Localization of oviductal glycoproteins within the zona pellucida and perivitelline space of ovulated ova and early embryos in baboons (Papio anubis)

The estrogen-dominated baboon oviductal epithelium synthesizes and secretes a family of oviduct-specific glycoproteins. The objective of this study was to determine if these glycoproteins become associated with ova and early embryos. Ovarian and oviductal eggs obtained from superovulated baboons 72 h post-hCG were subjected to an indirect immunofluorescent assay that used a polyclonal antibody prepared toward the baboon oviduct-specific glycoproteins. Oviductal ova as well as 2-cell and 4-cell embryos showed intense, specific fluorescence within their zonae pellucidae. Ovarian ova did not exhibit fluorescence. Oviductal eggs were also fixed and processed for peroxidase-antiperoxidase immunocytochemistry and colloidal gold immunoelectron microscopy to confirm the immunofluorescent data and to determine the subcellular distribution of the antigens. Oviductal ova as well as 2-cell and 3-cell embryos exhibited immunolabeling localized within the zona. Gold particles were distributed uniformly throughout the width of the zona. Occasional groupings of gold particles were observed within the zona. Also, in most eggs, immunoreactivity was observed associated with flocculent material in the perivitelline space as well as the vitelline membrane. Furthermore, immunogold labeling above background level was noted in the cytoplasm of the eggs, particularly in the blastomeres of 3-cell embryos. Collectively, these results indicate that baboon estrogen-dependent oviductal secretory glycoproteins become intimately associated with oviductal ova and with embryos.     

Gene expression of bovine embryos developing at the air-liquid interface on oviductal epithelial cells (ALI-BOEC)

We recently developed an air-liquid interface long-term culture of differentiated bovine oviductal epithelial cells (ALI-BOEC). This ex vivo oviduct epithelium is capable of supporting embryo development in co-culture up to the blastocyst stage without addition of embryo culture medium. However, blastocyst rates in co-culture were markedly lower than in conventional in vitro embryo production procedures. In the present study, we assessed target gene expression of ALI-BOEC derived embryos to test their similarity to embryos from conventional in vitro embryo culture. We screened previously published data from developing bovine embryos and selected 41 genes which are either differentially expressed during embryo development, or reflect differences between various in vitro culture conditions or in vitro and in vivo embryos. Target gene expression was measured in 8-cell embryos and blastocysts using a 48.48 Dynamic Array? on a Biomark HD instrument. For comparison with the ALI-BOEC system, we generated embryos by two different standard IVP protocols. The culture conditions lead to differential gene expression in both 8-cell embryos and blastocysts. Across the expression of all target genes the embryos developing on ALI-BOEC did not depart from conventional IVP embryos. These first results prove that gene expression in ALI-BOEC embryos is not largely aberrant. However, there was no clear indication for a more in vivo-like target gene expression of these embryos. This calls for further optimization of the ALI-BOEC system to increase its efficiency both quantitatively and qualitatively.     

Temporal effect of human oviductal cell and its derived embryotrophic factors on mouse embryo development.

Mouse embryos at different stages of development were cocultured with human oviduct cells or cultured in the presence of oviduct-derived embryotrophic factor-1, -2, and -3 (ETF-1, -2, and -3) for various amounts of time within the preimplantation period. Cocultures that included the period from 48 to 72 h post-hCG stimulated cell division and increased the cell numbers in the inner cell mass (ICM) of the exposed blastocyst. Exposure of embryos to oviductal cells from 96 to 120 h post-hCG increased the cell number in the trophectoderm (TE), blastocyst size, hatching rate, attachment, and in vitro spreading of the blastocyst. ETF-1 and ETF-2 affected embryos between 48 and 72 h post-hCG by increasing the number of cells in the ICM. In contrast, ETF-3 had a more profound effect on embryos that were exposed from 96 to 120 h post-hCG, where it mostly affected the development of TE cells, leading to higher hatching rate. Human oviductal cells improved mouse embryo development partly by the production of high molecular weight embryotrophic factors. These factors had differential effects on mouse embryo development.     

Presence of follicular fluid in the porcine oviduct and its contribution to the acrosome reaction

Two experiments were conducted to measure the quantity of follicular fluid entering the porcine oviduct following ovulation and to establish its influence on the sperm acrosome reaction in vivo. Prepubertal gilts treated with pregnant mare serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG) were used in both experiments. In experiment 1, each of 64 gilts was assigned at random to one of four treatment groups (n = 16 per group): I (preovulatory), surgery 38 hr post-hCG; II (ovulatory), (surgery 42 hr post-hCG; III (postovulatory), surgery 46 hr post-hCG; IV (ovulation blocked), surgery 46 hr post-hCG but also treated with indomethacin (INDO) at 24 hr. At surgery, both follicular and oviductal fluid were collected for determination of volume and progesterone (P4) concentration. In experiment 2, sperm were recovered surgically from the uterine horn, isthmus, and ampulla of gilts at 46 hr post-hCG either 1) inseminated and non-INDO-treated controls (n = 5) or 2) inseminated and INDO-treated at 24 hr (n = 4). Using P4 as a marker, it was calculated that only 0.51% +/- 0.10% of the available follicular fluid was present in the oviduct near the time of ovulation and that this amount had decreased 10-12-fold 4 hr later. Mean sperm concentration at 46 hr post-hCG was higher in the uterine horn than in the other two regions (P less than 0.05) but the percentage of acrosome-reacted sperm was greater in the ampulla (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Trophectoderm-specific expression of the X-linked Bex1/Rex3 gene in preimplantation stage mouse embryos

The Bex1/Rex3 gene was recently identified as an X-linked gene that is differentially expressed between parthenogenetic and normal fertilized, preimplantation stage mouse embryos. The Bex1/Rex3 gene appears to be expressed preferentially from the maternal X chromosome in blastocysts, but from either X chromosome in later stage embryonic tissues and adult tissues. To investigate whether differential expression of the Bex1/Rex3 gene between normal and parthenogenetic blastocyst stage embryos reflects genomic imprinting at the Bex1/Rex3 locus itself, or instead is the result of preferential inactivation of the paternal X chromosome or differences in timing of cellular differentiation, we examined in detail the expression pattern of the Bex1/Rex3 mRNA in normal preimplantation stage embryos, and compared its expression between androgenetic, gynogenetic, and normal fertilized embryos. Expression data reveal that the Bex1/Rex3 gene is initially transcribed at the 2-cell stage, transiently induced at the 8-cell stage, and then increases in expression again at the blastocyst stage. Very little expression is observed in isolated inner cell masses, indicating selective expression in the trophectoderm. Comparisons of Bex1/Rex3 mRNA expression between male and female androgenetic and control embryos and gynogenetic embros failed to reveal any significant difference in expression between the different classes of embryos at the 8-cell stage, or the expanding blastocyst stage (121 hr post-hCG). At the late blastocyst stage (141 hr post-hCG), expression was significantly lower in XY control embryos as compared with XX controls. Bex1/Rex3 mRNA expression did not differ between XX and XY androgenones at the blastocyst stage or between gynogenones and XX control embryos. Thus, the Bex1/Rex3 gene does not appear to be regulated directly by genomic imprinting during the preimplantation period, just as it is not regulated by imprinting at later stages. Apparent differences in gene expression may arise through the effects of trophectoderm-specific expression coupled with differences in timing of trophectoderm differentiation between the different classes of embryos and effects of preferential paternal X chromosome inactivation (XCI).     

Analysis of gene expression in mouse 2-cell embryos using fluorescein differential display: comparison of culture environments

The effect of the oviductal environment on gene expression in 2-cell mouse embryos was examined with mRNA differential display. Embryos used for experiments were cultured in modified Whitten medium with or without oviductal tissue until late 2-cell stage. The results of sequencing indicated that the genes for ATP synthase (ATPase 6), S:-adenosylmethionine decarboxylase (S:-AMDC) and nuclear autoantigenic sperm protein (NASP) were differentially expressed in embryos cultured in the oviductal environment (nonblocking culture condition). The ATPase 6 gene is encoded by mitochondrial DNA and is essential for the production of ATP. This indicates that the expression of ATP synthesis-related genes at the 2-cell stage may be required to maintain normal development in vitro. S:-Adenosylmethionine decarboxylase decarboxylates adenosylmethionine, which is a substrate of DNA methylation. The expression of S:-AMDC may be responsible for the low level of methylation of preimplantation development. As NASP is a histone-binding protein that is thought to be testis and sperm specific, its function in embryos remains unclear. On the other hand, the Tcl1 gene and a novel gene, the c-1 gene, were strongly expressed in embryos cultured without oviductal tissue (blocking culture condition). The expression patterns of these genes are quite similar. However, the detailed functions of these genes in embryos remain to be determined.     

Annealing control primer system identifies differentially expressed genes in blastocyst-stage porcine parthenotes

There is very little information available on stage-specific gene expression during early embryo development, particularly in the pig. Here, we accurately identified the genes that are specifically or prominently expressed in parthenogenetic porcine blastocysts as compared with 2-cell stage embryos. We accomplished this by using a PCR technology regulated by annealing control primers (ACPs). By utilizing 120 ACPs, a total of 46 expressed sequence tags (ESTs) of genes that are differentially expressed in blastocysts as compared with 2-cell stage embryos were cloned and sequenced. The cloned genes or ESTs all exhibited significant sequence similarity with known genes or ESTs of other species. Of the known genes, six genes [renin-binding protein (RNBP), BMDP, solute carrier family 25 (SLC25A6), MTHFD1, TRK-fused gene (TFG), spermidine synthase (SRM)] were selected and their stage-specific expression levels in porcine parthenotes were determined by real-time quantitative polymerase chain reaction at the 1-, 2-, 4-cell, morula and blastocyst stages. While RNBP, BMDP, SLC25A6, MTGFD1 and SRM were highly expressed only at the blastocyst stage, TFG was highly expressed at the 1-cell stage, then declined after genomic activation, high levels of expression being again detected at the morula and blastocyst stages. This analysis suggests that the ACP system is an effective tool for use in the identification of stage-specific genes in small numbers of porcine parthenotes. Examination of the genes differentially expressed in the blastocyst, which we have identified here, will provide insight into the molecular basis of preimplantation development.     

Phospholipid transfer protein (PLTP) mRNA expression is stimulated by developing embryos in the oviduct

In mammal, fertilization and early preimplantation embryo development occurs in the oviduct. Evidence is accumulating that the oviductal epithelia secrete various biomolecules to the lumen during the secretory phase of the estrus cycle to enhance embryo development. This secretory activity of the oviduct is under the regulation of steroid hormones. Observations also suggested that the gametes and embryos modulate the physiology and gene-expressing pattern of the oviduct. However, the underlying molecular changes remain elusive. We hypothesize that the developing embryos interact with the surrounding environment and affect the gene expression patterns of the oviduct, thereby modulating the oviductal secretory activity conducive to the preimplantation embryo development. To test this hypothesis, suppression subtractive hybridization (SSH) was used to compare the gene expressions in mouse oviduct containing transferred in vitro cultured preimplantation embryos with that of oviduct containing oocytes during the preimplantation period. We reported here the identification and characterization of phospholipids transfer protein (PLTP), which is highly expressed in the embryo-containing oviduct and localized at the oviductal epithelium by in situ hybridization. PLTP contains signal peptide putative for secretory function. More importantly, PLTP mRNA increases in the oviductal epithelia of pregnant, but not pseudo-pregnant mice when assayed by real-time PCR. Taken together, our data suggested that PLTP may play important role(s) during in vivo preimplantation embryo development. This molecule would be a target to delineate the mechanisms and the roles of oviductal secretory proteins on early embryonic development.     

Oviductal cell proteome alterations during the reproductive cycle in pigs

The mammalian oviduct plays a crucial role in events leading to the establishment of pregnancy. During the reproductive cycle, the reproductive system undergoes various changes, including alterations in the number of different cell types in the oviductal epithelium and changes in the height of oviductal cells. Maintaining the unique oviductal environment required for the fertilization and early embryonic development comes with an energy cost to the organism. Therefore, it is hypothesized that structural and functional changes to the oviduct during the reproductive cycle represent vital preparations for the development of suitable environments for conception and embryo support. Here, we aimed to identify the changes in protein expression profile that occur during the follicular and luteal stages of the reproductive cycle in oviductal epithelial cells. The porcine oviductal epithelial cell proteomes from the follicular and luteal stages of the reproductive cycle were contrasted after separation by 2-D gel electrophoresis. Several oviductal epithelial cell proteins were up- or down-regulated during the reproductive cycle. We checked the quantitative changes of two of these molecules during different stages of the reproductive cycle using Western blot analysis. Finally, a number of these proteins were identified using tandem mass spectrometry. The results demonstrated distinctive differences in the proteomic profiles of the oviduct between follicular and luteal phases of the reproductive cycle.     

Regulation of IGF-I and porcine oviductal secretory protein (pOSP) secretion into the pig oviduct in the peri-ovulatory period,and effects of previous nutrition

The mechanisms regulating oviduct function were investigated. In Experiment 1, porcine oviductal secretory protein (pOSP) mRNA, and pOSP and insulin-like growth factor (IGF-I) in oviductal flushings, decreased through the peri-ovulatory period. In Experiment 2, higher plasma steroids in oviductal veins, ipsilateral (INT), rather than contralateral (OVX), to the remaining ovary in unilaterally ovariectomized gilts, were associated with higher pOSP in INT oviductal flushings. In Experiment 3, oviduct function was assessed as part of a collaborative study in cyclic gilts. Feed restriction in the late, compared to the early, luteal phase reduced estradiol concentrations in oviductal plasma, pOSP mRNA in oviductal tissue, and IGF-I concentrations and pOSP abundance in oviduct flushings. Previous insulin treatment differentially affected oviduct function. These data provide the first direct evidence for effects of previous feed restriction and insulin treatment on the oviduct environment in the peri-ovulatory period, which may contribute to nutritional effects on embryonic survival.     

LEFTY2 expression and localization in rat oviduct during early pregnancy

In mammals, fertilization and preimplantation embryo development occurs in the oviduct. Cross-talk between the developing embryos and the maternal reproductive tract has been described in such a way as to show that the embryos modulate the physiology and gene expression of the oviduct. Different studies have indicated that transforming growth factor beta (TGF-β) can modulate the oviductal microenvironment and act as an autocrine/paracrine factor on embryo development. LEFTY2, a novel member of the TGF-β superfamily is involved in the negative regulation of other cytokines in this family such as nodal, activin, BMPs, TGF-β1 and Vg1. In previous studies, we have reported that LEFTY2 is differentially expressed in the rat oviduct during pregnancy. In this study, we describe the temporal pattern of LEFTY2 in pregnant and non-pregnant rat oviduct by western blotting, which showed higher levels of LEFTY2 on day 4 of pregnancy, a time at which the embryos are ending their journey along the oviduct. The cellular location of LEFTY2 was assessed by immunohistochemistry, which showed immunolabelling in the cytoplasm and at the apical surface of the oviductal epithelial cells. The oviductal fluid also presented a 26 kDa band, which corresponds to the biologically active form of this protein, at the preimplantation period of pregnancy, indicating LEFTY2 secretion to the lumen. As LEFTY2 is expressed at a high level just before the embryos pass to the uterus, its biological effect might be relevant and significant for the preimplantation stage of embryo development in the oviduct. The fact that embryos do not express LEFTY2 at this stage of development supports this hypothesis.     

Effects of media NaCl concentration and osmolarity on the culture of early-stage porcine embryos and the viability of embryos cultured in a selected superior medium

One- and 2-cell porcine embryos were obtained from oviductal flushes and cultured for 96 hours in media with varied osmolarity that resulted from alterations in NaCl and sorbitol content. The viability of experimental embryos cultured to advanced stages was determined by comparison with that of the controls, noncultured embryos transferred to recipient gilts. The data suggest that variation in embryonic development in the experimental media is related to the NaCl concentration rather than to osmolarity. Increased NaCl concentration impairs development of the embryos to the advanced morula/blastocyst stages (P<0.001). There was no difference in the pregnancy rate between the recipients of cultured (45%) and noncultured (57%) embryos on Day 25. There was, however, a higher embryonic survival rate (P<0.05) within the control gilts.