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a Departments of Obstetrics and Gynecology and Molecular and Integrative Physiology, Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City, Kansas 66160-7338
b Department of Biological Sciences, University of Idaho, Moscow, Idaho 83844-3051
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cyclooxygenase is the enzyme that converts arachidonic acid to PG. It exists as two isoforms: cyclooxygenase 1 (Cox1) and cyclooxygenase 2 (Cox2), which are encoded by separate genes [5, 6]. Cox1 is thought to be constitutively expressed whereas Cox2 is up-regulated in response to various stimuli including inflammatory agents, growth factors, cytokines, and as yet unidentified signals from implanting mouse and mink blastocysts [7–9]. These two isoforms also respond differently to various pharmacological agents that suppresses PG synthesis. Most nonsteroidal inhibitors of cyclooxygenases are better inhibitors of Cox1 than of Cox2 [10]. This may partially explain why indomethacin failed to affect endometrial vascular permeability during the periimplantation period in the previously cited ferret study [4]. Mutation of the gene coding for Cox2, but not Cox1, in mice leads to infertility [11–13]. In the absence of Cox2, both implantation and decidualization of the uterine stroma are impaired, thereby emphasizing the importance of this isoform and PG synthesis in implantation [13].
The Western spotted skunk is a small mustelid carnivore that breeds in the fall. The fertilized eggs rapidly develop to the blastocyst stage, pass into the uterus, and then enter into a prolonged state of embryonic diapause lasting about 200 days [14]. Renewed embryonic development, which begins when the blastocysts have reached a diameter of 1.2 mm, occurs in late March or early April [15]. Although the precise duration of blastocyst activation is unknown, the process requires several days for the blastocysts to increase in cell number, to acquire increased numbers of intracellular organelles, and to become fully expanded (2.0–2.2 mm) and capable of attaching to the uterine lining [16]. The uterus of this species simultaneously undergoes changes, some of which may be important in signaling renewed embryonic development or in creating a uterine environment conducive to resumption of blastocyst development. Examples of such changes include an increase in wet weight of the uterine cornua, loss of estrogen and progesterone receptors from the luminal epithelium, an increase in epidermal growth factor receptors, increased expression of leukemia inhibitory factor, and increased synthesis and secretion of uterine proteins [17–20]. Some of the changes in the uterus may be induced by signals from the blastocysts.
Mutation of the Cox2 gene has demonstrated the importance of PG for blastocyst implantation and decidualization of the uterus in mice, but the role of this important class of compounds in periimplantation uterine biology is less well understood in carnivores. Indomethacin reduced the number of implantation sites and appeared to delay implantation in ferrets [4]. It has been reported that Cox2 transcripts are induced in the uterus of mink by implanting blastocysts, as the localization of such transcripts and Cox2 protein was restricted to implantation sites [9]. Sudden changes in uterine weight can be due to edema, which occurs in response to stimuli that alter vascular permeability. Our previous reports of increased uterine weight during blastocyst activation in the spotted skunk suggest that isoforms of cyclooxygenase might be involved in uterine changes associated with preparation for implantation [17,20]. On the basis of studies in other species, we hypothesized that gene expression of Cox2, but not Cox1, would significantly increase during the periimplantation period and would be localized to those regions of the uterus where blastocysts were implanting.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Twenty-four Western spotted skunks (Spilogale putorius latifrons) that had bred in the wild were purchased from a USDA-licensed dealer in Oregon. The caging and maintenance of the animals followed the protocol reported previously [21] and was in accordance with the NIH Guide for the Care and Use of Laboratory Animals. After administration of an overdose of sodium pentobarbital, the uterus was removed via a midventral incision. The uterine cornua were dissected free from the corpus, oviducts, and mesenteries prior to flushing of one or both cornua with sterile saline to recover blastocysts. Uteri with visible uterine swellings (implantation chambers) were not flushed unless the swellings were < 3.5 mm and suspected to contain unattached blastocysts. The diameter of implantation chambers was measured with Vernier calipers, and the number of days postimplantation was estimated as previously described [22]. The cornua were flash frozen in liquid propane, weighed, and stored at -80°C until further processing. Blastocysts were measured with the aid of an ocular micrometer. Those with diameters of 1.1 mm or less were classified as being in diapause (delayed implantation). Blastocysts with diameters of 1.2–1.6 were classified as early activated, and those with diameters of 1.7 mm or greater were classified as activated. Protocols for all experiments involving animals described in this paper were reviewed and approved by the University of Idaho Animal Care and Use Committee.
Northern Blots
Total RNA was extracted from paired uterine horns of 13 spotted skunks in various stages of pregnancy by a modified guanidine thiocyanate procedure. Total RNA (6 µg) was denatured, separated by formaldehyde agarose gel electrophoresis, transferred and cross-linked to a nylon membrane, and prehybridized as previously described [8]. The blot was hybridized sequentially with antisense 32P-labeled cRNA probes for mouse Cox1, Cox2, and rpL7, a part of the ribosomal protein (a housekeeping gene). The probes had specific activities of about 1 x 109 dpm/µg and were prepared as previously described [8]. After each hybridization, the blot was washed using stringent conditions (68°C), and the hybrids were detected by autoradiography [23, 24]. The rpL probe was used to assess equal loading of sample in each lane.
In Situ Hybridization
Frozen sections (10 µm) of 11 skunk uteri collected during delayed implantation (n = 3), early activation (n = 3), activation (n = 2), and postimplantation (n = 3) were prepared. Uterine sections from an animal in each stage of pregnancy were mounted onto the same poly-L-lysine-coated slide. Several slides of all specimens were prepared and stored desiccated at -80°C until use. The sections were subsequently hybridized with 35S-labeled sense or antisense mouse cRNA probes for Cox1 and Cox2 as previously described [8]. After hybridization and washing, sections were incubated with RNase A (20 µl/ml) at 37°C for 20 min, and RNase A-resistant hybrids were detected by autoradiography using Kodak NTB-2 liquid emulsion (Eastman Kodak, Rochester, NY). Parallel sections hybridized with the sense probes served as negative controls. Slides were poststained with hematoxylin and eosin.
Statistical Analysis
Data for weight of paired uterine cornua are presented as the means ± SEM. These data were first analyzed by the Kruskal-Wallis one-way ANOVA on ranks and found to be significantly different (p < 0.001). A t-test was used to determine whether weight of uterine horns that contained 1.2-mm blastocysts differed from that of uterine horns containing 1.1-mm blastocysts.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Changes in wet weight of paired uterine horns collected at various times during embryonic diapause and the periimplantation period are plotted against the largest blastocyst flushed from each uterus in Figure 1. There was a significant increase (p = 0.029) in uterine weight at the time the largest blastocyst reached 1.2 mm in diameter. Preimplantation uterine weight reached a plateau when blastocysts expanded to 1.3 mm.
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Northern Blot Analysis of Cox1 and Cox2 mRNAs in the Periimplantation Skunk Uterus
Steady-state levels of Cox1 and Cox2 mRNAs in the skunk uterus during the periimplantation period were analyzed by visual inspection of Northern blot hybridization. Consistent with previous observations in the mouse [8], 2.8-kilobase (kb) and 4.7-kb transcripts were detected in uterine total RNA samples for Cox1 and Cox2 mRNAs, respectively. Cox1 mRNA was detected in uteri at all stages of pregnancy examined, but the levels did not exhibit much variation (Fig. 2). In contrast, little or no Cox2 mRNA could be detected in uteri with delayed-implanting blastocysts (Fig. 2, lanes 1–2), although this mRNA was detected at other stages of pregnancy examined (Fig. 2, lanes 3–10). Low levels of Cox2 mRNA were detected in uterine RNA of one skunk that had small (3.8 mm) uterine swellings and from which a fully activated zona-free preattachment blastocyst was flushed from the contralateral horn (Fig. 2, lane 3). In contrast, Cox2 mRNA was readily detected in the uterus of a skunk that had uterine swellings measuring 3.5–4.2 mm in diameter (Fig. 2, lane 4). The trophoblast in the larger implantation chambers of this animal should have begun to invade the luminal epithelium. Cox2 expression was increasingly up-regulated through Day 5 or 6 post-trophoblast attachment (Fig. 2, lanes 5–10).
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In Situ Hybridization of Cox1 and Cox2 mRNAs in the Periimplantation Skunk Uteri
No distinct hybridization signals for Cox1 mRNA were detected in skunk uteri containing delayed-implanting, early-activated, or fully expanded (2.0 mm) zona-encased blastocysts (Fig. 3, a–f). However, Cox1 mRNA was expressed in the trophoblast and perhaps in uterine glands of implantation chambers with 4-mm diameters representing uterine-embryonic development of about 24 h post-trophoblastic attachment (Fig. 3, g and h). No cell-specific hybridization signals were detected in the embryonic disc (data not shown) or sections of the interimplantation sites (Fig. 3, i and j). At the 48–72-h postattachment period, Cox1 mRNA was clearly detected in the necks of uterine glands but not in trophoblast of embryos in 7-mm implantation chambers (data not shown). No positive cell-specific signals were detected when sections were hybridized with the Cox1 sense probe (Fig. 4, a and b).
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With respect to Cox2 mRNA, no cell-specific hybridization signals could be detected in the uterus of animals containing blastocysts in diapause (Fig. 5, a and b) or early-activated blastocysts with 1.3-mm diameter (Fig. 5, c and d). However, modest levels of Cox2 mRNA accumulation were detected in uterine luminal epithelium of two skunks from which blastocysts measuring 1.5–1.6 mm in diameter (early activation) were flushed (data not shown). Low levels of hybridization signals were also visible in uterine glands in the lateral, antimesometrial region of the endometrium where early attachment would be expected to occur (Fig. 5, e and f). In contrast, intense hybridization signals were apparent in the trophoblast, but not in the embryonic disc, of this same animal. Blastocysts flushed from the contralateral uterine horn of this skunk measured 2.0 mm and were nearing full expansion. Very intense hybridization signals for Cox2 mRNA were detected in the cytotrophoblast, in invading syncytiotrophoblast, and in the neck regions of uterine glands of an animal with implantation sites 4 mm in diameter (Fig. 5, g and h). Intense signals were also detected in the luminal and glandular epithelia at the interimplantation sites of this animal (Fig. 5, i and j). No positive cell-specific hybridization signals were detected in sections hybridized with the Cox2 sense probe (Fig. 4, c and d).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Northern blot analysis of skunk uteri indicated that Cox2 mRNA expression is tightly regulated during various stages of pregnancy in contrast to that of Cox1. Little or no Cox2 expression was detected in uteri collected during delayed implantation as determined by Northern blot and in situ hybridization. Cox2 expression was first detected in the uterine luminal epithelium of two skunks that contained early-activated blastocysts (1.5–1.6 mm) but was not detected in the uterus of a skunk that contained 1.3-mm blastocysts. It is difficult to determine whether Cox2 expression in the luminal epithelium was induced by adjacent blastocysts, as the blastocysts were flushed from both specimens. The trophoblast of a fully expanded (2.0 mm) preattachment blastocyst exhibited relatively strong hybridization signals for Cox2 mRNA; in addition, a portion of the endometrium immediately adjacent to this blastocyst exhibited moderate expression. Because this is the region where attachment of the trophoblast will occur, we interpret these changes in Cox2 expression as being directly related to preparation for implantation. Cox2 mRNA was more distinctly expressed in the trophoblast of postattachment blastocysts and in the upper regions of uterine glands. Further, Cox2 mRNA was still expressed in postimplantation blastocysts in implantation chambers that were 9 to 10 mm in diameter or about 6 days postattachment. It may be noted that neither Cox1 nor Cox2 protein could be detected in the uterus or placenta of mink 12 days after blastocyst attachment [9] and that Cox2 expression is limited to the implantation chambers of the mink and mouse [8, 9]. These observations suggest that signal(s) from the blastocysts is responsible for the up-regulation of Cox2 expression that occurs during implantation. However, Cox2 mRNA was strongly expressed in the luminal and glandular epithelia in sections of uterus located between implantation chambers of the spotted skunk. This could possibly be due to diffusion of a potent or a more abundant signal emanating from skunk blastocysts. Alternatively, it might indicate that other factors are involved in up-regulating uterine expression of Cox2 in the skunk.
The abrupt increase in uterine weight was consistently observed in skunks in which at least one of the blastocysts had reached a diameter of 1.2 mm. This marked change in uterine weight occurred several days prior to the predicted time of the highly localized increases in endometrial vascular permeability. Previous studies have demonstrated that blastocysts with similar diameters exhibit increased RNA and protein synthesis as well as increased numbers of mitochondria and free ribosomes [15, 16, 25]. These changes are associated with blastocyst activation from diapause and resumption in development. Our data also indicate that the uterus likewise undergoes changes at the time the blastocysts are being activated. The most likely cause of increased uterine weight is increased fluid retention by this tissue. Our results do not reveal any marked change in cell-specific expression of either isoform of Cox that is temporally related to the onset of increased uterine weight prior to implantation. The cause and nature of this change in uterine physiology remains to be determined. Increased uterine and trophoblast expression of Cox2 clearly plays no role in decidualization of the uterine stroma in the spotted skunk or mink, because decidual tissue does not form in these species. On the other hand, expression of Cox2 in fully activated and early-implanting blastocysts suggests that the blastocysts may produce eicosanoids that induce highly localized increases in vascular permeability occurring at the time of trophoblastic attachment in the ferret and presumably in other mustelid carnivores. Products of Cox reportedly enhance cell adhesion to extracellular matrix and promote cell differentiation and mitogenesis [7], all of which occur during implantation. If secretion of PGs or other eicosanoids from the invading trophoblast enhance trophoblast-extracellular matrix interactions, that would facilitate implantation. Localized production of eicosanoids by the trophectoderm could conceivably stimulate differentiation and formation of syncytiotrophoblast and promote rapid proliferation of cytotrophoblast. Production of eicosanoids by the uterine epithelium and invading trophoblast might stimulate mitosis in uterine glands, thereby promoting their subsequent elongation, or promote angiogenesis. PGs promote the influx of serum proteins into the uterus [3]. An increase in serum proteins in uterine fluid of the spotted skunk is known to occur when the blastocysts reach 1.5–1.6 mm [26], which is precisely the stage at which Cox2 expression first becomes detectable in the uterus of the skunk.
Our data are partially consistent with our hypothesis that gene expression of Cox2, but not Cox1, would increase during the periimplantation period and would be localized to regions of the uterus where implantation occurs. Our results indicate that Cox2 expression is indeed up-regulated in both the uterus and trophoblast at the time of implantation; but it is also expressed in the luminal epithelium and necks of the glands in segments of uterus between implantation chambers. Our data also indicate that Cox1 is mostly constitutively expressed but that it also undergoes cell-specific up-regulation in the trophoblast and uterine glands during the first few days of implantation. Increased expression of Cox2 is temporally correlated with the influx of serum proteins into the uterus and the attachment reaction, both of which involve changes in vascular permeability.
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FOOTNOTES |
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2 Correspondence. FAX: 208 885 7905; rmead{at}uidaho.edu
Accepted: November 17, 1998.
Received: October 5, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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