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Biology of Reproduction 62, 1141-1145 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Tissue-Specific Localization of Cytochrome P450 Aromatase in the Equine Embryo by In Situ Hybridization and Immunocytochemistry1

Karen W. Walters2,a,b, C. Jo Corbina, Gary B. Andersonb, Janet F. Roserb, and Alan J. Conleya

a Departments of Population Health and Reproduction, b School of Veterinary Medicine and Animal Science, University of California, Davis, California 95616


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogen production by the preimplantation equine embryo is presumed to be important in maternal-conceptus communication in the mare. The synthesis of C18 estrogens from C19 androgens requires cytochrome P450 aromatase (P450arom) in the conceptus, but little information is available on the specific tissue location or potential developmental patterns of expression for the horse. The goal of this research was to localize P450arom in the equine conceptus by immunocytochemistry and in situ hybridization. Intact blastocyst-stage embryos were collected by nonsurgical flush on Days 12–15 of pregnancy, fixed in 4% paraformaldehyde, and paraffin-embedded. Aromatase protein was localized using rabbit anti-human placental aromatase antiserum with a detection system utilizing peroxidase and 3-amino-9-ethylcarbazole. For in situ hybridization, tissue sections were incubated with sense or antisense [35S]UTP-labeled cRNA probes prepared from equine aromatase cDNA. Aromatase protein and transcript were abundant in the extraembryonic trophectoderm but absent from embryonic ectoderm. No P450arom expression was detected in abembryonic endoderm or mesoderm. Aromatase expression was demonstrated in the endoderm beneath the disc (hypoblast). This pattern of P450arom expression in the equine blastocyst closely resembles that seen transiently in the porcine embryo, suggesting that regulatory mechanisms conferring tissue specificity may be conserved.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During early gestation, the equine conceptus synthesizes and secretes considerable quantities of estrogens [1–3], as well as estrogen conjugates [4]. The exact role of this embryonic estrogen production is not known, but it is presumed to participate in the synchronized maternal-conceptus communication necessary for the successful establishment of pregnancy in the mare. One role of estrogen may be to stimulate the extensive conceptus mobility needed for continuous interaction with the endometrium during the preimplantation period [5] to prevent luteolysis and maintain a receptive uterine environment [6, 7]. Studies in the pig suggest that estrogen synthesis by the blastocyst around Days 10–12 of pregnancy [8] may control conceptus mobility, preattachment spacing [9], and uterine prostaglandin F2{alpha} secretion [10], and prepare the uterine environment for implantation and nutritional support of the conceptus [11]. Steroid production by the porcine embryo is tightly controlled by the transient expression of the steroidogenic enzymes cytochrome P450 aromatase (P450arom) and 17{alpha}-hydroxylase (P450C17) in the trophoblast between Days 10 and 12 [12, 13]. In contrast, estrogen synthesis by the equine embryo begins as early as Day 6 [14] and continues to increase with embryo age and diameter [1, 4].

The synthesis of C18 estrogens from C19 androgens requires the presence of P450arom within the conceptus, but little information is available on specific tissue location or developmental patterns of expression during early gestation in the mare. Steroidogenesis throughout the trophoblast is suggested by the results of early histochemical studies showing 3ß-hydroxysteroid dehydrogenase (3ß-HSD) in the equine conceptus from Days 6 to 10 [14] and at Day 13 [15] of gestation. Aromatase activity in extraembryonic tissues of conceptuses collected on Days 20–52 after ovulation has been measured in vitro by incorporation of [3H]androstenedione into unconjugated estrogens [16]. In these postimplantation conceptuses, P450arom activity was highest in the yolk sac, with less activity in the allantochorion. These observations suggest that P450arom is present, and active, in the trophoblast and/or endoderm in conceptuses older than Day 20, but additional work is needed to determine the specific tissues responsible for estrogen biosynthesis in the younger, blastocyst-stage embryo. The goal of this work was to determine the specific localization of P450arom protein and mRNA in the early equine conceptus from Day 12 to Day 15 of gestation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Embryo Collection

Normally cycling light horse mares were teased daily, and those in estrus were palpated per rectum each day to monitor ovarian activity. After detection of a 30-mm or larger follicle, mares were bred every other day until ovulation. Ultrasonography was used from Day 10 postovulation onward to determine pregnancy status and record embryonic size. Intact, blastocyst-stage embryos were collected by nonsurgical flush using 500 ml prewarmed (37°C) PBS as previously described [4]. Ten embryos were collected from six mares on Days 12 (n = 2), 13 (n = 4), 14 (n = 2), and 15 (n = 2) of pregnancy. All procedures on animals followed protocols approved by the University of California, Davis, Animal Care and Use Committee.

Tissue Preparation

Immediately after collection, embryos were fixed in freshly prepared 4% paraformaldehyde (pH 7.4; 4°C) for 1 h, dehydrated through a series of ethanols (30–70%), and paraffin-embedded by standard procedures. Serial sections of each embryo were cut to 5-µm thickness and mounted on poly-L-lysine-coated slides. Equine testes were collected from routine castrations performed at the Veterinary Medical Teaching Hospital, University of California, Davis, fixed in 4% paraformaldehyde, and paraffin-embedded.

Immunocytochemistry

Serial sections of embryos, inclusive of both embryonic disc and trophoblast, were stained for the presence of P450arom protein using rabbit anti-human placental aromatase antiserum (courtesy of Dr. N. Harada, Fujita Health University, Toyoake, Japan). Tissue sections were processed by standard protocol using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). Briefly, after deparaffinization and rehydration, tissues were rinsed in PBS containing 0.5% Triton X (PBS-X), blocked for 20 min at room temperature with 10% normal goat serum, and incubated with a 1:1000 dilution of anti-aromatase antiserum at 4°C for 18 h. Tissues were subsequently rinsed (PBS-X) and incubated with biotinylated goat anti-rabbit linking antibody (30 min) and then with an avidin-biotin-peroxidase complex (30 min). Immunostaining was visualized using a commercial peroxidase kit (Vectastain Elite AEC; Vector Laboratories). Tissues were developed for 10–30 min with 3-amino-9-ethylcarbazole (AEC) and counterstained using Mayer's hematoxylin. Normal rabbit preimmune serum (1:1000) was substituted for the primary antibody for negative controls. Testicular sections from a 4-yr-old stallion were used as a positive control tissue. All slides were mounted with Crystal Mount (Biomedia Corporation, Foster City, CA) or Dako Faramount (Dako Corporation, Carpinteria, CA). Sections were stained with hematoxylin and eosin Y by standard protocol for morphological assessment.

In Situ Hybridization

Equine-specific [35S]UTP-labeled sense and antisense cRNA probes were prepared from the 5' region of an equine aromatase cDNA (courtesy of Dr. J. Sirois [17]). A 650-base pair fragment was subcloned into pSport (Gibco BRL, Rockville, MD) and utilized for in vitro transcription following standard protocols. Linear template was prepared by restriction digestion of plasmid DNA at 37°C for 2 h with BamHI or PstI for use, respectively, with T7 or SP6 RNA polymerase (Pharmacia Biotech Inc., Piscataway, NJ). For in vitro transcription, 1 µg of linearized DNA template was incubated with 120 µCi [35S]UTP (DuPont NEN Research Products, Boston, MA), transcription buffer, 10 mM dithiothreitol (DTT), ribonuclease inhibitor (RNasin), and 500 µM rNTPs. After 2-h incubation at 37°C, deoxyribonuclease (DNase) I and tRNA were added for 15 min. Probes were purified over G50 spin columns (5 Prime->3 Prime, Boulder, CO).

Fresh sections of whole blastocysts were prepared and processed for in situ hybridization as previously described [18], with minor modifications. Tissue sections were deparaffinized with xylene, rehydrated through graded ethanols (95–30%) to PBS, incubated 15 min at 37°C in Proteinase K (1 µg/ml in 0.1 M Tris, 50 mM EDTA), postfixed for 10 min at 4°C in 4% paraformaldehyde, acetylated with 0.1 M triethanolamine (pH 8.0) for 10 min, and dehydrated through graded ethanols. Slides were allowed to air-dry for 1 h before sense or antisense probes were applied. Probes were diluted in hybridization buffer to 40 x 106 dpm/ml, denatured for 3 min at 85°C, and applied to tissue sections. Hybridization was performed overnight at 50°C in a humidified chamber. After posthybridization washes of increasing stringency and RNase A (20 µg/ml) digestion, slides were dehydrated, dried, and coated in NBT-2 nuclear track emulsion (Eastman Kodak Co., Rochester, NY). After 7–10 days exposure at 4°C, slides were developed with D-19 developer (Eastman Kodak Co.) and counterstained with toluidine blue.

Photography

Tissues were visualized and photographed under brightfield and darkfield illumination with a Leica DM RB microscope (Milton Keynes, Bucks, UK).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
At the time of collection, all embryonic vesicles were spherical, with an outer capsule and visible embryonic disc. Vesicle diameter, measured by ultrasonography, ranged from 8 mm (Day 12) to 19 mm (Day 15). Morphologically, embryos in this age group were similar. The most predominant tissue was bilaminar trophoblast consisting of columnar trophectoderm with sparse endoderm lining the yolk sac. The nuclei of trophectodermal cells were generally aligned along the basal side of the cell. The endodermal cells were often dislodged toward the blastocoel, or lost completely during processing. The embryonic disc was clearly distinguishable from the trophoblast, and in older embryos, outgrowth of the mesoderm was evident.

Immunocytochemistry

Expression of P450arom was highly tissue-specific with no change in staining pattern from Day 12 to Day 15. Strong positive immunostaining was evident throughout the extraembryonic trophectoderm in all embryos but was absent from the embryonic disc (Fig. 1, A and B). The most intense positive staining was observed in the bilaminar trophoblast distal to the disc, with less intensity in the trophectoderm of more proximal trilaminar areas. No staining was observed in abembryonic endoderm or mesoderm; however, P450arom staining was observed in the endoderm just beneath the embryonic disc (hypoblast; Fig. 1C). In fact, the transition from disc to trophoblast was clearly distinguished by the marked change in cell-specific aromatase expression (Fig. 1D). Sections treated with preimmune rabbit serum were negative (Fig. 1C, insert). Positive staining was Leydig cell-specific in control testis as previously described [19].



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  		FIG. 1. Immunocytochemical localization of P450arom in the equine blastocyst. Day 13 embryo (A) sectioned through the embryonic disc (d), with positive immunostaining in the trophectoderm (t) but no staining in the endoderm (e) or mesoderm (m). B) Bilaminar trophoblast magnified 4 times (A) showing positive cytoplasmic staining in the trophectoderm (t). C and D) Day 12 blastocyst in the region of the embryonic disc (d) with visible endoderm (e). The transition from disc to trophoblast is marked by differential staining in the trophectoderm (closed arrow) and endoderm (open arrow). Bars = 100 µm (A), 50 µm (B–D)

In Situ Hybridization

In situ hybridization with an equine-specific antisense cRNA probe confirmed the pattern of aromatase expression observed with immunocytochemistry (Fig. 2, A and B). The strongest mRNA hybridization signal was observed in the distal, bilaminar trophoblast with less intense hybridization in the trophectoderm of trilaminar areas. Strikingly, no hybridization was seen in the embryonic disc or in extraembryonic endodermal and mesodermal tissues. Hybridization in the hypoblast was not clearly distinguishable above background. Tissue-specific expression patterns did not change with embryonic age. No hybridization above background was observed with the sense probe (Fig. 2C), and positive control testis showed Leydig cell-specific hybridization (data not shown).



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  		FIG. 2. In situ hybridization of P450arom 35S-antisense cRNA in the Day 13 embryo visualized under brightfield (A) and darkfield (B) illumination. Areas of embryonic disc (d), trophectoderm (t), endoderm (e) and mesoderm (m) are labeled. No hybridization signal was present in the disc, endoderm, or mesoderm (white arrows). C) Negative control hybridization with 35S-sense cRNA. Bars = 100 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study provide the first, definitive tissue-specific localization of P450arom within the blastocyst-stage equine embryo. Our observation that P450arom protein and transcript are abundant in the equine trophoblast on Days 12–15 of pregnancy is consistent with the significant production of estrogen by intact [4] or dispersed [2, 20, 21] embryos of the same age, and it further defines the cells responsible for this prodigious steroid production. The pattern of P450arom expression we observed in the horse conceptus was similar to that in the preimplantation porcine embryo [13], although the regulation of expression with respect to developmental stage appears to be different. Similar spatial distribution patterns have also been reported for interferon-{tau} in the trophoblast of ruminant species [22, 23]. The physiologic significance of the similar spatial distribution patterns of these important conceptus-secreted factors remains to be determined.

Aromatase expression in the horse conceptus does not appear to be temporally regulated from Day 12 to Day 15 of the preimplantation period as it is around Day 12 in the pig. Positive immunostaining and in situ hybridization for P450arom were evident in all ages of equine embryos examined. These results are consistent with the observation that estrogen production continues to increase with embryonic age [1, 4]. In contrast, clear temporal and spatial regulation of P450arom and P450C17 in the pig blastocyst has been confirmed by both Northern analysis [12] and immunocytochemical localization [13], which provided the following data. Staining for both P450arom and P450C17 was intense in 7- to 10-mm porcine blastocysts but was not observed in 2- to 4-mm blastocysts, and was less intense or absent in later stage 20-mm and filamentous embryos. In positively staining porcine embryos, P450arom was observed throughout the trophoblast and in the endoderm. Endodermal staining of the greatest intensity was seen in the layer under the embryonic disc (hypoblast). No staining was observed in the mesoderm. The temporal regulation of P450arom and P450C17 coincides with observed patterns of estrogen synthesis and suggests that regulation of these enzymes is the critical step in control of estrogen biosynthesis by the porcine blastocyst [5].

The expression pattern of P450arom in the Day 12–15 equine embryo did not change with blastocyst development but showed clear tissue specificity. In horse conceptuses, expression of P450arom protein and message was evident throughout the trophectoderm, with endodermal staining only directly under the embryonic disc. While staining for P450arom protein was strong in the hypoblast, the results of in situ hybridization were not as clear. The lack of discernable signal may indicate that aromatase message is absent or at very low levels in this tissue, but more likely represents the limitations of resolution using radiolabeled probes. The strong expression of P450arom in the trophectoderm, but lack of expression in the extraembryonic endoderm, is consistent with in vitro results [21]. Percoll separation of cells from Day 12–15 embryos identified two populations of cells with distinct steroid secretion patterns. A low-density population, presumed to be endodermal cells, was found to secrete low levels of estrogen and higher levels of progesterone. The higher-density cells, presumed to be mainly ectodermal, secreted higher levels of estrogen. The high rate of estrogen production by ectodermal cells is well supported by the localization of P450arom throughout the trophectoderm. The lack of aromatase expression in the extraembryonic endoderm precludes estrogen formation by these cells in isolation. The expression of P450arom in the endoderm associated with the embryonic disc suggests that these cells do produce estrogen, as has been reported for this cell population in the pig [24, 25]. The role of this cell-specific estrogen production remains to be elucidated.

Localization of P450arom to the trophoblast in the horse conceptus appears to follow the expression pattern of steroidogenic enzymes in the pig and interferon-{tau} in ruminant embryos. In these species, the role of these conceptus-derived proteins around the critical events of elongation, implantation, conceptus mobility, and maternal recognition of pregnancy (MRP) has been well established [26]. Although the role of estrogen in MRP in the horse is controversial, the potential remains for estrogen or a metabolite to participate in this dialogue. The continuous and prodigious expression of P450arom in the Day 12–15 equine embryo also suggests a significant role for conceptus-derived estrogen in the mare but points to a less rigid temporal regulation. Perhaps the most important role of conceptus-derived estrogen is to maintain mobility of the embryo [5] so that it may signal the maternal endometrium for nutritional support and participate in pregnancy recognition. The equine conceptus is somewhat unusual among mammalian embryos in that it remains free-floating in the uterine lumen for an extended period of time, traversing both horns and the uterine body daily [27], and this mobility is essential for delivery of conceptus-secreted factors that prevent uterine release of prostaglandin F2{alpha} and maintain a viable pregnancy [6, 7].

In conclusion, we have demonstrated tissue-specific regulation of P450arom enzyme in the preimplantation horse conceptus, confirming that the trophectoderm and hypoblast are the primary tissues responsible for estrogen production during this critical period of early pregnancy. The similarity of P450arom distribution between horse and pig embryos suggests the potential for conserved mechanisms for regulation of spatial expression. Divergent patterns of P450arom expression with respect to embryonic age and developmental stage point to more species-specific mechanisms, perhaps reflecting differences in the uterine environments. Future work is focused on the mechanisms governing trophoblast-specific gene expression.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Ken Taylor, Hilary Ward, and the student stallion managers for horse breeding and embryo collection, and Diane Nayden for expert guidance in tissue preparation.


   FOOTNOTES
 
First decision: 12 November 1999.

1 This project was supported in part by the Center for Equine Health, University of California, Davis, with funds provided by the Oak Tree Racing Association, the State of California satellite wagering fund, and contributions by private donors. Back

2 Correspondence: Karen W. Walters, Department of Population Health and Reproduction, School of Veterinary Medicine, 1114 Tupper Hall, University of California, Davis, CA 95616. FAX: 530 752 4278; kwwalters{at}ucdavis.edu Back

Accepted: December 7, 1999.

Received: October 25, 1999.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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