Apoptotic endometrial caspase-3 mediated phospholipase a2 activation, a critical component in programing uterine receptivity

The objective of this study was to determine the consequence of uterine apoptotic caspase-3 activation on day 1 post coitus (dpc) in the pregnant mouse. We previously demonstrated that during pregnancy uterine caspase-3 activation isolated to the myometrial compartment is largely non-apoptotic and controls uterine quiescence. In this study we determined that uterine caspase-3 activation on 1 dpc may play a critical role in regulating endometrial PGE2 synthesis though iPLA2 activation. These analyses provide novel insight into the molecular mechanisms that regulate previously reported increases in endometrial PGE2 synthesis in very early pregnancy, that act to enhance uterine receptivity. We have identified the site and impact of that uterine apoptotic caspase-3 activation utilizing uteri isolated from non-pregnant control animals at estrous and diestrous and from control pregnant mice at 1-19 dpc. In addition, uteri were isolated from non-ligated controls (GD), unilateral (UL) and bilateral ligated (BL) uterine horn mouse models at 1, 3 and 6 dpc. Uteri were examined for apoptotic indices, such as caspase-3 activation and TUNEL staining. Immunohistochemical analysis was performed to identify the site of apoptotic caspase-3 activation. The presence of the truncated form of phospholipase A2 (tiPLA2) was examined as a measure of apoptotic caspase-3 mediated iPLA2 activation. Our analysis determined that apoptotic caspase-3 and iPLA2 activation were limited to the endometrial compartments of the control and unilateral uteri on 1dpc and were not found in the bilateral ligated uterine horn on 3 or 6 dpc. Our data indicates that the presence of a conceptus on 1 dpc triggers an increase in endometrial apoptotic caspase-3 mediated iPLA2 activation. iPLA2 when activated causes the hydrolysis of fatty acids resulting in arachidonic acid release and production of PGE2, which in early pregnancy has been demonstrated to act in a leutoprotective manner, prolonging progesterone synthesis and promoting uterine receptivity.


55 Introduction
56 Despite many advances in assisted reproductive technologies (ART), 57 implantation rates continue to remain low. We know that the process of 58 implantation requires a reciprocal interaction between blastocyst and 59 endometrium, involving both the embryo, with its intrinsic molecular program of 60 cell growth and differentiation, and the progressive differentiation of endometrial 61 cells to accomplish a state of appropriate uterine receptivity. However, it remains 62 unresolved why implantation failure occurs. Failure to implant is thought to occur 63 as a consequence of impaired embryo developmental potential, deficiency in 64 uterine receptivity and/or a diminished embryo-uterine dialogue. Therefore, 65 gaining a better understanding of the molecular events that govern uterine 66 receptivity and implanation is warranted. By understanding the activity and 67 function of the factors involved, it may be possible to use them as predictors of 68 endometrial receptivity or embryo quality to maximize implantation rates in future 69 ART cycles.

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Previous analysis has demonstrated in several animal models that 71 an increase in uterine prostaglandin PGE2 biosynthesis in very early pregnancy 72 plays a role in the maintenance of the corpus luteum and luteal function [1].
73 Uterine PGE2 biosynthesis in very early pregnancy has been demonstrated 74 conclusively to increase ovarian progesterone synthesis through binding its EP2 75 receptors on the corpus luteum [2,3]. These events have been demonstrated to 76 increase ovarian progesterone production in early pregnancy, promoting uterine 5 77 receptivity and implantation. However, the trigger for this early uterine 78 prostaglandin biosynthesis prior to implantation remains unknown.

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Our current analysis suggests that a spike in apoptotic caspase-3 activity 80 isolated to the endometrial compartment on 1dpc may trigger this leutoprotective, 81 prostaglandin dependent processes through truncation and activation of iPLA2. Signaling Technology and anti-iPLA2 1:500 (Cat 07-169-i) from Millipore.

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Immunoreactivity was detected using HRP-conjugated secondary antibody

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Nuclei were counterstained blue with haemotoxylin for 2 minutes.

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As can be observed caspase-3 activation on 1 dpc in the UL mouse model is 264 strikingly isolated to the endometrial compartment in both the P and NP uterine

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Apoptosis is isolated to the endometrial compartment in early gestation.

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TUNEL staining (green) was performed to identify the site of apoptotic caspase-3 275 activity in mouse uterine tissues isolated from our bilateral ligated mouse model

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Nuclei were counterstained with Dapi (Blue). As can be observed apoptotic

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This current study was initiated by our observation of a significant pre-294 implanation surge in active caspase-3 levels on 1 dpc in the pregnant mouse 295 model (Figure 1), which rapidly declines to barely detectable levels on 2 dpc, 296 prior to a mid gestational resurgence (5-17 dpc 313 Figure 2 confirms that similar to term and postpartum uterine caspase-3,

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uterine pre-implantation caspase-3 activity is also apoptotic in nature, (as