The likely functions of uterine secretions, often termed histotroph, in the nurture of the early conceptus are reviewed. Particular emphasis has been placed on the pig in which the uterus synthesizes and secretes large amounts of protein in response to progesterone. In this species, which possesses a non-invasive, diffuse type of epitheliochorial placentation, the secretions provide a sustained embryotrophic environment which is distinct from that of serum. A group of basic proteins dominates these uterine secretions after Day 11 of pregnancy and its best characterized member is uteroferrin, an iron-containing acid phosphatase with a deep purple colour. Evidence has accumulated to suggest that uteroferrin, rather than functioning as an acid phosphatase, is involved in transporting iron to the conceptus. Three basic polypeptides which are found noncovalently associated with uteroferrin have been shown to be antigenically closely related to one another and to have arisen by post-translational processing from a common precursor molecule. Their function is unknown. A group of basic protease inhibitors has been identified which bear considerable sequence homology to bovine pancreatic trypsin inhibitor (aprotinin) and may control intrauterine proteolytic events initiated by the conceptuses. The last basic protein so far characterized is lysozyme which is presumed to have an antibacterial role. Finally, two low molecular weight (M r ∼ 18 000) acidic polypeptides have been purified and have sequence homology to a plasma retinol binding protein. Like uteroferrin, these proteins may be responsible for transport of an essential nutrient to the conceptus.
R. M. Roberts and F. W. Bazer
S. M. M. Basha, F. W. Bazer, and R. M. Roberts
Summary. Endometrial explants were removed from uteri of animals pregnant and pseudopregnant (5 mg oestradiol valerate on Days 11–15) for 60 days, from the gravid and non-gravid horns of unilaterally pregnant pigs at Day 60 of pregnancy and from non-pregnant animals at Day 12 of the oestrous cycle. The tissues were cultured in the presence of l-[35S]methionine for 24 h, and the tissues and medium were then analysed separately by two-dimensional polyacrylamide gel electrophoresis. Radioactive polypeptides were identified by autoradiography of dried gels. Tissues from all except the cyclic animals released an identical group of polypeptides into the culture medium: the major radioactive products were 4 acidic polypeptides of low molecular weight and several basic proteins, which included the purple phosphatase uteroferrin, and lysozyme. In separate experiments explants from 3 unilaterally pregnant pigs were cultured with l-[3H]leucine, and, on a fresh weight basis, the tissue from the non-gravid horn released significantly less radioactive macromolecular material into the medium in 24 h than did tissue from gravid horns. It therefore appears that although the nature of the secretion produced by the pregnant uterus is a consequence of maternal hormonal regulation alone, the tissue underlying a conceptus is quantitatively more active than that from unoccupied regions of a uterus.
M. T. Zavyt, R. M. Roberts, and F. W. Bazer
Summary. The activities of uteroferrin, measured as acid phosphatase (AP), and an aminoacylpeptidase (AA) were measured in uterine flushings collected from gilts on Days 6, 8, 10, 12, 14, 15, 16 and 18 of the oestrous cycle and pregnancy (N = 37). Changes in AP (P < 0·05) were associated with day for both specific and total AP in non-pregnant and pregnant gilts. For pregnant and non-pregnant gilts, AP activity was greatest between Days 14 and 16 and then decreased to Day 18.
The AA specific activity increased (P < 0·01) between Days 10 and 12 of the oestrous cycle and pregnancy, but neither effects of pregnancy nor day by pregnancy status interaction were detected. The AA total activity was greater for pregnant gilts (P < 0·01). These data suggest an inhibitory effect of oestrogens of blastocyst origin on synthesis and/or secretion of uteroferrin, but not AA.
P. J. Hansen, F. W. Bazer, and R. M. Roberts
Summary. In one experiment, ovariectomized gilts were treated with corn oil (vehicle), progesterone, oestradiol-17β or both steroids. While oestradiol treatment did not stimulate enzyme activity in uterine flushings relative to vehicle-treated animals, gilts treated with progesterone had elevated amounts of all enzymes measured. Progesterone was less effective when co-administered with oestradiol-17β. Enzymes were not equally stimulated by progesterone. For example, there was a 909-fold increase in acid phosphatase activity in uterine flushings and a 304-fold increase in β-N-acetylglucosaminidase, but only a 10-fold increase in β-glucosidase. Endometrial explants from gilts synthesized and secreted radiolabelled β-N-acetylglucosaminidase, suggesting that at least some lysosomal enzymes enter the uterus through secretory processes. In other experiments, changes in β-N-acetylglucosaminidase in uterine fluids of mares and ewes treated with hormonal regimens similar to those given to the gilts were evaluated. Treatment with the combination of progesterone and oestrogen stimulated accumulation of the enzyme relative to that in vehicle-treated animals. The biochemical properties of porcine β-N-acetylglucosaminidase were examined in detail. Properties of the uterine enzyme were similar to reported values for lysosomal hexosaminidase. These included molecular weight (82 000–89 000), pH optimum (pH 4·4), presence of two isomers (isoelectric points of 5·5 and 8·0) and ability to hydrolyse substrates for glucosaminidase and galactosaminidase. We conclude that steroids induce the accumulation of lysosomal enzymes in the uterine lumen. The degree of stimulation differed between enzymes, suggesting that those enzymes stimulated to the greatest extent may play an important role in pregnancy.
K. L. Adams, F. W. Bazer, and R. M. Roberts
Summary. A retinol binding protein(s) of molecular weight about 17 000 has been demonstrated in uterine secretions from pigs in the luteal phase of the oestrous cycle. This protein was induced in ovariectomized sows treated with progesterone or progesterone plus oestradiol, but not in sows given oestradiol or corn oil. The vitamin A content of secretions from progesterone-treated animals also increased relative to those in controls. The apparent K d of the binding protein for retinol was 2·6 × 10−6 m. The protein had some affinity for retinoic acid and oleic acid, but did not bind retinyl esters or retinal. The protein probably comprises 5% or less of the total fraction of low molecular weight proteins induced by progesterone. A similar protein was found in allantoic fluid of pregnant animals, suggesting that, like uteroferrin, it serves to transport a water-insoluble nutrient from the maternal uterine endometrium to the conceptus.
H. Francis, D. H. Keisler, and R. M. Roberts
Summary. We examined the effect of recombinant bovine interferon-αI 1 (rboIFN-αI1) or recombinant bovine trophoblast protein-1 (rbTP-1) on protein synthesis by endometrial explants from Day-13 cyclic ewes and studied the ability of rboIFN-αI1 injected i.m. to influence subsequent protein scretion by endometrial tissue explants.
In Expt 1, ewes were injected with either 2 mg rboIFN-αI1 or vehicle alone at 12-h intervals beginning on Day 11 of the oestrous cycle and ending on the morning of Day 13; 8 h after the last injection, ewes were hysterectomized and endometrial explant cultures were prepared. Explants were cultured for 24 h in leucine-deficient medium supplemented with 250 μCi l-[3H]leucine per culture. For Expt 2, additional explants were prepared from Expt 1 controls. Explants were cultured in the presence of 0, 20 or 200 ng/ml of either rboIFN-αI1 or rbTP-1 for 24 h in leucine-deficient medium supplemented with 250 μCi l-[3H]leucine per culture. Secreted proteins were analysed by two-dimensional electrophoresis and fluorography. There was a marked enhancement of a 70 kDa acidic protein, p70, in explants cultured in the presence of rboIFN-αI1 or rbTP-1. This polypeptide is a product of the gravid uterine horn from Day 14 to Day 20 of pregnancy and is a useful marker of the action of interferon-α (IFN-α) on endometrium. Enhanced production of p70 also occurred in ewes injected i.m. with rboIFN-αI1. These results are consistent with the hypothesis that injected rboIFN-αI1 exerts its effects on such variables as interoestrus interval and maternal recognition of pregnancy by actions on the uterus.
Keywords: cattle; interferon-α; pregnancy; protein; sheep; trophoblast
J. D. Godkin, F. W. Bazer, and R. M. Roberts
Summary. Primary cell cultures were established from cells derived from dissociated Day-14 and -16 sheep and pig blastocysts. The appearance of cells in culture from both species was similar. Cultures contained a variety of cells with distinct morphologies, some were small and compact and formed clumps and multiple layers while others were large, flat and formed a monolayer. Within 4 h of culturing small floating fluid-filled spheres of cells were observed in the medium; some of these increased in size to > 1 cm diameter over 1–2 weeks. In addition, fluid-filled domes of cells arose from the underlying monolayer. Contractile cells became evident after about 8 days and some became organized into large patches of contracting tissue. Two-dimensional polyacrylamide gel electrophoresis and fluorography were performed on proteins released into the medium by confluent monolayers, floating spheres and floating cells that failed to attach during the first 24 h. All cultures produced as major products proteins with electrophoretic mobilities identical to certain fetal plasma proteins. In general, cultures did not produce proteins characteristic of short-term cultures of whole conceptuses harvested at Days 14–16. In cultures established from sheep blastocysts only the cells that failed to attach produced ovine trophoblast protein-1, a major ploypeptide produced by the trophectoderm of the sheep conceptus between Days 13 and 21 of pregnancy.
G. P. Robert, J. M. Parker, and S. R. Henderson
Summary. The proteins in saline washings from uteri of women at different stages of the menstrual cycle were examined by polyacrylamide gel electrophoresis at pH 4·5 and pH 8·9, isoelectric focusing in polyacrylamide, and immunoelectrophoresis. The components present were mainly serum proteins but small amounts of uterine– specific proteins were also detected.
Measurement of the activities of several glycosidases in uterine washings revealed that α-l-fucosidase, β-N-acetylgalactosaminidase and β-N-acetylglucosaminidase were elevated when compared with serum.
H. M. Kubisch, J. J. Hernandez-Ledezma, M. A. Larson, J. D. Sikes, and R. M. Roberts
Bovine zygotes produced by in vitro maturation–in vitro fertilization (IVM–IVF) were examined for their potential to serve as recipients of transgenes. Pronuclei, which were maximally visible at about 22 h after IVF, were injected with a SV40–LacZ construct (pSVON). Injected zygotes had lower cleavage rates (49.1%, n = 1162, P < 0.01) than did either noninjected controls (87.4%, n = 1420) or noninjected zygotes in which pronuclei were not visible (67.6%, n = 803). Zygotes that were injected into their pronuclei cleaved as well as zygotes injected cytoplasmically. At 48 h after injection, when most embryos had reached the four- and eight-cell stages, more zygotes in the pronuclear group (22.7%, n = 125) stained positively for LacZ than did zygotes in the cytoplasmic group (8.0%, n = 125). A group of zygotes injected into the pronucleus with pSVON was cultured for 9 days. More morulae (10.8%, n = 134) than blastocysts (3.2%, n = 31) expressed the LacZ gene, indicating that silencing of expression occurred as development progressed. Another group of zygotes was injected with a β-actin–LacZ gene construct (pbActinLacZ) and, of the embryos assayed at 48 h, 10.6% (n = 255) stained positively. At 9 days, 36.3% of morulae (n = 91) and 21% of blastocysts (n = 33) expressed the transgene. Almost all putative transgenic embryos injected with either construct showed a mosaic pattern of LacZ expression, with an average of only 2–3 cells staining at the eight-cell stage and the majority of cells in positive blastocysts showing no evidence of expression.
M. T. Zavy, D. C. Sharp, F. W. Bazer, A. Fazleabas, F. Sessions, and R. M. Roberts
Summary. Uterine secretions were obtained on Days 4, 8, 12, 14, 16, 18 and 20 of the oestrous cycle and early pregnancy. Acid phosphatase activity was significantly affected by day of the cycle, reaching a maximum at Days 12–14 during the luteal phase and then declining to almost undetectable levels by Day 20. In pregnant animals, activity continued to increase beyond Day 14. Two-dimensional polyacrylamide gel electrophoresis showed that albumin was a major component. However, a number of unique proteins of non-serum origin appeared in mid-cycle but had disappeared by Day 20. One of these was a basic protein indistinguishable in electrophoretic properties from the uterine acid phosphatase of the pig, uteroferrin, which is believed to be involved in iron transport from the uterine endometrial epithelium to the conceptus. These same polypeptides, including the putative uteroferrin, were also present in uterine flushings from pregnant animals until Day 20, and in flushings from ovariectomized mares treated with progesterone but not in those given only oestradiol-17β. Flushings from all ovariectomized animals contained a non-serum, acidic polypeptide (pI 5·3) of molecular weight 70 000. One basic polypeptide (molecular weight ~17 000) appeared by Day 4 of the oestrous cycle and disappeared by Day 16 but was maintained during pregnancy until Day 20. It was absent, however, in flushings from a Day 45 pseudopregnant mare. Like the sow, therefore, the mare possesses a number of proteins associated with cyclic changes in steroid hormones during the oestrous cycle and early pregnancy.