsule is linked to the maintenance of pregnancy. Six pregnancies, confirmed by ultrasound, were terminated by prostaglandin injection on day 14, prior to the time of embryo fixation. These “defective” conceptuses were collected at day 17, and the structure and molecular properties of their capsules were compared to those of day 17 conceptuses collected from 5 normal pregnancies. Defective capsules were not significantly different from normal capsules in terms of dry weight, amino acid composition, and content of neutral and amino sugars. However, defective capsules failed to show the loss of sialic acid normally occurring around the time of embryo fixation. Analysis of the capsular mucins following trypsin digestion was carried out by radioactive labeling with ³H on sialyl-oligosaccharides and ¹²⁵I on tyrosine residues, followed by fast protein liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Differences in the trypsin fragmentation patterns indicated increased susceptibility of the defective capsules to proteolysis. We conclude that there is a temporal association between desialylation of the equine capsule and embryonic survival, and that failure to desialylate alters the properties of the capsule. Mol. Reprod. Dev. 46:286–295, 1997. © 1997 Wiley-Liss, Inc.     

The Equine PeptideAtlas: A resource for developing proteomics‐based veterinary research

Progress in MS‐based methods for veterinary research and diagnostics is lagging behind compared to the human research, and proteome data of domestic animals is still not well represented in open source data repositories. This is particularly true for the equine species. Here we present a first Equine PeptideAtlas encompassing high‐resolution tandem MS analyses of 51 samples representing a selection of equine tissues and body fluids from healthy and diseased animals. The raw data were processed through the Trans‐Proteomic Pipeline to yield high quality identification of proteins and peptides. The current release comprises 24 131 distinct peptides representing 2636 canonical proteins observed at false discovery rates of 0.2% at the peptide level and 1.4% at the protein level. Data from the Equine PeptideAtlas are available for experimental planning, validation of new datasets, and as a proteomic data mining resource. The advantages of the Equine PeptideAtlas are demonstrated by examples of mining the contents for information on potential and well‐known equine acute phase proteins, which have extensive general interest in the veterinary clinic. The extracted information will support further analyses, and emphasizes the value of the Equine PeptideAtlas as a resource for the design of targeted quantitative proteomic studies.     

Alterations in cellular proteome and secretome upon differentiation from monocyte to macrophage by treatment with phorbol myristate acetate: Insights into biological processes

Monocyte and macrophage are mainly involved in immune response and inflammatory processes. Monocytes circulate in the bloodstream and migrate to various tissues where they can differentiate to macrophages. However, the molecular basis of biological processes involved in this cellular differentiation remains ambiguous. This study was to investigate alterations in cellular and secreted proteins after this differentiation phase. Macrophage was differentiated from U937 human monocytic cell line by treatment with 100 ng/ml phorbol myristate acetate (PMA) for 48 h. Cellular and secreted proteins extracted from PMA-treated cells (macrophages) were compared with those of untreated cells (monocytes) using 2-DE (n = 5 gels/condition; stained with Deep Purple fluorescence dye). Quantitative intensity analysis revealed 81 and 67 protein spots whose levels were significantly altered in cellular proteome and secretome. These proteins were subsequently identified by Q-TOF MS and/or MS/MS analyses. The altered levels of cellular elongation factor-2 (EF-2) and secreted α-tubulin were confirmed by Western blot analysis. Global protein network analysis demonstrated that these altered proteins were involved in cell death, lipid metabolism, cell morphology, cellular movement, and protein folding. Our data may provide some insights into molecular mechanisms of biological processes upon differentiation from monocytes to macrophages.     

Gestational profiles of rat placental lactogen-II (rPL-II) and growth hormone (GH) in maternal and fetal serum, amniotic fluid, and placental tissue.

Rat placental lactogen-II (rPL-II) and growth hormone (rGH) in maternal and fetal serum, amniotic fluid, and placental tissue were measured by a homologous radioimmunoassay during the last half of pregnancy. rPL-II appeared first in maternal circulation and the placental tissue on day 11 of pregnancy. The maternal serum rPL-II concentration increased progressively and reached the peak value (684 +/- 76 ng/ml) on day 19, and declined thereafter up to term. rPL-II content in the tissue had a similar pattern to the maternal serum profile of rPL-II, while its concentration in the tissue increased dramatically on day 12 and remained high until day 19. Fetal serum rPL-II was detected on days 17 and 18, though its concentration was much lower (ranged between 3-10 ng/ml) than that of maternal serum. rPL-II in amniotic fluid was also detectable only on days 12-14 of pregnancy, and the peak value on day 13 was 22% of the maternal serum rPL-II concentration. The rGH concentration increased gradually as pregnancy advanced with a decline on the day before parturition. Although rGH in fetal serum increased on day 20 with a decline on the following day, it was slightly detectable in amniotic fluid on the last two days of pregnancy. The molecular profile of rPL-II in amniotic fluid and maternal serum of day 13 pregnant rats were examined by Western blotting. Anti-rPL-II serum detected two proteins with molecular weights (mol wt) of 19.5K and 20.5K in amniotic fluid and one protein with a mol wt of 20.5K in maternal serum under nonreducing conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Desialylation of core type 1 O-glycan in the equine embryonic capsule coincides with immobilization of the conceptus in the uterus

During the second and third weeks of pregnancy, the equine conceptus expands rapidly while it is enclosed within a glycan capsule. Around day 16 of gestation, the conceptus loses its mobility in the uterus by a process termed 'fixation', coinciding with various changes in the capsule. Here, we compared the structure of the carbohydrate moieties expressed by the capsule during pre- and post-fixation periods. The glycan structures were studied by chemical analyses in combination with mass spectrometry. Capsule material from conceptuses collected before fixation (days 13-16) was observed to carry a sialylated core type 1 O-linked glycan, Neu5Ac-(2-->3)-Gal-(1-->3)-GalNAc-(1-->Ser/Thr. By comparison, analysis of post-fixation capsules (days 17-19) revealed a desialylated core type 1, Gal-(1-->3)-GalNAc-(1-->Ser/Thr. The equine embryonic capsule also furnished 4-substituted GlcNAc, 4-substituted Glc and 2,3,4,6-tetrasubstituted Glc residues, the concentrations of which did not change between pre- and post-fixation stages. The loss of sialic acid from the sialylated core type 1 in the capsule appears to be directly related to successful fixation of the conceptus, and thus critical to the continuance of pregnancy in horses.     

Equine oocyte in vitro maturation: influences of sera, time, and hormones.

Objectives of the present research were to determine the influences of types of media, sera, time and hormones on equine oocyte in vitro maturation (IVM). The following types of media and sera were evaluated: Menezo's B2 medium (B2), modified Tissue Culture Medium 199 (TCM), Defined Medium (DM), fetal calf serum (FCS), mare serum collected on the first day of estrus (MS), and mare serum collected on the day of ovulation (MSO). Resultant oocyte maturation was compared with the control: DM with bovine serum albumin (BSA). Effect of culture time (0, 15, and 32 hr) and the following hormones on oocyte IVM were evaluated: none, bovine luteinizing hormone (bLH; 1, 10, 100 micrograms/ml), equine luteinizing hormone (eLH; 100 micrograms/ml), bovine follicle-stimulating hormone (FSH; 5 micrograms/ml), and equine chorionic gonadotropin (eCG; 1 and 100 IU/ml). Cumulus expansion in the media and sera experiments was 50% (DM with BSA), 80% (TCM, B2, and DM with MS or MSO), and 100% (FCS with any medium). The proportion of metaphase II (MII) oocytes was significantly (P less than 0.05) increased the percentage of MII oocytes as compared with 0 hr of culture. Cumulus expansion in the hormone experiments was 80% (none, bLH, and eLH), and 100% (eCG and FSH). Freshly prepared bLH significantly (P less than 0.05) inhibited nuclear maturation of equine oocytes. In summary, 15 hr of culture was sufficient time for equine oocyte IVM and all combinations of medium, serum, and hormone addition were equally effective in achieving IVM except fresh bLH and DM with BSA.     

Equine zona pellucida and capsule: Some physicochemical and antigenic properties

The capsule which surrounds the pre-attachment equine embryo has been compared with the zona pellucida (zp) that it replaces, as well as with the rabbit blastocyst coverings, by means of physicochemical and immunological methods. Trypsin solution at pH varying between 7.5 and 9.0 completely solubilized the capsule, as did Na borohydride. However, solutions of pH 2.0 or 12.0, urea, high temperature (65°C, 60 min or 80°C, 30 min), mercaptoethanol and dithiothreitol were able to solubilize the zp but not the capsule at the concentrations used. Indirect immunofluorescence on cryostat sections and whole mounts of fresh or frozen-thawed material showed that 1) common antigens are shared by equine, porcine and bovine zp; 2) day 7 to day 15.5 capsule reacted with anti-capsule-serum but not with anti-zp-serum except for a few patches on the surface of the capsule; 3) anti-capsule-serum, but not anti-zp-serum, reacted with the capsular material recovered along with a broken day 27.5 conceptus; 4) anti-capsule-serum does not react with rabbit blastocyst coverings; 5) anti-capsule antibodies can be absorbed from the anti-capsule-serum by uterine proteins from either pregnant or non-pregnant mares; and 6) the capsule does not contain mouse laminin-like material.     

Efficiency of superstimulatory protocol P-36 associated with the administration of eCG and LH in Nelore cows

Recent work with P-36 demonstrates that the replacement of the last two doses of Follicle-Stimulating Hormone (FSH) with equine chorionic gonadotropin (eCG) increases embryo yields. However, it is unclear if the positive effect of eCG is related to its FSH-like activity, LH-like activity, or both. This study aimed to verify the replacement of eCG with pLH on the last day of superstimulatory treatment. Twenty-five Nelore cows were allocated to four groups: P-36 (control), P-36/eCG, P-36/LH2, and P-36/LH4. All animals underwent four treatments in a crossover design. The control group cows were superstimulated with decreasing doses of porcine Follicle-Stimulating Hormone (pFSH, 133 mg, im). In the P-36/eCG, P-36/LH2, and P-36/LH4 groups, the last two doses of pFSH were replaced in the former group by two doses of eCG (200 IU each dose, im) and in the latter two groups by two doses of pLH (1 and 2 mg each dose, im), respectively. Donors received fixed-time artificial insemination 12 and 24 hours after pLH. Embryo flushing was performed on D16. Data were analyzed by ANOVA (Proc Mixed, SAS). There was a trend of decreasing ovulation rate when comparing groups LH2 and eCG (P = 0.06). However, there was no significant difference in the mean number of viable embryos among groups P-36 (3.3 ± 0.7), P-36/eCG (4.5 ± 0.5), P-36/LH2 (3.7 ± 0.8), and P-36/LH4 (4.2 ± 1.0). It is concluded that the replacement of eCG by pLH on the last day of superstimulatory treatment can be performed with no significant variation in the production of viable embryos.     

Unwrapping the hydrolytic system of the phytopathogenic fungus Phoma exigua by secretome analysis

The present work describes the secretome profiling of a phytopathogenic fungus, Phoma exigua by liquid chromatography coupled tandem mass spectrometry (LC–MS/MS) based proteomics approach to highlight the suites of enzymes responsible for biomass hydrolysis. Mass spectrometry identified 33 proteins in the Phoma secretome when grown on α-cellulose as the sole carbon source. The functional classification revealed a unique extracellular enzyme system mainly belonging to the family of glycosyl hydrolase proteins (52%). This hydrolytic system consisted of cellulases (endo-1,4-β-glucanase, cellobiohydrolase I, exoglucanase, and β-glucosidase), hemicellulases (1,4-β-xylosidase and endo-1,4-β-xylanase) and other hypothetical proteins including GH3, GH5, GH6, GH7, GH11, GH20, GH32 and GH54. The synergistic action of this enzyme cocktail was assessed by the saccharification of alkali treated wheat straw. Since the Phoma secretome has limited β-glucosidase activity, it was supplemented with commercial β-glucosidase. After supplementation, this enzyme complex resulted in high yields of glucose (177.2 ± 1.0 mg/gds), xylose (209.2 ± 1.5 mg/gds) and arabinose (25.2 ± 0.3 mg/gds). The secretome analysis and biomass hydrolysis by P. exigua revealed its unique potential as a source of hydrolytic enzymes for lignocellulosic biomass hydrolysis.     

The secretome of Pichia pastoris in fed-batch cultivations is largely independent of the carbon source but changes quantitatively over cultivation time

The quantitative changes of the secretome of recombinant Pichia pastoris (Komagataella phaffii) CBS7435 over the time-course of methanol- or glucose-limited fed-batch cultures were investigated by LC-ESI-MS/MS to define the carbon source-specific secretomes under controlled bioreactor conditions. In both set-ups, no indication for elevated cell lysis was found. The quantitative data revealed that intact and viable P. pastoris cells secrete only a low number of endogenous proteins (in total 51), even during high cell density cultivation. Interestingly, no marked differences in the functional composition of the P. pastoris secretome between methanol- and glucose-grown cultures were observed with only few proteins being specifically affected by the carbon source. The ‘core secretome’ of 22 proteins present in all analysed carbon sources (glycerol, glucose and methanol) consists mainly of cell wall proteins. The quantitative analysis additionally revealed that most secretome proteins were already present after the batch phase, and depletion rather than accumulation occurred during the fed-batch processes. Among the changes over cultivation time, the depletion of both the extracellularly detected chaperones and the only two identified proteases (Pep4 and Yps1-1) during the methanol- or glucose-feed phase appear as most prominent.     

Permeability of the equine embryonic capsule to ethylene glycol and glycerol in vitro

Poor survival of cryopreservation by equine expanded blastocysts may involve low penetration of the embryonic capsule by cryoprotective agents (CPAs). This study characterized the permeation and accumulation rates of the CPAs ethylene glycol (EG) and glycerol (GLY) across isolated capsule in vitro, using a dual-chambered Valia-Chien permeation apparatus. Pieces of Days 14 to 18 ±1 capsules separated media in the “donor” chamber containing either 1.5 M EG (n = 6), 0.74 M EG (n = 5), 0.87 M GLY (n = 7), or 0.15 M NaCl (saline, SAL) (n = 6), from the “recipient” chamber. Concentrations of CPA, determined by gas chromatography, allowed calculation of the capsule's apparent permeability (P_app) to those CPAs. Permeation of capsule by 1.5 M EG was significantly more rapid than by 0.87 M GLY, or 0.74 M EG; permeation by both CPAs was significantly slower than by SAL. Accumulation of CPA in the recipient chamber depended more on initial donor chamber concentration, rather than type, of CPA. Accumulation rates for CPAs and SAL were linear only when capsule was present, demonstrating that their permeation through capsule was more complex than simple diffusion. Successful cryopreservation of equine expanded blastocysts has been previously linked to lengths of step-wise exposures to CPAs. Based on the present results, we inferred that alternative CPAs, more capable of permeating the capsule, or alternative methods of ensuring CPA entry into the cells, may also be required.     

Bottom‐up proteome analysis of E. coli using capillary zone electrophoresis‐tandem mass spectrometry with an electrokinetic sheath‐flow electrospray interface

The Escherichia coli proteome was digested with trypsin and fractionated using SPE on a C18 SPE column. Seven fractions were collected and analyzed by CZE‐ESI‐MS/MS. The separation was performed in a 60‐cm‐long linear polyacrylamide‐coated capillary with a 0.1% v/v formic acid separation buffer. An electrokinetic sheath‐flow electrospray interface was used to couple the separation capillary with an Orbitrap‐Velos operating in higher‐energy collisional dissociation mode. Each CZE‐ESI‐MS/MS run lasted 50 min and total MS time was 350 min. A total of 23 706 peptide spectra matches, 4902 peptide IDs, and 871 protein group IDs were generated using MASCOT with false discovery rate less than 1% on the peptide level. The total mass spectrometer analysis time was less than 6 h, the sample identification rate (145 proteins/h) was more than two times higher than previous studies of the E. coli proteome, and the amount of sample consumed (<1 μg) was roughly fourfold less than previous studies. These results demonstrate that CZE is a useful tool for the bottom‐up analysis of prokaryote proteomes.     

Recombinant human follicle-stimulating hormone alters maternal ovarian hormone concentrations and the uterus and perturbs fetal development in mice

Gonadotropins are routinely administered to produce multiple oocytes for clinical in vitro fertilization (IVF) treatment, laboratory research, and livestock industries. Studies in mice have shown gonadotropin stimulation using equine chorionic gonadotropin (eCG) affects the endometrium, implantation, and fetal development. Evidence from clinical studies also indicates that stimulation with recombinant human follicle-stimulating hormone (rhFSH) may be detrimental to the endometrium and implantation rates. We investigated the effect of rhFSH in mice on maternal plasma hormone concentrations and uterine gene and protein expression and the effect of a stimulated maternal environment on pregnancy. Adult females were stimulated with rhFSH or eCG, followed by human chorionic gonadotropin (hCG). On day 4 of pseudopregnancy, mice either had embryos transferred to the uterus or were killed, and blood and uterine samples were collected. Pregnancy outcomes were examined on day 15. Gonadotropin stimulation increased plasma progesterone concentrations on day 4 compared with controls, whereas estradiol concentrations were unaffected. Stimulation also reduced uterine leukemia inhibitory factor (Lif) mRNA, but the expression of estrogen and progesterone receptors (Esr1 and Pgr), homeobox gene Hoxa10, and Vegf mRNA were unchanged. Furthermore, distribution of uterine PGR protein expression was altered by stimulation, but LIF protein was unchanged. Stimulated embryo transfer recipients had lower pregnancy rates than controls, and fetuses from the rhFSH group had reduced weight, length, and maturity. These results demonstrate that gonadotropin stimulation with rhFSH or eCG alters the preimplantation maternal environment, which results in reduced pregnancy rates and fetal development in the mouse.