QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) A corrigendum has been published Alert me when this article is cited Alert me if a correction is posted 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 Song, H. Articles by Dey, S. K. Search for Related Content PubMed PubMed Citation Articles by Song, H. Articles by Dey, S. K.

Cytosolic phospholipase A₂ is crucial for ‘on-time’ embryo implantation that directs subsequent development

¹ Departments of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160-7336, USA
² Departments of Obstetrics and Gynecology, Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA
³ Department of Pediatrics, University of Kansas Medical Center, Kansas City, KS 66160-7336, USA
⁴ Department of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-2279, USA
⁵ Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA

View larger version (26K): [in a new window]   Fig. 1. Ovulation and fertilization rates in Pla2g4a–/– mice. The rate of ovulation and fertilization in wild-type and Pla2g4a–/– mice was examined on day 2 of pregnancy on C57BL/6J (A) and CD1 (B) genetic backgrounds. The numbers within the bars indicate the number of mice with ovulation/total number of mice. Results of ovulation are mean±s.e.m. Statistical significance was evaluated using unpaired t-test and 2-test, respectively (*P<0.01).

View larger version (69K): [in a new window]   Fig. 2. Implantation and decidualization in Pla2g4a–/– mice. (A) The number of implantation sites was examined on days 5 and 6 of pregnancy in wild-type and Pla2g4a–/– mice by the blue dye method. The numbers above the bars indicate the number of mice with implantation sites/total number of mice (unpaired t-test, *P<0.001). The uteri of mice with a few or without implantation sites were flushed to recover unimplanted blastocysts. The mice without implantation sites or blastocysts were excluded from the experiments. (B) A representative photomicrograph of blastocysts recovered from Pla2g4a–/– mice on day 5 of pregnancy (1000 hours) is shown. Note blastocysts with (arrow) or without zona pellucida. (C) Representative photographs of uteri with implantation sites (blue bands) on days 5 and 6. Note very few or no implantation sites on day 5, but unevenly spaced implantation sites on day 6 in Pla2g4a–/– mice. Arrowhead and arrow indicate ovary and implantation site, respectively. Brackets indicate crowding of implantation sites. (D) Decidualization. Wild-type or Pla2g4a–/– mice received intraluminal oil infusion on day 4 of pseudopregnancy. On day 8, uterine weights were recorded. Fold increases denote comparison of weights between infused and non-infused uterine horns. The numbers above the bars indicate the number of responding mice/total number of mice. No significant difference in decidualization was noted between wild-type and Pla2g4a–/– mice (unpaired t-test; P>0.05).

View larger version (78K): [in a new window]   Fig. 3. Expression of Pla2g4a in wild-type uteri during implantation and expression of implantation-specific genes in Pla2g4a–/– uteri. (A) Comparison of expression of Pla2g4a with Ptgs1 and Ptgs2 in the mouse uterus during implantation. In situ hybridization of Pla2g4a, Ptgs1 and Ptgs2 on days 4-6 (09:00 hours) of pregnancy is shown. (B) In situ hybridization of Hegfl, Ptgs2, and Lif in uteri of wild-type and Pla2g4a–/– mice on day 5 of pregnancy (10:00 hours). Note aberrant expression of Hegfl, Ptgs2 and Lif in the luminal epithelium and/or underlying stroma surrounding the blastocyst in Pla2g4a–/– mice. Arrows indicate the location of blastocysts. ge, glandular epithelium; le, luminal epithelium; s, stroma; myo, myometrium.

View larger version (58K): [in a new window]   Fig. 4. Uterine status of PGs and expression of genes encoding sPLA2s, and restoration of normal implantation in Pla2g4a–/– mice by PGs. (A,B) The levels of PGs in uteri of wild-type and Pla2g4a–/– mice on days 4 and 5 of pregnancy, respectively. PGI2 was measured as 6-keto-PGF1. N.D., not detectable (unpaired t-test, *P<0.05; **P<0.01; n=4-5). (C) Expression of genes encoding sPLA2 isoforms in various wild-type mouse tissues by RT-PCR. Heart (H), intestine (I), kidney (K), liver (Li), lung (Lu), spleen (S) and testis (T) tissue samples were used as controls along with uterine (U) samples obtained on day 4 of pregnancy. Actb, mouse ß-actin. (D) In situ hybridization of Pla2g10 (sPLA2-X) in uteri of wild-type mice on days 4 and 5 of pregnancy. Note uterine expression of Pla2g10 similar to that of Pla2g4a and Ptgs1 on day 4 (compare with Fig. 3A). The arrow indicates the location of a blastocyst. ge, glandular epithelium; le, luminal epithelium; s, stroma; myo, myometrium. (E) Restoration of normal implantation in Pla2g4a–/– mice. Pla2g4a–/– mice were injected with saline or PGE2 plus cPGI twice (10:00 and 18:00 hours) on day 4 and implantation sites were examined on day 5 (10:00 hours). The numbers above the bars indicate the number of Pla2g4a–/– mice with implantation sites/total number of Pla2g4a–/– mice used (unpaired t-test; *P<0.001). The mice without implantation sites or blastocysts were excluded from the experiments. (F) Representative photographs of day 5 uteri of Pla2g4a–/– mice given the vehicle or PGs on day 4 of pregnancy. Note increased number of implantation sites with prominent blue reaction after PG treatment. Brackets indicate crowding of implantation sites.

View larger version (79K): [in a new window]   Fig. 5. Defective postimplantation developments in Pla2g4a–/– mice. (A) A composite photograph of uteri in wild-type and Pla2g4a–/– mice on day 12 of pregnancy. Resorption sites were often noted (arrows) and many implantation sites were closely apposed and even conjoined (brackets) to each other in Pla2g4a–/– mice. (B) Median weights of implantation sites and their embryos on day 12. The implantation sites without embryos (resorption sites) were excluded from this computation. (C) Photographs of embryos isolated from implantation sites of one representative wild-type and two Pla2g4a–/– mice on day 12. Note retarded and asynchronous development of embryos in Pla2g4a–/– mice. (D) Histological examination of day 12 implantation sites in Pla2g4a–/– mice. Feto-placental units from Pla2g4a–/– mice were examined on day 12. Embryos and placentas show defective development with a preponderance of trophoblast giant cells. Arrowheads and an arrow indicate trophoblast giant cells and degenerating embryo, respectively. (c,d) Higher magnifications of a,b, respectively. la, labyrinthine trophoblast; sp, spongiotrophoblast; dec, decidua. (E) Distribution of embryonic weights on day 12 (n=60-75). The horizontal orange lines represent median values of embryonic weights. Pla2g4a–/– mice were given the vehicle or PGs at 10:00 and 18:00 hours on day 4 of pregnancy and killed on day 12. Note a reduction in numbers of retarded embryos in the PG-treated group. (F) Representative photographs of conjoined embryos in a placenta (a,c) and three embryos in the same decidual envelope (b,d) from Pla2g4a–/– mice on day 12. (c) A histological section of (a) with two embryos; embryos shown in (d) are from (b). Yellow arrows indicate the source of the embryos from the decidual envelope.

View larger version (32K): [in a new window]   Fig. 6. Deferred implantation leading to retarded development and poor pregnancy outcome in wild-type mice. (A) Implantation of day 4 wild-type blastocysts transferred into wild-type recipients on day 4 (n=5) or 5 (n=7) of pseudopregnancy. The numbers above the bars indicate the number of implantation sites per total number of blastocysts transferred. Implantation sites were recorded 48 hours later by the blue dye method. Implantation rate was similar between the two groups. (B) Postimplantation developments of wild-type blastocysts transferred into wild-type recipients on day 4 (n=3) or 5 (n=4) of pseudopregnancy. Implantation sites were examined 8 days later after blastocyst transfer equivalent to day 12 of pregnancy. While resorption and retarded feto-placental growth were frequent in recipients that received blastocyst transfers on day 5, vastly normal development was noted in those receiving blastocyst transfer on day 4. Normal implantation sites (IS) represent sites with normally developing embryos, while retarded IS represents resorption sites and IS with retarded embryo development. On day 12, the number of normal IS was significantly higher in day 4 recipients than in day 5 recipients (18/23 versus 10/42; 2-test; P<0.001). (C) The pregnancy outcome of wild-type blastocysts transferred into wild-type recipients on day 4 (n=13) or 5 (n=11) of pseudopregnancy. The numbers above the bars indicate the number of pups delivered at term/total number of blastocysts transferred (2-test; *P<0.01). The number of pups born was significantly lower for mothers receiving blastocyst transfers on day 5. (D) Decidualization in wild-type mice. Mice received intraluminal oil infusion on day 4 or 5 of pseudopregnancy. Uterine weights were recorded 4 days later. Fold increases denote comparison of weights between infused and non-infused uterine horns. The numbers above the bars indicate the number of responding mice/total number of mice. No significant difference in decidualization was noted between these two groups (unpaired t-test; P>0.05).