Culture of zygotes increases TRP53 [corrected] expression in B6 mouse embryos, which reduces embryo viability.

The expression of TRP53 in blastocysts that had been cultured from the zygote stage in vitro for 90 h was compared with that in blastocysts collected from the uterus in C57BL6 (B6) and in F1 hybrid (B6CBF1) strain mice. In both strains, there was little TRP53 detected in blastocysts collected from the uterus. There was some increased expression in cultured embryos from B6CBF1 mice and marked increased expression in cultured B6 blastocysts. In cultured B6 embryos, there was obvious accumulation of TRP53 within the nuclear region of embryonic cells. Cultured B6 zygotes had significantly poorer rates of blastocyst formation and of capacity to undergo implantation or form viable fetuses than cultured zygotes from B6CBF1 mice or B6 blastocysts collected from the uterus. Trp53-/- zygotes (B6 background) were significantly more likely to form blastocysts than sibling wild-type embryos, with Trp53+/- embryos having an intermediate level of viability (P<0.01). On transfer of blastocysts to recipient females, Trp53-/- blastocysts were more likely to form viable fetuses than wild-type or heterozygous sibling blastocysts when the embryos resulted from culture of zygotes (P<0.001). This shift in viability did not occur when embryos were only subjected to 24 h of culture from the compacted embryo stage. Culture in vitro in the B6 strain caused a marked increase in the expression and nuclear accumulation of TRP53. This expression was a significant cause of the loss of viability that occurs on culture of zygotes from this strain in vitro.

Components of a platelet-activating factor-signaling loop are assembled in the ovine endometrium late in the estrous cycle.

Pulsatile release of uterine prostaglandin F(2alpha) (PGF(2alpha)) induces luteolysis in ruminants. Exogenous PAF is well known to cause PGF(2alpha) release from the ovine uterus. This study examines whether the components of a PAF-signaling loop exist in sheep at the time luteolysis is normally initiated. Day 14 of the cycle was the first day the uterus responded to an infusion of PAF, inducing a significant short-term increase in circulating levels of the PGF(2alpha) metabolite. There was a significant increase of PAF concentration (P < 0.001) in the endometrium and PAF release by tissue explants (P < 0.001) from day 10 to day 16 of the cycle. Endometrial tissue PAF receptor mRNA expression was induced (P < 0.01) by estradiol and progesterone treatment of animals, and transcripts were present between days 10 and 16 of the estrous cycle. Western analysis of endometrial tissue showed marked upregulation of PAF receptor protein expression from day 14 of the cycle, and immunolocalization studies showed that the receptor expression was predominantly around the endometrial glands. PAF:acetylhydrolase was primarily located within the lumen of the endometrial glands. The study shows that a PAF-signaling loop was assembled within the ovine endometrium at the time that PGF(2alpha) pulsatility was first observed.

Evidence for the autocrine induction of capacitation of mammalian spermatozoa.

Mammalian spermatozoa require a maturational event after ejaculation that allows them to acquire the capacity for fertilization. This process, known as capacitation, occurs spontaneously in simple defined medium implicating a potential role of autocrine induction. This study shows that the ether phospholipid 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine (PAF) meets the criteria for an autocrine mediator of capacitation. Sperm released PAF after their dilution into capacitation medium and expressed a receptor for PAF on their membranes. PAF stimulated changes in the motility of sperm and enhanced fertilization in vitro. These actions were inhibited by a PAF receptor antagonist (UR-12519) and by extracellular recombinant PAF:acetylhydrolase (an enzyme that degrades PAF to a biologically inert form). Seminal plasma contained an acid-labile PAF:acetylhydrolase, whereas capacitation was inhibited by an acid-labile factor within seminal plasma, implicating this factor as a potential decapacitation factor within seminal plasma. Sperm from a PAF receptor knock-out mouse strain failed to express the receptor and displayed a significantly (p < 0.01) reduced rate of capacitation, as assessed by the spontaneous onset of the acrosome reaction in vitro. When used for in vitro fertilization, sperm from PAF receptor knock-out mice gave a significantly lower rate of fertilization (21.5%) than did wild-type sperm (66.7%). The study shows for the first time the operation of an autocrine loop that induces capacitation in sperm in vitro and shows that this loop acts in concert with other mediators of capacitation to promote efficient fertilization.

Identification and regulation of the platelet-activating factor acetylhydrolase activity in the mouse uterus in early pregnancy.

Platelet-activating factor (PAF) is a product of the embryo and the endometrium in early pregnancy. The actions of PAF may be regulated by its degradation and this is largely achieved by the enzyme PAF acetylhydrolase (PAF:ah; EC 3.1.1.47). The present study characterized the PAF:ah in the endometrium and uterine fluid of mice during early pregnancy. The enzyme activity from uterine endometrium and luminal fluids had the same biochemical characteristics as the plasma form of the enzyme. The three sources of enzyme activity (i) had an apparent native molecular mass greater than 10(6) Da, but this was reduced after detergent treatment and purification to 60-65 kDa; (ii) bound to cholesterol hemisuccinate agarose matrix; and (iii) were found in the high density lipoprotein-enriched fraction after density gradient ultracentrifugation. In castrate females, oestradiol-17beta (E2) caused a dose-dependent increase in the activity of the enzyme in endometrium and luminal fluid. Progesterone (P4) inhibited the E2-induced increase in PAF:ah in uterine tissue. Treatment with E2 alone caused an increase in endometrial PAF:ah activity within 24 h, which declined within 48 h. In luminal fluid, the same treatment caused increased activity within 24 h, peaking after 48 h of treatment and then declining. In E2-treated castrate females, mRNA for an intracellular (but not plasma) form of PAF:ah was detected, yet the intracellular form was not detected biochemically. The results suggest that most of the enzyme activity was not produced locally, but probably resulted from the influx of the plasma form of the enzyme.

Platelet-activating factor may act as an endogenous pulse generator for sheep of luteolytic PGF2alpha release.

Pulsatile release of uterine prostaglandin F2alpha (PGF2alpha) induces luteolysis in ruminants. However, the mechanism(s) that initiates and maintains luteolysis has not been defined. The present study tested the hypothesis that the endogenous PGF2alpha pulse generator is uterine-derived platelet-activating factor (PAF). Ovariectomized ewes were given exogenous progesterone (P), estradiol (E), or both (P+E, mimicking the normal luteal phase). Only ewes treated with steroids released PAF into the uterine lumen and had increased PAF:acetylhydrolase activity in the uterine lumen. Steroid treatment also influenced the capacity of the uterus to release PGF2alpha in response to exogenous PAF. PAF infusion did not affect plasma PGF2alpha metabolite (PGFM) levels in control (no steroid treatment) ewes but increased plasma PGFM levels in P+E ewes (P < 0.001) and ewes treated with P or E alone (P < 0.05). Infusion of PAF followed by or coincident with oxytocin (OT) acted in a synergistic manner to increase plasma PGFM levels. Repeated infusion of PAF into the uterus at 1-h intervals induced tachyphylaxis of the PGFM response to PAF; however, sensitivity of the uterus to PAF returned spontaneously by the 6th h. Interferon-tau (IFN-tau) inhibits pulsatile release of PGF2alpha during pregnancy to prevent luteolysis. Exogenous recombinant ovine IFN-tau (50 microgram) inhibited the uterine response to PAF alone or the combined effects of PAF and OT. These results indicate that uterine PAF fulfills many of the criteria for an endogenous PGF2alpha pulse-generator: steroid induction of PAF production and uterine responsiveness to PAF-induced release of PGF; synergistic stimulation of PAF-induced PGF release by OT; inhibition of PAF effects by IFN-tau; and PAF's ability to induce pulses of PGF with a periodicity during a period of chronic exposure of the uterus to PAF.