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Pregnancy |
Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada N1G2W1
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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E1) by trophoblast varied with development; it favored E2 in older conceptuses, more in bilaminar than trilaminar tissues. Some more polar products were also noted, with loss of tritium seen as [3H]2O at SPE, and confirmed by HPLC in a second system with authentic reference steroids. Almost all radioactivity in the endometrium was present as E2 in both free and sulfate fractions. It was concluded that local metabolism of E2 is quantitatively significant and may play an important role in the actions of the large amounts of estradiol produced by the early equine conceptus.
conceptus, early pregnancy, endometrium, equine, estradiol, estrogen metabolism, placenta, pregnancy, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Steroidogenesis in the equine embryo appears to begin as early as Day 6 after ovulation, based on histochemical evidence of 3ß-hydroxysteroid dehydrogenase activity [1]. By Day 12, the equine conceptus produces significant quantities of estrogens [2–4]. This activity increases subsequently and is reflected in the concentrations of estrogens in yolk-sac fluid, especially after Day 20 [5]. A more transient production of estrogens occurs in the pig [8, 9] in accord with the limited duration of expression of cytochrome P450 aromatase (P450arom) activity in porcine blastocysts [10]. In contrast, estrogen synthesis by the conceptus of the mare is sustained [2, 5]. Estrogens serve as the antiluteolytic factor in the maternal recognition of pregnancy in the sow [11] but may not do so in the mare [12]. They may make an essential contribution to proper maternal/conceptus communication in the epitheliochorial placenta, which is characteristic of the mare [13]. A simple apposition of the trophoblast and the uterine epithelium from the time of implantation means that the demand for nutrients and oxygen must be met by diffusion across this interface, where efficiency may depend critically on permeability. Perhaps estrogens, acting through a vascular permeability factor (e.g., the vascular endothelial growth factor, VEGF) as agent [14, 15], provide a means to this end. One of the earliest reported actions of estrogens is the increase in uterine edema, resulting from increased vascular permeability in the mouse [16].
The present study was aimed at providing more complete information on the extent of local metabolism of estrogens by the early equine conceptus and endometrium and, as a second aim, to present more complete data on the amounts of E2 and E1 and their sulfoconjugated forms (E2S and E1S) in yolk-sac fluid. The stages chosen for examination of estrogen metabolism (Days 12, 15, and 18) were selected to represent the beginning and end of the early period of increase in diameter of the conceptus (Days 11– 16) and shortly after the time of fixation (Day 16), when the migration of the conceptus within the uterus comes to an end [17]. Such knowledge is deemed essential for subsequent studies on the role of estrogen production in the early stages of pregnancy and its relation to early pregnancy loss in the mare. Preliminary reports on some of our data have appeared as abstracts [18, 19].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nonradioactive steroids were purchased from Steraloids Inc. (Newport, RI). The radioactive steroids obtained from NEN Life Sciences Products Inc. (Boston, MA) were [2,4,6,7-3H]-estradiol-17ß (71.0 Ci/mmol) and [2,4,6,7-3H]-estrone (74.1 Ci/mmol). [4-14C]-E1 (53.4 mCi/mmol) was from the Radiochemical Centre, Amersham, U.K. Solvents from Caledon Laboratories, Ltd. (Georgetown, ON, Canada) were glass-distilled, reagent grade, and acetonitrile (190) was used for HPLC. All other chemicals were analytical grade from Fisher Scientific (Toronto, ON, Canada) unless otherwise indicated.
Collection of Conceptuses and Endometrial Tissue
Standardbred and Thoroughbred mares (n = 11 each season, for 3 yr) from the research herd at the University of Guelph were used to recover conceptuses under conditions approved by the University Animal Care Committee. The timing of ovulation was monitored by transrectal ultrasonography at intervals of 2, or occasionally 3, days and was deemed to have occurred on Day 0, midway between examinations showing an intact follicle and a corpus luteum. Thus, ages of conceptuses were subject to an error of ±1 or ±1.5 days. Conceptuses were collected from mares by transcervical uterine lavage, with Dulbecco PBS, pH 7.4, using a technique based on one described previously [20]. The mares were sedated with xylazine hydrochloride (Rompun; BAY-VET, Etobicoke, ON), administered i.v., usually 0.3 mg/kg body weight. The flush fluid (1000 ml) was infused by gravity flow and drained into a beaker to recover the conceptus. Endometrial biopsy samples were taken immediately thereafter. Yolk-sac fluid was obtained from most conceptuses by rupture in a dry Petri dish and aspiration into a syringe with a 20-G hypodermic needle. However, samples from conceptuses older than 20 days were taken by direct needle aspiration. Sixty-three conceptuses, ranging in age from 11 to 26 days, were used to provide yolk-sac fluids. The fluids were stored at –20°C until the time of assay. The amount from each conceptus was determined by measuring the volume of aspirated fluid wherever possible or, otherwise, calculated from ultrasonographic measurements taken immediately before uterine flushing. Eleven conceptuses were used to study estrogen metabolism at Days 12 (n = 5), 15 (n = 3), and 18 (n = 3).
Radioimmunoassay of Estrogens in Yolk-Sac Fluid
Steroids in the yolk-sac fluid were recovered by taking an aliquot (
1 ml), which was diluted with distilled water to a volume of 5 ml for solid-phase extraction (Waters C18 Sep-Pak column; Canada Waters Limited, Mississauga, ON), as described previously for equine plasma [21]. Unconjugated and conjugated steroids were eluted from the primed columns with 5 ml of diethyl ether and 5 ml of methanol, successively. The ether and methanol eluates were evaporated separately under nitrogen at <45°C and the dried ether residue (unconjugated fraction) was used to measure E2 and E1. The dried methanol (conjugate) fraction was acid solvolyzed overnight at 45°C with trifluoroacetic acid:ethyl acetate (1:100; v/v) to obtain free steroids from sulfoconjugated estrogens by means of a second Sep-Pak column. The dried residues were dissolved in methanol and aliquots taken for radioimmunoassays (RIA) of the estrogens, as reported previously [22].
Tissue Preparation and Incubations
After removal of yolk-sac fluid, the trophoblastic tissues were divided into 2–4 equal portions (about 15–20 mg wet weight) depending on amounts available. For Day 18, the trophoblast was divided into bilaminar (trophectoderm and endoderm) and trilaminar (trophectoderm, mesoderm, and endoderm) components by dissection, and these were incubated separately. Lesser amounts of tissue from the Day-12 conceptuses precluded incubations in duplicate and so additional conceptuses were used to provide sufficient replication. The tissues were washed with PBS and dispensed into small flasks containing 2.5 ml TCM 199 to which [3H]-E2, [3H]-E1 (1 x 106 cpm), or [14C]-E1 (0.25 x 106 cpm) was added for a 2-h incubation in a shaking waterbath at 37°C under 5% CO2 in air. Similarly, the biopsy samples of endometrium were incubated, in duplicate, as minced tissues (30–60 mg wet weight). As an inactivated tissue control in some experiments for Days 15 and 18, samples from trophoblast (n = 5) and endometrium (n = 2), in TCM 199, were placed in boiling water for at least 10 min before incubation with the radiolabeled estrogens.
Analytical Procedures
Steroids in the media were recovered by solid-phase extraction (SPE) as described above. Unconjugated and conjugated steroids were eluted with 5 ml diethyl ether and 5 ml methanol in succession. The conjugated material underwent two hydrolytic steps, in series, to yield a sulfate and a glucuronidate fraction obtained in each case as free steroids from Sep-Pak columns [23]. The amounts of radioactive material recovered from each incubation, each hydrolysis step, and in each Sep-Pak fraction were determined by liquid scintillation counting in 5 ml of Ecolite cocktail (ICN, Costa Mesa, CA). In addition, an indirect assessment of metabolism involving loss of the tritium-label at carbons 2, 4, 6, and/or 7 was made by collecting the initial (flow-through) and the following (water-wash) fraction from the SPE. These aqueous fractions were combined and, after treatment with charcoal and centrifugation as done in radioimmunoassay, the measurement of radioactivity in the supernatant was assumed to reflect the formation of [3H]2O from oxygenation at the tritium-labeled carbon sites of the estrogen molecule. Further confirmation was obtained from distillation of the supernatants from the charcoal treatment, for two incubations of bilaminar trophoblast from Day-18 conceptuses, with almost complete recovery of radioactivity in the distillate (data not shown).
Unconjugated and hydrolyzed steroids in the fractions derived serially from Sep-Pak columns were examined by HPLC. Profiles of steroid metabolic products were generated using a binary solvent gradient of acetonitrile:water (from 28:72 to 90:10, over 35 min) on a Waters HPLC column and system (Waters Corp, Milford, MA), at a flow rate of 0.7 ml/ min, and absorbance monitored at 280 nm. A radiodetector (Packard 505TR; Packard Instrument Co., Meriden, CT) was used for a direct scan of the effluent for radioactivity (cpm). The areas recorded (as percentage of total radioactivity) for detected peaks were taken for quantitative comparisons of the products formed. A second HPLC system, with methanol in the gradient, was used to further identify the material in some peaks of the chromatograms. The ternary gradient started with a methanol:acetonitrile:water mix of 46.8:4.2:49.0 that was changed to 70:15:15 over a 30-min period using Waters gradient curve #7, at a flow rate of 0.5 ml/min. Identification of the radiolabeled metabolites was then based on their coelution with authentic reference steroids from the two column matrices.
Statistical Analysis
Scatter plots were used to present the data for total amounts of E2, E1, and their sulfates in yolk-sac fluid. Log transformation of the data for estrogens was done, to accommodate the extreme ranges in values, before fitting regression lines using SigmaPlot5.05. Comparisons for the amounts of radioactivity seen as steroid conjugates from incubations were made by one-way ANOVA with Bonferroni multiple comparison post test performed using the Instat Version for Windows 95 (GraphPad Software, San Diego, CA). P values < 0.05 were considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Estrogens in Yolk-Sac Fluids
The amounts of estrogens (concentrations x volumes) found in yolk-sac fluid from conceptuses collected over the period of our steroid metabolism studies, and slightly beyond, are shown in Figure 2. Values for the total amounts of the estrogens ranged from 0.2 to 128.5, 0.1 to 76.6, 0.26 to 158.3, and 0.28 to 720.9 ng, respectively, for E2, E1, E2S, and E1S in conceptuses of 5–32-mm diameter (volumes = 0.07–17.15 ml). Estrone sulfate clearly became the dominant form as development progressed. The rates of increase for E2 and E1 production in the early stages were similar, but a plateau was reached earlier for E2 than for the other estrogens. This was reflected by the concentrations in the yolk-sac fluid (data not shown), where the daily increments in concentrations of Days 11–16 were similar for E2 and E1 (0.66 and 0.55 ng/ml, respectively) but differed markedly between those for E2S and E1S (0.31 and 2.63 ng/ml). Mean values (± SEM) for the concentrations (ng/ml) of endogenous substrates on the days of the metabolism studies (Days 12, 15, and 18; n = 11, 13, and 7, respectively) were 2.03 ± 0.45, 4.93 ± 0.63, and 4.89 ± 0.73 for E2; 2.59 ± 0.43, 4.66 ± 1.26, and 7.77 ± 1.41 for E1; 1.17 ± 0.23, 1.75 ± 0.21, and 3.69 ± 0.77 for E2S; 3.79 ± 0.69, 10.31 ± 1.75, and 29.92 ± 3.42 for E1S. These values include data from other conceptuses not used in the metabolism studies.
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Conjugation of Steroids
Conjugation of estrogens by the trophoblast was markedly different from that by endometrial samples (Table 1). The data showed an increase in conjugation after Day 12 for the trophoblast and formation of greater amounts by the bilaminar than by trilaminar tissues on Day 18. Levels of conjugation by the endometrium were higher than by the trophoblast, especially on Day 12, even allowing for the larger amounts of endometrium used. There seemed to be greater quantities formed from E1 than from E2 as substrate, but this was statistically significant only for Day 18. It was also noted that conjugation of E2 by the endometrium was significantly lower on Day 18 than on Day 12. Incubations of boiled tissues from conceptuses on Days 15 and 18 resulted in about 3% of the radioactivity appearing in the conjugate fraction for [3H]-E2 and [3H]-E1. No subtractions of these amounts were made for the data in Table 1.
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Within the conjugate fractions for the trophoblast, the predominant form in all cases was the sulfate from incubations with either E2 or E1, with values of about 60% for each (e.g., Day 18 bilaminar, 60.9 ± 3.6, n = 3, from E2; and 59.7 ± 2.2, n = 3, from E1). Using a bacterial ß-glucuronidase preparation [23], recovery of radioactivity from the remaining, nonsolvolyzed fraction (about 40%) was estimated for Day 15 (n = 2) and for Day 18 (bilaminar trophoblast only, n = 2), where sufficient amounts of radioactivity were available. The mean values for yields of glucuronidates for duplicate incubations with E2 and E1, respectively, were 28.1% and 24.7% for Day 15 and 29.4% and 25.6% for Day 18. No further examination of the remaining radioactivity (about 28% of total conjugated material) was made for other forms of conjugation. Only estrogen sulfates (>95%) were seen in media from endometrial incubations with both E2 and E1.
HPLC Profiles from Estrogen Metabolism
All profiles presented are examples from two or more replicates for each substrate in each age group. Each profile closely represented that of its replicate(s) (data not shown). Chromatography of unconjugated steroids showed extensive metabolism by trophoblast tissue of a Day-12 conceptus for each estrogen as substrate (Fig. 3). E2 was converted to E1 in large measure; conversely, E1 was metabolized to E2 to a lesser extent. Several minor peaks were seen as products from each substrate.
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At Day 15, the profiles from metabolism of E2 and E1 were remarkably similar to each other (Fig. 4). However, they showed a much lower formation of E1 from E2 than noted for trophoblast tissues on Day 12. On the other hand, E1 was converted mainly to E2. This resulted in a similar ratio for the distribution of radioactivity between E2 and E1 regardless of the estrogen used as substrate. It was also noted that two lesser peaks had similar retention times (Rt) to those of reference standards of 6
- (or 6ß-) OH-E2 and 6-oxo-E2 run separately (Fig. 3A). These peaks made up about 6–8% each of the total radioactivity in the profiles and were present in about the same proportions from either E2 or E1 as substrate.
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Incubation of trophoblast tissues from the Day-18 conceptuses resulted in markedly different metabolic profiles for the bilaminar and trilaminar components, with either E2 or E1 as substrate (Fig. 5). The amounts of E1 formed from E2 were 14% and 57% for the bilaminar versus trilaminar tissues, respectively. About 5% of the radioactivity was noted in each of the polar peaks corresponding to Rt of 6-OH-E2 and 6-oxo-E2, but only for the bilaminar tissues. Differences between the two tissue types were again expressed when E1 was the substrate. The resulting profiles for the metabolites of E1 were almost identical to those seen with E2 as substrate. It was concluded that E2 was the major estrogen resulting from metabolism in bilaminar tissue regardless of which substrate was used. A more even distribution between E2 and E1 occurred with the trilaminar material.
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Evidence for the formation of 6-oxygenated estrogens was strengthened by further chromatography of the metabolites of [3H]-E2 and [3H]-E1, from incubation of Day-18 bilaminar tissue, with authentic reference steroids as internal standards. In the first gradient system (acetonitrile/water), two peaks of radiolabeled material were found, which corresponded to those of the 6-OH-E2 and 6-oxo-E2 standard compounds (Table 2), but separation of 6-OH- from 6ß-OH-E2 was barely adequate (Rt = 3.2 and 3.6 min). In the second gradient system, with methanol, the two epimers were clearly separated (Rt = 3.9 and 6.6 min; Table 3).
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Because losses of [3H], detected as [3H2O] at the SPE step, were between 30% and 50% for Day-18 bilaminar tissue, the relative amounts of 6-oxygenated estrogens formed were not fully reflected at chromatography. A comparison of [4-14C]-E1 with [3H]-E1 as substrates was made in an additional experiment. The results of chromatography in the first system (HPLC-1) showed clearly that the proportion of radioactivity that appeared as polar metabolites (6-oxygenated estrogens) was three times higher for the [14C]-E1 incubation (Table 2), when total areas under the peaks were compared for retention times <10 min (139.0 versus 46.4 arbitrary units). With the second solvent system (HPLC-2), evidence for both 6- and 6ß-OH-E2 as well as for 6-oxo-E2 was obtained in the 14C-labeled material (Table 3). In fact, the data suggest that the 6ß-epimer is the major form resulting from 6-hydroxylation by the bilaminar trophoblast.
Profiles of the sulfoconjugated steroids from incubations of E2 with Day-18 endometrial tissues are shown in Figure 6. With >95% of the radioactivity in a conjugated form from these incubations, it was seen that almost all of the radioactivity was in the sulfate fraction and was present as E2S, with only trace amounts of E1S. This was also true for the lesser amounts of free steroids, where E2 was again predominant.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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secretion in the maternal recognition of pregnancy in the pig [11]. In this regard, it may be noted that the concentrations of prostaglandins E2 and F2 in the yolk-sac fluid in the horse remain constant [25] over the same period that the amounts of estrogens are seen to rise in our study. Nevertheless, it seems likely that estrogen secretion by the early equine conceptus is a requirement for the maintenance of pregnancy. For example, studies with exogenous steroids in anestrous mares suggest that conceptus-derived estrogens are likely to be the cause of the increased proliferative activity in some components of the endometrium during early pregnancy [26]. The high content of estrogens in the yolk-sac fluid we have reported lends strong support for this view. The actual amounts of E2 that are available to act on the conceptus and endometrium at this critical stage depend on several factors in addition to the production of E2 by the conceptus itself. Our findings indicate that 17ß-hydroxysteroid dehydrogenase (17ß-HSD) enzymes in the trophoblast make a significant contribution to the bioavailability of E2 to the tissues, as evidenced by marked changes in the extent to which E1 is converted to E2 over the period of rapid expansion of the blastocyst. How these changes favoring E2 formation in the trophoblast can be reconciled with the higher concentrations of E1S in yolk-sac fluid in the older conceptuses is unanswered, but might lie in a possible difference in 17ß-HSD activity toward the free and conjugate forms. It should be noted, however, that our experimental approach did not address the difficult question of metabolic clearance rates for estrogens in the conceptus. No clear explanation for differences in estrogen metabolism between the bilaminar and trilaminar tissues can be given at this point, but it may relate to differences in their cellular components.
Estrogen sulfates were the major form of conjugation found in the yolk-sac fluid and on incubation of the tissues, especially the endometrium. The biological significance of this estrogen sulfotransferase (EST) activity for early equine pregnancy has yet to be demonstrated, but it would have a bearing on the bioavailability of estrogen. Although we did not include incubations with radiolabeled estrogen sulfates to test for sulfatase activity, our findings strongly suggest that the dominant activity is EST, leading to relatively reduced amounts of free E2. Recently, a porcine uterine EST has been characterized [27]. Also, EST rather than sulfatase is the more widely expressed enzyme in various human peripheral tissues, including the syncytiotrophoblast of the placenta [28]. However, it remains to be demonstrated whether estrogen sulfates are active in their own right and not formed simply to protect tissues from possible excessive estrogenic stimulation or even to serve as a latent precursor for the active free steroid [29].
Some actions of estradiol are dependent not only on the local level of the estrogen in the target tissues and on the presence of its receptors but also, in part, on its metabolism within the target cells themselves [6]. These estrogen metabolites may have unique roles as local mediators and contribute to the overall action of estradiol. It is for this reason that we have examined the metabolism of estradiol in the early equine conceptus and endometrium. Evidence from the HPLC profiles and from losses as great as 30–50% of [3H] on incubation of radiolabeled E2 and E1 clearly shows that a significant amount of oxidative metabolism of the estrogens does occur in the trophoblast. As a consequence of the removal of tritium from E2 and E1, the radioscan for the HPLC profiles cannot fully reflect the amounts of some of the oxygenated metabolites formed. Use of [14C]-E1 as substrate allowed not only a better appreciation of the extent of the formation of these metabolic products but also helped toward establishing their identity; formation by the equine trophoblast of both 6-OH-E2 and 6ß-OH-E2 as well as 6-oxo-E2 can reasonably be assumed from our chromatographic findings. The presence of metabolites with oxygenated functions at position C6 (6-OH and 6-oxo-E2 and -E1) may have particular relevance insofar as they have been referred to as impeded estrogens, from their depression of estrogen-induced uterine growth [30]. To the best of our knowledge, no such studies have been reported for the early stages of pregnancy in the mare.
Estrogen biosynthesis by preattachment embryos, as seen in the mare and pig, has been documented for other species with epitheliochorial placentation [31, 32]. Furthermore, a recent report revealed by immunocytochemistry that steroid synthesis enzymes were present in the uninucleate trophoblast in the camelid placenta at Days 14 and 30 after ovulation [33]. This would allow the conversion of cholesterol to estrogen. It was suggested that the capacity for steroidogenesis close to the fetomaternal interface may maintain high concentrations of steroids at this position and indicate a need for localized placental estrogen for a successful camelid pregnancy. Immunocytochemical localization of the aromatase enzyme in the trophoblast of the early equine conceptus from Day 12 to Day 15 [34] could represent a similar requirement for local estrogen synthesis in the pregnant mare.
Estrogens might promote the establishment of pregnancy in the mare through the agency of various growth factors. A relationship between estrogens and growth factors in early pregnancy in the mare has yet to be explored except for the largely negative findings for IGF-1 [35]. Vascular endothelial growth factor/permeability factor, an endothelial cell mitogen and permeability factor, acts as an intermediary in a variety of estrogen-responsive tissues. It has been shown that E2-induced VEGF gene expression in human endometrial cells depends on both estrogen receptors, ER and ERß [36]. The rapidly developing vascularization of the mesoderm of the equine conceptus, illustrated in Figure 1, strongly suggests that VEGF may be required for this progressive modification of the yolk-sac wall and implies that estrogens could well play a role in this regard. With regard to permeability, the equine conceptus remains spherical, with linearly increasing diameter between Days 11 and 16 [17] despite the hypotonicity of its yolk-sac fluid [24]. For this reason, it may be important to look for factors that can influence permeability rather than, or in addition to, playing a mitogenic role in the epithelium. Such a factor might be found in the discovery of a tissue-specific growth factor that induced proliferation and fenestration in capillary endothelial cells of endocrine glands, the so-called endocrine-gland-derived vascular endothelial growth factor (EG-VEGF) [37, 38]. This permeability-enhancing factor is restricted mainly to steroidogenic tissues, including the placenta. Thus, EG-VEGF could serve a dual role as a permeability-enhancing factor and a mitogenic agent in the trophoblast, which functions in some respects as an endothelial tissue as well as producing estrogens. Moreover, the recent demonstration of estradiol regulation of aquaporin-2 in the mouse [39–41] may have relevance in the mare, especially given the demonstrated role of aquaporins in blastocyst expansion [41]. In all of this, our findings suggest a need to consider the role of local estrogen metabolism within the reproductive tissues when examining the possible biological actions of the large quantities of estrogens present in, and formed by, the conceptus at this early stage of pregnancy, which includes the critical events of conceptus expansion and fixation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: J.I. Raeside, Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1. FAX: 519 767 1450; jraeside{at}uoguelph.ca
Received: 20 February 2004.
First decision: 13 March 2004.
Accepted: 25 May 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5-3ß-hydroxysteroid dehydrogenase and steroid hormone content in early preimplantation horse embryos. Folia Histochim Cytobiol 1985 23:81-84 by the uterine endometrium. Prostaglandins 1977 14:397-400[CrossRef][Medline] production by equine conceptuses and concentrations in conceptus fluids and uterine flushings recovered from early pregnant and dioestrous mares. Reproduction 2002 123:261-268[Abstract]This article has been cited by other articles:
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  L.A. Silva, E.L. Gastal, M.A. Beg, and O.J. Ginther Changes in Vascular Perfusion of the Endometrium in Association with Changes in Location of the Embryonic Vesicle in Mares Biol Reprod, March 1, 2005; 72(3): 755 - 761. [Abstract] [Full Text] [PDF]
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