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This Article Abstract Full Text (PDF) All Versions of this Article: 68/2/401    most recent biolreprod.102.009589v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cheng, J.-G. Articles by Stewart, C. L. Search for Related Content PubMed PubMed Citation Articles by Cheng, J.-G. Articles by Stewart, C. L. Agricola Articles by Cheng, J.-G. Articles by Stewart, C. L.

Loss of Cyclooxygenase-2 Retards Decidual Growth but Does Not Inhibit Embryo Implantation or Development to Term

^a Cancer and Developmental Biology Laboratory, National Cancer Institute at Frederick, Frederick, Maryland 21702

	ABSTRACT

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Previous reports have described that female mice deficient in cyclooxygenase-2 (COX2) are largely infertile because of failure to ovulate, poor fertilization, and defective implantation and decidualization. In the present study, we reinvestigated reproduction in these mice and found they do show a reduction in the numbers of ovulated and fertilized eggs. However, we did not observe any substantial effect on embryo implantation frequencies or an inability of COX2-deficient females to support embryo development to weaning. Pseudopregnant COX2-null recipients do not show any alteration in the timing of implantation following blastocyst transfer, but they do show a delay in the initial rate of decidual growth after implantation that lags by approximately 24 h compared to that in heterozygous or wild-type recipients. These results support previous findings that COX2 has a role in mediating the initial uterine decidual response but is not essential to sustaining decidual growth and embryo development throughout the remainder of pregnancy.

decidua, female reproductive tract, implantation

	INTRODUCTION

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An essential step in the reproduction of all mammals is embryo implantation. At implantation, the blastocyst establishes a more intimate association with the maternal uterus, a process essential for the embryo's continued development. Numerous studies have revealed that implantation depends on the uterus becoming receptive to the blastocyst and that uterine receptivity is a transient state during the reproductive cycle. In rodents, receptivity is primarily regulated by the ovarian steroid hormones estrogen and progesterone. Both hormones act directly and indirectly in regulating proliferation and differentiation of the uterine tissues in preparation for embryo implantation [1, 2]. The indirect actions of the steroid hormones on the uterine tissues are mediated by the production of, and the response of cells to, locally produced growth factors and cytokines [3]. For instance, in the mouse, uterine expression of the interleukin-6 family member leukemia inhibitory factor (LIF) is stimulated by estrogen and is essential to inducing a receptive state in the uterus [4, 5]. In addition to LIF, other factors, based on the spatial and temporal patterns of their expression, have also been suggested to mediate implantation. Among these are the cyclooxygenases (COX1 and COX2; gene symbols Ptgs1 and Ptgs2, respectively), which are rate-limiting enzymes regulating the synthesis of prostaglandins. Of the two enzymes, COX2 has been shown by gene targeting in the mouse to mediate the uterine response to implanting embryos [6]. A series of studies showed that in Ptgs2-null female mice, the frequency of embryo implantation was greatly reduced and decidualization was impaired. Treatment of Ptgs2-null female mice with the prostacyclin-analogue cPGI at the time of blastocyst transfer markedly improved rates of implantation [7].

While analyzing links between the LIF and COX2 deficiencies on implantation, we reinvestigated embryo implantation in Ptgs2-deficient female mice. Using mice derived from the same original stock as those used in the previous studies, we found that contrary to those reports, wild-type embryos implant in Ptgs2-deficient uteri at frequencies comparable to those found in heterozygous or wild-type recipients. The uterine response, as measured by the increase in decidual weights on subsequent days following implantation, occurs at rates comparable to those found in wild-type female mice, although the mean weight lagged by approximately 24 h. Furthermore, in the COX2-null mice, embryo development proceeds to term and, subsequently, to weaning. Loss of COX2 in some mouse lines may only act to retard the rate at which the uterus initially decidualizes in response to the implanting embryo. Thus, COX2 is not essential for either postimplantation development to term or, subsequently, to weaning.

	MATERIALS AND METHODS

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Dr. J. Dinchuk (DuPont Merck, Wilmington, DE) kindly provided the Ptgs2 +/- male and female mice. They were maintained in our facility in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (NIH Publication no. 86-23, 1985). The mice were maintained by sib-sib breeding and were not outcrossed to any other strain. Genotyping by Southern blot analysis was performed exactly as previously described [8]. Pseudopregnant female +/- and -/- Ptgs2 mice were derived by mating to vasectomized males of proven sterility, with Day 1 of pregnancy being equivalent to the day of plug. On Day 3, blastocysts obtained from previously mated wild-type mice (B6C3H x B6C3H) were surgically transferred to each uterine horn (10–12 blastocysts/mouse). No additional hormones or factors were provided. Subsequently, on days equivalent to Days 6–10 of pregnancy, the recipients were killed and the uteri isolated. Implantation sites (i.e., deciduas) were counted and then dissected free of the surrounding uterine muscle, with each decidua then being individually weighed. Some of the recipient female mice were allowed to complete their pregnancy and give birth to the transferred embryos. The embryos that were born were allowed to complete their postnatal development to weaning. All results were subjected to statistical analysis (Student t-test).

Histology

Uteri were dissected and fixed in neutral buffered formalin, embedded and sectioned (thickness, 6 µm), and stained with hematoxylin and eosin.

	RESULTS

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As previously reported, Ptgs2-/- female mice from our colony were largely infertile because of defective ovulation and fertilization. The Ptgs2-/- female mice mated to wild-type males produced only 2 fertilized eggs from a total of 21 eggs recovered from 6 females, whereas 32 fertilized eggs were recovered from 5 Ptgs2+/- females, 1 of whom was not pregnant [6, 9].

To analyze the effect of COX2 loss on implantation, we mated Ptgs2-/- female mice to vasectomized males of proven sterility. The Ptgs2+/- mice were used as controls. On the morning of Day 3 of pregnancy (Day 1 day of plug), five to six wild-type blastocysts were transferred to each uterine horn. The female mice were allowed to recover and then examined for the presence of implantation sites and decidualization on Days 6–10 of pregnancy.

In contrast to previous reports, we consistently found recognizable implantation sites and deciduas in the uteri of Days 7–10 pregnant Ptgs2-/- recipients (Fig. 1A). The mean percentage number of implantations per mouse was slightly higher in the Ptgs2+/- recipients compared to those in the Ptgs2-/- recipients (67% vs. 78%, P > 0.1). The decidual weights were lower in Ptgs2-/- recipients than in the heterozygous recipients on the same day after mating (Fig. 1C). Compared to the mean weights from the heterozygotes, the mean decidual weight in the Ptgs2-/- female mice on any day lagged by approximately 24 h, with such a lag persisting to Day 10 of gestation. Subsequently, we allowed the recipient mice to complete their pregnancy. Both Ptgs2-/- recipients and heterozygotes successfully delivered viable pups, although the majority of pups from the Ptgs2-/- recipients were delivered 1 day later (Day 21) than those from the Ptgs2+/- mice (Day 20). Both genotypes successfully nursed the pups to weaning (Table 1).

View larger version (40K): [in this window] [in a new window]   FIG. 1. A) Uteri dissected from COX2-deficient and heterozygous female mice following the transfer of blastocysts on Day 3 of pregnancy. In the upper panel, uteri from +/- mice on Day 7 of pregnancy show typical implantation sites in the form of decidua. The lower uterus from a Day 7 COX2-null mouse also shows implantation sites in the form of smaller decidua (arrows). The lower panel shows uteri from Day 8 COX2+/- and -/- mice showing the smaller size of decidua in the COX-/- recipients. B) Histological section through the uterus from a Day 5 pregnant COX2-/- recipient. The embryos transferred on Day 3 at approximately 1100 h have implanted, and the endometrium is decidualizing. The uteri were removed and fixed at 1100 h on Day 5 (i.e., 48 h after transfer). C) Mean increase in decidual weights from Day 6 to Day 10 of pregnancy in both COX2+/- and -/- recipients. The increase in decidual weights in the COX2-/- recipients lags that in the +/- recipients by approximately 24 h. The differences were highly significant (P < 0.001)

The retarded decidual growth was caused either by a delay in the onset of implantation or by the initial decidual growth proceeding at a slower rate. To distinguish between these alternatives, we transferred blastocysts to both Ptgs2-/- and +/- pseudopregnant recipients on Day 3 of pregnancy. Forty-eight hours later (equivalent to the morning of Day 5 of pregnancy), the recipients were injected via the tail vein with a Pontamine blue solution to identify implantation sites [10]. The uteri were then isolated and processed for histological analysis. Analysis of the uteri in the Ptgs2-/- recipients revealed that implantation sites were present, as indicated by localized accumulation of the blue dye. Subsequent histological analysis revealed that decidualization of the uterine endometrium was also proceeding, indicating that the time at which implantation started probably did not differ between the Ptgs2-/- and +/- recipients (Fig. 1B). Consequently, decidual growth during the first 24–48 h after implantation is slowed in the Ptgs2-/- mice. Thereafter, decidual growth proceeds at a normal rate (Fig. 1C).

	DISCUSSION

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In the present study, we report that COX2 deficiency does not always result in a high rate of implantation failure and defective decidualization. We compared the rates of embryo implantation in Ptgs2+/- and -/- mice following the surgical transfer of wild-type embryos to pseudopregnant recipients. In the recipients of both genotypes, the blastocysts implanted and developed successfully to term. The frequency of embryo implantations was quite high (67–78%), although a slight but significant reduction (P > 0.1) was observed in the number of implantation sites in the null females compared to the heterozygotes, possibly because of their poorer health, because Ptgs2 null mice develop kidney problems [8].

The reason for the great difference in female reproduction between our colony and that reported previously [6] is, however, unclear. We maintained our Ptgs2 colony on a mixed (C57Bl6/JX129S7/SvEvBrd) background as previously described [8]. Different breeding protocols may have been established between the two laboratories, which could have resulted in the segregation of a critical modifier gene, affecting the requirement for COX2 during the implantation period. In the previous reports, implantation frequencies were determined primarily by looking for implantation sites using the injection of blue dye within 24 h following embryo transfer [6]. In the present study, we waited at least 48 h before looking for implantation; thus, the previous reports may have missed the difference by curtailing their analysis at an earlier time point. This, however, does not explain the discrepancy in a subsequent report describing the partial rescue of the decidual response by injection of cPGI and progesterone, because we did not have to raise circulating progesterone levels by injection to sustain postimplantation development in the COX2-deficient female mice [7]. It is therefore conceivable that the reproductive differences between the two colonies may have arisen by the inadvertent segregation of an as-yet-unidentified modifier gene.

Nevertheless, the initial delay in decidual growth is intriguing and supports the observation that COX2 is required at the start of decidualization, because within 24–36 h, decidual growth is restored to a normal rate. What role COX2 has at the start of decidual growth is not understood. One possibility is that COX2 is required to initiate some of the angiogenic changes in the architecture of the uterine vasculature that occur with implantation and on which decidualization of the endometrium depends [11]. Many different factors are involved in mediating angiogenesis, with vascular endothelial growth factor (VEGF) being essential [12, 13]. VEGF is also required for embryo implantation, because blocking its activity inhibits decidualization and embryo implantation [14]. Therefore, COX2 may be required for the induction of VEGF expression during the initial stages of decidualization, because Ptgs2-/- fibroblasts show greatly reduced levels of VEGF [15]. Other factors, such as fibroblast growth factors or interleukin-1, that are also expressed in the uterus at implantation [16–19] may compensate for COX2 deficiency by increasing VEGF and/or its receptor levels, thus rescuing angiogenesis and restoring decidual growth to its normal rate.

In conclusion, COX2 is not an absolute requirement for blastocyst implantation and decidualization in the mouse. Loss of COX2 activity can retard the rate at which decidualization proceeds within the first 24 h of implantation. Thereafter, both decidual and embryonic development proceeds, apparently normally, and results in the birth of viable offspring.

	ACKNOWLEDGMENTS

 
We would very much like to thank Lori Sewell for excellent assistance in managing our mouse colony and Dr. Joe Dinchuk (DuPont Merck) for providing us with the COX2 mice

	FOOTNOTES

 
¹ Correspondence. FAX: 301 846 7117; stewartc{at}ncifcrf.gov

Received: 23 July 2002.

First decision: 7 August 2002.

Accepted: 22 August 2002.

	REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 68/2/401    most recent biolreprod.102.009589v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cheng, J.-G. Articles by Stewart, C. L. Search for Related Content PubMed PubMed Citation Articles by Cheng, J.-G. Articles by Stewart, C. L. Agricola Articles by Cheng, J.-G. Articles by Stewart, C. L.