Auswirkung einer postovulatorischen Alterung auf molekulare Parameter und die Entwicklungskompetenz muriner Oozyten

Die postovulatorische Alterung wird durch eine verzögerte Fertilisation nach der Ovulation hervorgerufen und führt zu einem zeitabhängigen Qualitätsverlust der Oozyte. Dieser macht sich durch eine verringerte Fertilisations- und Implantationsrate, ausgelöst durch verschiedene veränderte zelluläre Prozesse, bemerkbar (Miao et al. 2009). Ziel dieser Arbeit war es, molekulare Parameter der postovulatorisch gealterten Oozyte sowie daraus resultierender 2-Zell-Embryonen weitergehend zu analysieren. Zunächst wurde durch qRT-PCR der Poly(A)-Anteil von 11 Maternaleffektgenen nach postovulatorischer Alterung untersucht. Dabei konnte festgestellt werden, dass nach 24 h Alterung der Poly(A)-Schwanz von 6 maternalen Effektgenen im Vergleich zu Kontroll-Oozyten signifikant verkürzt war. Um diese Reduktion zu validieren und quantifizieren, wurde ein extension Poly(A) Test etabliert welcher die exakte Länge des Poly(A)-Schwanzes eines Transkripts misst. Dieser Test wurde repräsentativ für die Gene Dnmt1 und Zar1 durchgeführt. Dabei konnte für Dnmt1 eine Deadenylierung und für Zar1 keine Änderung der Länge des Poly(A)-Schwanzes nach 24 h postovulatorischer Alterung nachgewiesen werden. Diese Versuchsergebnisse bestätigten die Resultate der qRT-PCR und zeigten zudem, dass eine postovulatorische Alterung Einfluss auf die Poly(A)-Schwanzlänge von Maternaleffektgenen hat. Außerdem wurde durch immunhistologische Methoden eine signifikante Abnahme der Trimethylierung am H3K9 nach postovulatorischer Alterung ermittelt. Um zu untersuchen, ob die postovulatorische Alterung auch einen Effekt auf die frühe embryonale Entwicklung hat, wurden IVF Studien mit gealterten Oozyten durchgeführt. Dabei zeigte sich, dass nach 4, 6 und 8 h postovulatorischer Alterung keine Veränderungen in der relativen Anzahl an 2-Zell-Embryonen im Vergleich zu den Kontrollen detektiert werden konnten. Allerdings wurde durch mikroskopische Analysen festgestellt, dass die Anzahl an 2-Zell-Embryonen mit sichtbaren Spermien im perivitellinen Raum nach 8 h postovulatorischer Alterung im Vergleich zu den Kontrollen signifikant erhöht war. Dies deutet auf eine veränderte Struktur der Zona pellucida nach postovulatorischer Alterung hin. Die Analyse der zygotische Genomaktivierung (ZGA) in 2-Zell-Embryonen zeigte, dass eine postovulatorische Alterung von 4 h zu einer vorzeitigen ZGA führte, während nach 6 und 8 h Alterung eine signifikant verzögerte ZGA auftrat. Zusammenfassend zeigen die Befunde der vorliegenden Arbeit, dass eine postovulatorische Alterung von Oozyten zu deutlichen molekularen Veränderungen in der Oozyte sowie auch in den daraus resultierenden Embryonen führt. Dies könnte schwerwiegende Folgen für die weitere Entwicklung des Embryos haben.Postovulatory aging occurs if fertilization of the ovulated oocyte is delayed. During this delay, unfertilized oocytes undergo a time-dependent deterioration in quality, which results in lower fertilization- and implantation rates. The molecular mechanisms causing this decrease in oocyte competence comprises cell degradation including oxidative stress, chromosome anomalies and epigenetic changes (Miao et al. 2009). The aim of this study was to analyze molecular parameters of postovulatory aged oocytes and effects on their development to 2-cell embryos. For this, the poly(A)-content of 11 maternal-effect genes was investigated after postovulatory aging by qRT-PCR. Shortening of the poly(A)-tail was observed for 6 maternal-effect genes after 24 h postovulatory aging compared to controls. In order to validate this reduction, an extension poly(A) test was established to measure the exact lengths of the poly(A)-tails of the two representative maternal-effect genes Dnmt1 and Zar1. A reduction of poly(A)-tail length was observed for Dnmt1 whereas no changes in poly(A)-tail length for Zar1 were seen after 24 h postovulatory aging. This confirmed the qRT-PCR results and proved that postovulatory oocyte aging may affect the poly(A)-tail length of maternal-effect genes. Additionally, a significant reduction of trimethylation of histon H3(Lysin 9) was observed in postovulatory aged oocytes. To investigate possible effects of postovulatory oocyte aging on their development to the 2-cell embryos after fertilization, IVF studies were conducted. No differences in the relative amount of 2-cell embryos were shown after 4, 6 or 8 hours postovulatory oocyte aging compared to control oocytes. However, a significant increase of sperms in the perivitelline space of 2-cell embryos after 8 hours postovulatory aging was observed, which indicated an altered structure of the zona pellucida after postovulatory aging. Analysis of zygotic genome activation (ZGA) was examined in 2-cell embryos after fertilization of postovulatory aged oocytes. After 4 hours postovulatory aging an increase in ZGA was observed in the resulting 2-cell embryo, whereas after 6 and 8 hours postovulatory aging ZGA significantly decreased in the 2-cell embryos. In summary, it was shown that postovulatory aging strongly affects molecular parameters in the oocyte and the resulting 2-cell embryo and could have major implications of further development of the embryo

Pre- and Postovulatory Aging of Murine Oocytes Affect the Transcript Level and Poly(A) Tail Length of Maternal Effect Genes

Dankert D, Demond H, Trapphoff T, et al. Pre- and Postovulatory Aging of Murine Oocytes Affect the Transcript Level and Poly(A) Tail Length of Maternal Effect Genes. PLoS ONE. 2014;9(10): e108907.Maternal effect genes code for oocyte proteins that are important for early embryogenesis. Transcription in oocytes does not take place from the onset of meiotic progression until zygotic genome activation. During this period, protein levels are regulated posttranscriptionally, for example by poly(A) tail length. Posttranscriptional regulation may be impaired in preovulatory and postovulatory aged oocytes, caused by delayed ovulation or delayed fertilization, respectively, and may lead to developmental defects. We investigated transcript levels and poly(A) tail length of ten maternal effect genes in in vivo-and in vitro- (follicle culture) grown oocytes after pre- and postovulatory aging. Quantitative RT-PCR was performed using random hexamer-primed cDNA to determine total transcript levels and oligo(dT)(16)-primed cDNA to analyze poly(A) tail length. Transcript levels of in vivo preovulatory-aged oocytes remained stable except for decreases in Brg1 and Tet3. Most genes investigated showed a tendency towards increased poly(A) content. Polyadenylation of in vitro preovulatory-aged oocytes was also increased, along with transcript level declines of Trim28, Nlrp2, Nlrp14 and Zar1. In contrast to preovulatory aging, postovulatory aging of in vivo-and in vitro-grown oocytes led to a shortening of poly(A) tails. Postovulatory aging of in vivo-grown oocytes resulted in deadenylation of Nlrp5 after 12 h, and deadenylation of 4 further genes (Tet3, Trim28, Dnmt1, Oct4) after 24 h. Similarly, transcripts of in vitro-grown oocytes were deadenylated after 12 h of postovulatory aging (Tet3, Trim28, Zfp57, Dnmt1, Nlrp5, Zar1). This impact of aging on poly(A) tail length may affect the timed translation of maternal effect gene transcripts and thereby contribute to developmental defects

Sequence and position of CPEs of ME-genes within 150 bp of the PAS.

<p>*Position of PAS-sequence lying closest to 3′ end of the transcript.</p><p>Sequence and position of CPEs of ME-genes within 150 bp of the PAS.</p

Quantification of poly(A) tail length for <i>Dnmt1</i> and <i>Zar1</i> by ePAT.

<p>24 h postovulatory-aged, in vivo-grown oocytes were analyzed by extension poly(A) test (ePAT). A) Gel electrophoresis of the product shows a decrease of poly(A) tail length for <i>Dnmt1</i> in aged oocytes compared to controls, whereas poly(A) tail length of <i>Zar1</i> remains widely stable. These results were quantified by capillary electrophoresis for <i>Zar1</i> (B) and <i>Dnmt1</i> (C). Indicated is the fluorescence intensity (FU) of amplicon lengths (in base pairs) for aged oocytes (red line) and controls (blue line).</p

Follicle morphology, morphokinetics, and hormone concentrations in conditioned medium of preantral follicle culture.

<p>A) Follicle characteristics, culture survival and maturation of control (<i>n</i> = 1230), preovulatory-aged (PreOA; <i>n</i> = 411) and postovulatory-aged (PostOA; <i>n</i> = 613) oocytes. B-D) Antral stage follicle grown in vitro for 12 d (B) and cumulus-oocyte complexes on day 13 after in vitro ovulation in control oocytes (C) and after postovulatory aging (D) for 12 h. E, F) Altered granulosa cell characteristics after preovulatory aging at day 15 of culture; follicles with an increased accumulation of mural granulosa cells and an apparent follicle compaction (E), and a degenerating follicle with dispersed granulosa cells and a nearly denuded oocyte from day 15 of culture (F). G) Estrogen and (H) progesterone levels (mean ± SEM) in conditioned culture medium prior to and past hormonal stimulation by rhCG/rEGF in the different experimental groups (* <i>P</i><0.05, ** <i>P</i><0.01).</p

Expression levels and poly(A) content of ME genes in preovulatory-aged oocytes.

<p>The normalized fold change (mean ± SEM) of preovulatory-aged oocytes compared to control oocytes (dotted line) of total transcript (black bars) and polyadenylated transcript (white bars) is shown. A) After preovulatory in vivo aging, oocytes show a significant decline in total transcript levels for <i>Brg1</i> and <i>Tet3</i>. Comparison of total with polyadenylated transcript levels reveals that poly(A) content of ME gene mRNA tends to increase during preovulatory in vivo aging. B) Total transcript amounts of <i>Trim28</i>, <i>Nlrp2</i>, <i>Nlrp14</i> and <i>Zar1</i> decreased significantly after preovulatory aging in vitro. A similar trend was observed for <i>Nlrp5</i>. Several genes investigated showed a tendency towards a relative increase in poly(A) content compared to total transcript levels, which was most evident for <i>Zar1</i> (t: <i>P</i><0.10, * <i>P</i><0.05).</p

Timeline of pre- and postovulatory aging in vivo (A, B) and in vitro (C, D).

<p>A, B) For in vivo maturation of oocytes, follicle maturation was stimulated by PMSG on day 0. Ovulation was induced by hCG 48 h later. Control oocytes were collected from the ampullae the next morning. For preovulatory in vivo aging, ovulation was delayed by the GnRH antagonist cetrorelix for 3 d (A). Oocytes for postovulatory aging were collected at the same time as controls and cultured in M2 medium for further 12 or 24 h (B). C, D) For in vitro growth and maturation of oocytes, preantral follicles were cultured for 12 d in the presence of rLH and rFSH to the antral follicle stage. Ovulation was induced with rhCG/rEGF and control oocytes were collected after 18 h. To obtain preovulatory-aged oocytes, ovulation was induced with rhCG/rEGF on day 15 of follicle culture instead of day 12 (C). For postovulatory aging, ovulation was triggered with rhCG/rEGF and oocytes were incubated for additional 12 h before collection (D).</p