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a Department of Animal Science, University of Missouri, Columbia, Missouri 65211
b Agricultural Research Service, United States Department of Agriculture, Columbia, Missouri 65211
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Analysis of in vivo- and in vitro-derived control embryos revealed a decline in cyclin B1 transcripts from 5 to 33 h post-4-cell cleavage (P4CC). The quantity of cyclin B1 for the in vivo-derived embryos at 5 and 33 h P4CC was 11.26 and 4.54 attomol/embryo, respectively (P < 0.03), while the in vitro-derived embryos had 20.18 and 7.52 attomol/embryo, respectively (P < 0.03). Treatment with alpha-amanitin from 5, 10, 18, or 25 h P4CC to 33 h P4CC resulted in cyclin B1 quantities that did not differ from those in the 33-h control embryos, irrespective of time spent in the inhibitor. These findings suggest that maternal cyclin B1 transcript degradation occurred over the 4-cell stage with no detectable embryonic cyclin B1 transcripts produced.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The relationship between regulation of the MZTT and cell cycle control within the early embryo is complex to say the least. In Xenopus, the mid-blastula transition appears to be the time when cleavage control switches from maternal to zygotic [4]. This transition occurs following 12 rapid, highly synchronous cleavages and is characterized by a switch to slow asynchronous divisions.
In Drosophila, the timing of the MZTT is slightly different from that in Xenopus. Edgar [5] reports that a significant switch from maternal to zygotic cell cycle control occurs at the 14th interphase. Nurse [6], along with Edgar and Lehner [7], reports that the transition at the 14th interphase appears to be controlled by depletion of the maternal cdc25 phosphatase, STRING. STRING is a key regulator of the maturation promotion factor (MPF) complex responsible for the G2 to M shift in the cell cycle. MPF is composed of two subunits, p34cdc2 and cyclin B1 [8]. It has been established that the degradation of an additional maternal cdc25 phosphatase, TWINE, also drives the transition from maternal to zygotic control [5]. However, the degradation of maternal STRING is inhibited by alpha-amanitin, a potent RNA polymerase II and III inhibitor [5]. This information implies that zygotic transcription of certain gene(s) is required for maternal mRNA degradation.
The MZTT is thought to occur at the 2-cell stage in the mouse [1, 2, 9]. In addition to the onset of mouse embryonic transcription beginning at the 2-cell stage, Yokoi et al. [10] report a major mRNA degradation event occurring over the same period. A drop in maternal mRNAs to 10–20% of initial levels was observed. Nothias et al. [2] add that approximately 90% of maternal mRNA degradation was completed during the 2-cell stage with synthesis of some proteins from maternal transcripts continuing to the 8-cell stage. These observations tend to suggest that both the rate and timing of maternal RNA degradation play a role in MZTT timing in the mouse.
In other mammals, the timing of the onset of the MZTT varies. The MZTT occurs at the 4-cell stage in both pigs [11] and humans [12], whereas in cattle, sheep [9], and rabbits [13] the transition is at the 8- to 16-cell stage. In the domestic cat, MZTT occurs over the 5- to 8-cell stage [14].
As mentioned above, cdc25 phosphatase acts on MPF in Drosophila at the G2 to M transition of the cell cycle. The series of events and participants at the G2 to M transition appear to be universally conserved among species. Activation of the MPF dimer occurs when p34cdc2 is dephosphorylated by cdc25 at certain tyrosine and threonine residues [8]. In addition, the levels of cyclin B1 vary over the course of the cell cycle with a set threshold level necessary to achieve dimer formation with p34cdc2 at the onset of mitosis [15]. Therefore, both cdc25 phosphatase and cyclin B1 can be thought of as positive regulators of the G2 to M cell cycle transition as their activities are absolutely required for MPF formation and function.
Pope and First [16] report that one of the major periods of in vivo porcine embryonic mortality occurs over the first 40 days of development. Some of this loss may be due to problems with embryonic transcription initiation over the MZTT and the subsequent posttranscriptional difficulties that result. These possibilities, coupled with the difficulties associated with porcine IVM-IVF technology such as polyspermy [17], low rates of male pronuclear formation [3], and decreased rates of embryonic development [18], led us to explore the relationship between in vivo and in vitro embryonic loss with cell cycle control over the MZTT. Specifically, a quantitative reverse transcription-competitive polymerase chain reaction (RT-cPCR) technique was used to study cyclin B1 transcript levels, whose role following translation is that of a universal positive regulator of mitotic entry.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All chemicals and reagents were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted.
Animals
Experiments involving the use of animals were conducted in accordance with protocols approved by the University of Missouri Animal Care and Use Committee. Landrace crossed gilts in this study had cycled at least twice before being used as a source of viable in vivo-produced embryos. Gilts were checked for signs of estrus once per day, with the onset of estrus denoted as Day 0. Gilts in estrus were bred using artificial insemination (AI), and the resulting embryos were flushed early on Day 2 according to previously published procedures [18]. All recovered embryos were pooled together for later random distribution to replicates. The uterine flush and in vitro embryo culture medium was Whitten's (WM) plus 0.3% BSA [19].
Embryo In Vitro Culture and Timing
In vivo-derived 2-cell embryos were cultured in vitro according to standard protocols [19]. To monitor when individual 2-cell embryos cleaved, embryos were briefly checked every 2 h. To minimize environmental fluctuations and associated slowdown of in vitro embryonic development during cleavage checks, four steps were taken. First, the culture plates (Falcon 1008; Fisher Scientific, St. Louis, MO) containing 2-cell embryos were kept on water-filled culture flasks (Falcon 3111; Fisher Scientific) in the incubator. When the cleavage check occurred, the entire plate/flask system was removed to help minimize temperature fluctuations, and cleavage status was determined. Second, all dissecting microscopes used for embryo viewing were equipped with a heated stage (Cryogenic Concepts, Bethlehem, PA) set at 39°C. Third, the time of the cleavage check was kept to a minimum, typically less than a few minutes. Fourth, all embryos were cultured in 50-µl drops of WM under light mineral oil.
When embryos cleaved to the 4-cell stage, the time was noted, and the embryos were individually moved to a different drop of medium and randomly assigned to one of two treatment groups: a control group and a group treated with alpha-amanitin (RNA polymerase II inhibitor). The control group represented individual embryos cultured in WM as described above for either 5, 10, 18, 25, or 33 h post-4-cell cleavage (P4CC). These times represent early, mid, and late periods within the 4-cell stage for in vivo-derived embryos cultured in vitro.
The inhibitor-treated embryos were cultured in WM+20 µg/ml alpha-amanitin under the same conditions as the control group. The actual time of exposure to the inhibitor varied based on the time point to which individual embryos were assigned. The times of alpha-amanitin exposure for the individual embryos were 1) from 5 to 33 h P4CC, 2) from 10 to 33 h P4CC, 3) from 18 to 33 h P4CC, and 4) from 25 to 33 h P4CC.
After timed culture of single embryos in the two treatment groups, each embryo was rinsed through diethylpyrocarbonate-treated PBS (DEPC-PBS) three times. The individual embryos were then transferred to 500-µl microfuge tubes (Fisher Scientific) containing 10 µl DEPC-PBS, flash frozen in liquid nitrogen, and stored at -80°C until poly(A) RNA could be isolated, usually within 2 days. Each replicate consisted of the 9 individual 4-cell embryos from the two treatment groups plus RT-cPCR controls (see below).
In Vitro-Derived Embryos
Porcine ovaries were collected at a local abattoir (Excel Co., Marshall, MO) and transported to the laboratory in saline plus antibiotics [20] at 37°C. Oocytes were aspirated from 4- to 7-mm follicles. After IVM-IVF [20], 1-cell embryos were cultured in WM as described above until cleavage to the 4-cell stage occurred. Upon cleavage to the 4-cell stage, individual embryos were assigned to the same control and alpha-amanitin treatment groups as described for the in vivo-derived embryos. The timing for the in vitro-derived embryos and subsequent handling prior to poly(A) RNA isolation was as described above.
In Vivo-Derived Metaphase II (MII) Oocytes, 2-Cell Embryos, and Late Compact Morulae (LCM)
In addition to the 4-cell-stage embryos used in this study, three other groups of in vivo-derived embryos/oocytes were collected for RT-cPCR. The groups included MII oocytes, early 2-cell embryos, and LCM. The developmentally competent MII-arrested oocytes were collected on Day 2 after standing estrus on Day 0. The 2-cell embryos were collected early on Day 2, and the LCM were collected on Day 4.5 following estrus detection and use of the AI protocols described above. After surgical collection, individual oocytes/embryos were rinsed in DEPC-PBS, flash frozen, and stored as described above with minimal time in vitro. These three groups were chosen to represent reference points during early porcine development outside the MZTT.
Poly(A) RNA Isolation
Individual in vivo- or in vitro-derived embryos/oocytes were subjected to poly(A) RNA isolation via the Hybond-messenger affinity paper (Hybond-mAP; Amersham, Arlington Heights, IL; now Amersham Pharmacia Biotech, Piscataway, NJ) technique [21]. Briefly, individual embryos in DEPC-PBS were incubated for 2 h at room temperature with a 3- to 4-mm2 piece of Hybond-mAP in guanidium isothiocyanate (GITC) lysis solution (4 M GITC; 0.1 M Tris-HCl, pH 7.4; 1 M beta-mercaptoethanol; all in DEPC-H2O). After incubation, the Hybond-mAP was placed on Whatman filter paper (Fisher Scientific), and the aqueous contents of the microvial were slowly spotted onto the membrane. The Hybond-mAP was next washed twice in 0.5 M NaCl + 0.1 M Tris-HCl, pH 7.4, in DEPC-H2O. Two additional washes in 0.5 M NaCl in DEPC-H2O and two final rinses in 70% ethanol followed the initial wash. The Hybond-mAP was then allowed to air dry for a few minutes before the immediate initiation of reverse transcription (RT).
Embryo Reverse Transcription
To help minimize potential sources of variation, all RT and cPCR mixes described below were derived from large (> 15 ml) mixes prepared ahead of time, aliquoted into smaller volumes, and frozen until needed. Hybond-mAP with attached poly(A) RNA from individual oocytes/embryos was used in RT procedures. The RT reactions were carried out using a PTC-100 Peltier effect thermocycler with a nonheated lid (MJ Research, Watertown, MA) under conditions of 42°C for 45 min followed by 95°C for 5 min. Individual RT reactions consisted of the following components: 5 mM MgCl2 (Fisher Scientific), single-strength buffer B (10 mM Tris-HCl, pH 8.3, 50 mM KCl; Fisher Scientific), 2.5 µM random hexamers (Promega, Madison, WI), 1 mM each dNTP (Amersham), 20 IU RNase inhibitor (Perkin Elmer, Branchburg, NJ), and 50 IU murine leukemia virus reverse transcriptase (Perkin Elmer). The final individual RT reaction volume of 20 µl was achieved by adding Milli-Q water (MQ-H2O; Millipore Corp., Bedford, MA). A 50-µl light mineral oil overlay was added to each reaction.
In addition to each group of 9 embryos per replicate undergoing the poly(A) RNA isolation procedure and RT, an additional set of 3 RT-cPCR controls per replicate underwent the same procedure. Control 1 was a positive RT control and consisted of Day 17 porcine embryo RNA. Control 2 was a DNA contamination control identical to control 1 except for omission of reverse transcriptase from its master mix. Control 3 was a negative control and contained only DEPC-PBS. After completion of RT, all cDNAs from individual time points and controls were stored at -40°C until future use in cPCR.
Clones
We isolated a 551-base pair (bp) cyclin B1 clone (GenBank accession no. L48205) from Day 17 porcine embryo total RNA by reverse transcription-polymerase chain reaction (RT-PCR) using the cyclin B1 upper and lower primers listed below. The cyclin B1 primer sequences were designed from human sequences available [22]. The 551-bp product was gel purified and subcloned using the pGEM-T Vector System (Promega). After isolation and purification, the identity of the clone was confirmed using nonradioactive silver sequencing (Promega) in three independent subclones. The 551-bp cyclin B1 clone was shown to be 93.5% homologous to a human cyclin B1 clone (accession no. M25753).
The 551-bp cyclin B1 clone was used as the cDNA source for the digoxigenin-11-dUTP (DIG)-labeled probes employed in the nonradioactive Southern hybridization procedures described below. In addition, the nested cyclin B1 primer sequences and associated 503-bp amplified product, as well as the 435-bp competitor discussed below, were derived from the sequence of the original 551-bp clone. The nested primers were used in RT-cPCR because of an observed increase in amplification robustness over the original primer set.
Primers
The following primers were synthesized (Gibco-BRL; Gaithersburg, MD) and were used in all experiments. Sequences are read 5' to 3': cyclin B1 upper, TCC AAG CCC AAT GGA AAC AT; cyclin B1 lower, ATG CTC TCC GAA GGA AGT GC; cyclin B1 nested (N) upper, CTG TGC CCC TGC AGA AGA AT; cyclin B1 N lower, GTA GAG GGC GAC CCA GAC AA; cyclin B1 ``40-mer," GTA GAG GGC GAC CCA GAC AAT CAC AAA GGC AAA GTC ACC AA.
Competitor Production and Use
The 435-bp truncated cyclin B1 competitor was generated and used as described in Figure 1. The use of the cyclin B1 40-mer [23] was the key to the production of the competitor, which was capable of being coamplified with the full-length 503-bp cyclin B1 product using one set of primers (N upper and lower). Once a product the projected size of the competitor was observed in PCR (435 bp), the fragment was subcloned and purified, and identity was confirmed as described above. The competitor was then diluted from a stock of known concentration, and the working stock concentration of 200 attograms (ag)/µl was used in each individual cPCR. The use of a truncated, homologous cyclin B1 DNA competitor was ideal for these experiments because of its stability and equivalent amplification efficiency as compared with the 503-bp species [24].
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Standard-Curve Dilution Series
Using the subcloned 503-bp cyclin B1 product of known concentration as starting material, a series of 10-fold, then log dilutions, was made. The log dilutions were used in a range that would correspond under cPCR conditions to that of the amplification observed for the individual embryos. Of the standards diluted, a log dilution series with concentrations from 27 to 800 fg/µl gave the best amplification profile over the exponential range of the cPCR. When these standards were coamplified with 200 ag/µl of the truncated competitor, a linear relationship resulted that corresponded well with values obtained for individual embryos coamplified with 200 ag/µl of competitor during cPCR (Fig. 2).
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Embryo cPCR
Competitive PCR was carried out on embryos from each replicate and standards using a second isolated PTC-100 Peltier effect thermocycler with heated lid (MJ Research). For each time point within a replicate, 3 different volumes of cDNA were used in individual cPCRs along with a consistent concentration of competitor (200 ag/µl). Total individual cPCR volume was 25 µl; it consisted of 20 µl cPCR master mix plus either 5, 2.5, or 1.25 µl of embryo cDNA from the above-described RT. An additional 2.5 or 3.75 µl of "filler" master mix (cPCR master mix minus enzymes) was added to those reactions where 2.5 or 1.25 µl, respectively, of cDNA was added to the cPCR master mix for a total volume of 25 µl. Therefore, since each embryo time point was represented by three individual cPCRs, the total number of embryo reactions per replicate was 27 plus the 3 controls. The 30 embryo cPCRs were coamplified in conjunction with 11 standard-curve cPCRs (standards and ± controls) for a total of 41 reactions. These 41 reactions represent one complete replicate.
The conditions of the cPCR over the predetermined exponential portion of the amplification profile for both 503- and 435-bp cyclin B1 were as follows: 1 cycle of 95°C, 4 min; 35 cycles of 95°C for 30 sec, 60°C for 30 sec, and 72°C for 1 min; 1 cycle of 72°C, 5 min. Each cPCR consisted of the following components: 1.25 mM MgCl2, single-strength buffer B (10 mM Tris-HCl, pH 8.3; 50 mM KCl), 1.25 IU Taq Polymerase (Fisher Scientific), 30 pmol each cyclin B1 N upper and lower primers, 200 ag cyclin B1 truncated 435-bp competitor (Fig. 1), and MQ-H2O. A 50-µl light mineral oil overlay was added on top of each reaction.
Standard-Curve cPCR
Since both embryo and standard-curve cPCRs for a single replicate were coamplified, the reaction conditions were the same. Components of each individual standard cPCR were 3.75 mM MgCl2, single-strength buffer B (9.5 mM Tris-HCl, pH 8.3; 45 mM KCl), 0.9 IU Taq polymerase, 50 pmol each N cyclin B1 upper and lower primers, 0.3 mM each dNTP, 200 ag truncated 435-bp cyclin B1 competitor, standard cDNA, and MQ-H2O for a final volume of 25 µl. A 50-µl oil overlay was added to each reaction. The production, dilution, and concentration range of the 503-bp standards used in each cPCR were described above.
Agarose Gel Electrophoresis
After completion of cPCR, loading dye (Promega) was added to each reaction, and a portion of each reaction was loaded into a 2.5% agarose/synergel (Gibco, Gaithersburg, MD/Diversified Biotech, Boston, MA) matrix. The matrix contained ethidium bromide (0.5 µg/ml; Fisher Scientific) for ultraviolet (UV) viewing. Horizontal gel electrophoresis (model H4; Gibco) was performed on all cPCR samples.
The matrix containing the cPCRs and molecular weight markers (PGEM; Promega) was run in single-strength Tris-acetate-EDTA electrophoresis buffer for 8 h at 50 V to allow for adequate separation of 503-bp cyclin B1 from 435-bp truncated competitor. After completion of electrophoresis, the gel was viewed under UV illumination, and a digital record was made using the Gel Print 2000I (Genomic Solutions Inc., Ann Arbor, MI). The area containing the cPCR bands was cut from the gel in preparation for Southern hybridization.
Southern Hybridization
Southern hybridization was used to confirm the identity of cyclin B1 products generated in RT-cPCR. The procedure has been previously described [25]. The nylon membrane used for the transfer was Magnacharge (Micron Separations Inc., Westborough, MA). The procedure used to prepare the membrane and matrix for blotting, along with the hybridization of the DIG-labeled 551-bp cyclin B1 probe to the membrane, is described in the Genius System User's Guide (Boehringer-Mannheim, Indianapolis, IN). Random prime labeling was used to produce the nonradioactive DIG-labeled 551-bp cyclin B1 probe, and hybridization at a concentration of 20 ng/ml was carried out at 65°C for at least 8 h.
The final step of the Southern hybridization procedure was colorimetric detection (Genius System). This procedure used an anti-digoxigenin-alkaline phosphatase (anti-DIG) to recognize the 551-bp cyclin B1 probe. The colored bands were then captured as a digital image by densitometric analysis as described below.
Computer Analysis of Southern Blots
Digital membrane images were analyzed in the x, y, and z axes using the Molecular Analyst (MA) software package (Bio-Rad Laboratories, Hercules, CA) following densitometry (Bio-Rad; Model GS-700) with a personal computer (Gateway 3100; 266-mHZ processor; Sioux Falls, SD). With use of the MA software, individual bands representing both 503-bp and truncated cyclin B1 competitor could be examined for all samples. After local background subtraction, ratios for each individual embryo as well as the standards were formulated for 503-bp cyclin B1 cDNA:competitor. These ratios could then be used to generate the quantity of cyclin B1 poly(A) RNA present for each embryo at a specific time point as described below.
Standard-Curve Generation and Use
Averaging the 503-bp cyclin B1:competitor ratio from all replicates for each specific cyclin B1 standard generated standard-curve values. After log transformation of each standard concentration, the average ratio along with the log-transformed concentration of each standard was used to generate a line of best fit and its equation (Fig. 2). Next, the embryo ratios from each time point were used in the standard-curve equation to generate a relative concentration for each. Of the embryo ratios generated, only those that fell within the ± 10% range of the low and high standard-curve ratio values, respectively, were used in determination of final embryo cyclin B1 RNA values at each specific time point. These relative concentrations were then adjusted to compensate for the difference in volume of cDNA used in the cPCR and volume of cPCR product loaded into the agarose/synergel matrix. The resulting compensated cyclin B1 transcript values were then capable of being compared, since all were equivalent to 1 embryo.
Additional criteria that had to be met before a specific embryo ratio was used in final cyclin B1 poly(A) RNA concentration determination included the following. 1) Parallelism: An uncompensated embryo value (i.e., not multiplied by appropriate dilution factors) had to correspond to at least one of the other two values for that particular time point within the replicate before being accepted. For example, the uncompensated value for the 1.25-µl cDNA dilution used in cPCR for a specific time point had to be approximately one half the value of the 2.5-µl dilution and/or one fourth the value of the 5-µl cDNA used in the cPCR. If two or more values exhibited parallelism, then the average of the adjusted dilution values would be used in cyclin B1 transcript calculation. In the situation in which no parallelism was observed for a specific time point, then the middle 2.5-µl cDNA dilution value was used, since instances in which the 5- and 1.25-µl dilutions exhibited either out-competition or under-competition, respectively, occurred. 2) Abnormal competition: In a few situations in which the full-length 503-bp embryo-derived cyclin B1 cDNA totally out-competed the truncated competitor in the 5-µl cPCR for a specific time point and parallelism was not observed, the ratio obtained from the most dilute cDNA (i.e., 1.25 µl), which exhibited normal competition (fell within the range of the standard curve), was used in cyclin B1 RNA quantity determination. The typical range of ratios observed for embryo unknowns was from approximately 0.7 to 3.5 (Fig. 2). When abnormal competition occurred, the ratio would be an inflated value, ranging from 5- to 10-fold greater. In a few cases, individual embryos (6 total from both in vivo and in vitro sources) failed to meet the above criteria and were not used in embryo time point calculations.
Statistical Analysis
The cyclin B1 values generated for each individual embryo were analyzed by calculating least significant differences using the General Linear Models feature of the Statistical Analysis System (SAS Institute, Cary, NC) in one-way ANOVA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Through use of the cyclin B1 standard curve, values for both in vivo and in vitro treatment groups (control and alpha-amanitin) within each replicate were generated. Upon comparison of the values obtained for each time point in both in vitro- and in vivo-derived control and alpha-amanitin-treated embryos, a similar relationship was observed (Fig. 3). In both the in vivo (Fig. 3A) and in vitro (Fig. 3B) control embryos, the highest level of cyclin B1 poly(A) RNA expression was at 5 h P4CC (11.26 and 20.18 attomol/embryo, respectively), and the lowest level was at 33 h P4CC (4.54 and 7.52 attomol/embryo, respectively; P < 0.03). The 10-, 18-, and 25-h P4CC in vivo control embryo time points showed a somewhat reduced level of cyclin B1 poly(A) RNA in comparison to that of the 5-h time point (7.63, 7.42, and 6.51 attomol/embryo, respectively). The same time points in the in vitro control embryos exhibited a greater relative decline in value as compared to the in vivo group (11.83, 9.91, and 9.40 attomol/embryo, respectively), with 18 h being the first time point to exhibit a reduced level of cyclin B1 (P < 0.03).
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The alpha-amanitin-treated in vivo and in vitro embryos both exhibited a low, maintained level of cyclin B1 over the time points studied, irrespective of the time spent in the inhibitor. The values for the in vivo alpha-amanitin embryos beginning inhibitor treatment at 5, 10, 18, and 25 h P4CC were 5.49, 4.48, 5.86, and 6.18 attomol cyclin B1 per embryo, respectively, while the in vitro embryos had levels of 7.67, 9.92, 8.58, and 9.47 attomol cyclin B1 per embryo, respectively. None of the alpha-amanitin-treated in vivo- or in vitro-derived embryos displayed a significantly lower level of cyclin B1 transcript as compared to their respective 33-h control groups (Fig. 3, A and B).
In Vivo-Derived MII Oocyte, 2-Cell, and LCM Embryo Reference cPCR
After cPCR of MII oocytes (n = 7) and 2-cell (n = 7) and LCM (n = 7) embryos, the ratios obtained from comparing the embryonic-derived 503-bp cyclin B1 to the 435-bp competitor were too large (data not shown) to be analyzed using the standard curve (Fig. 2). Essentially, the quantity of embryonic cyclin B1 cDNA was large enough in the 3 groups to cause a total out-competition of the introduced competitor. The ratios obtained from these samples greatly exceeded the maximum acceptable standard-curve value, with many approaching a value of 7 or greater (data not shown).
Sperm Cyclin B1 Poly(A) RNA Content
In vivo-derived 4-cell porcine embryos have a large, variable number of sperm (200 or more; unpublished results) attached to the zona pellucida (zp). This observation led us to use RT-PCR to check for the presence of cyclin B1 transcripts in the numerous sperm attached to the zp. After collection of in vivo embryos as described above, individual zp with attached sperm were carefully excised from the accompanying blastomeres of 4-cell embryos using 25-gauge needles. The excised zp were placed in groups of 1, 2, or 3, with 3 replicates of each obtained. Poly(A) RNA was extracted, and RT-PCR with appropriate controls was carried out as described above. No competitor was used in the reaction since cyclin B1 poly(A) RNA presence or absence was in question, not quantity. After Southern hybridization with cyclin B1 probe, no bands from any of the replicates mentioned above were visible (data not shown). From these findings it was concluded that no detectable amounts of cyclin B1 transcript were present in porcine spermatozoa or the zp to which they were attached.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The quantitative RT-cPCR methodology used in this study measured all cyclin B1 transcripts, irrespective of origin, in the developing 4-cell embryo. Problems associated with the incorporation of [3H]uridine into early porcine embryos [26] kept us from using this more direct method of measuring embryonic transcript production and therefore differentiating between maternal- and embryonic-derived cyclin B1 transcripts. Even with this limitation imposed on our system, the combination of a highly sensitive, repeatable, and quantitative RT-cPCR technique along with the use of an RNA polymerase inhibitor on embryos for varying lengths of time allowed for an accurate assessment of attomolar quantities of cyclin B1 transcripts over the MZTT.
Our findings of a steady decrease in cyclin B1 transcript levels as one progresses from early to late in the 4-cell stage was not expected. One would expect an increase in cyclin B1 transcript levels sometime during the 4-cell stage, since embryonic gene activation at the MZTT is known to occur over this period [11]. In addition, it is widely accepted that cyclin protein levels change as cell cycle progression occurs [27]. Cyclin B1 protein accumulates through interphase, with highest levels at G2/M, and an immediate decline at the metaphase/anaphase transition [22].
Historically, protein levels have been thought to follow mRNA levels for a specific gene. However, recent work suggests this is not always true. Mitra and Schultz [28] found that changes in protein concentrations of mouse oocyte cell cycle participants, such as cyclin B1, wee1, cdc2, and cdc25C, were not reflected by similar changes in their respective transcript concentrations. These authors state that posttranscriptional regulation such as translational efficiency most likely regulate the protein levels observed. Indeed, Memili et al. [29] discuss how early preimplantation development is highly regulated at the posttranscriptional level. These authors found an active transcriptional complex, which is alpha-amanitin insensitive, at the 2- to 4-cell stage of bovine embryos that is not required for progression of embryonic development. This "early" transcriptional complex is highly regulated at the posttranscriptional level and precedes the start of the alpha-amanitin-sensitive MZTT at the 8- to 16-cell stage.
Other work [30, 31] completed in the mouse showed a minor activation of the zygotic transcriptional machinery at the 1-cell stage. These studies showed posttranscriptional control to be present in the 1-cell embryo. At the 1-cell stage, the zygotic gene activation was shown to be a minor event, with the MZTT reached at the 2-cell stage where major activation along with significant maternal RNA degradation occurred. These findings along with those of Nothias et al. [2], who found translation of maternal transcripts to proteins occurring as late as the 8-cell-stage mouse embryo, add to the evidence of the complex posttranscriptional control active during the early stages of development, particularly at the MZTT.
The data presented above from cattle and mice, detailing the posttranscriptional control of a wide variety of transcripts and their protein products, may help to explain why no significant increase in cyclin B1 transcript levels was observed in the 4-cell porcine embryos used in this study. Even though preparations for the next mitosis are under way in the 4-cell porcine embryo, the quantity of cyclin B1 transcript may not reflect this due to posttranscriptional control. In addition, cyclin B1 may be one of the maternally derived transcripts active after the initiation of the MZTT as others have shown [2].
The use of MII oocytes, early 2-cell embryos, and LCM as reference points outside of the 4-cell stage resulted in out-competition of the truncated competitor by the oocyte/embryo-derived cDNA in all cases. Even though no actual values could be calculated from these three groups, the results indicate the presence of substantially larger pools of cyclin B1 poly(A) RNA than observed over the 4-cell stage. One interpretation of these results is that higher levels of maternally derived cyclin B1 transcript were present before the MZTT at the MII oocyte and 2-cell embryo stages, while the LCM stage displayed higher levels of embryonically derived transcripts following completion of the MZTT.
The time points studied over the MZTT represented early, mid, and late periods within the 4-cell stage when both in vivo- and in vitro-derived porcine embryos are cultured in vitro. The length of the porcine 4-cell stage during in vitro culture with WM varies quite a bit, but has been shown to be up to 50 h in length [19]. With use of the techniques outlined above to minimize environmental fluctuations, the overall length of the 4-cell stage in our experiments was approximately 38–44 h (data not shown). Embryos cultured to 33 h P4CC never cleaved to the next cell stage, while embryos cultured for periods longer than 33 h gave more variable results, with some progressing to the 8-cell stage earlier than others.
The treatment of embryos in alpha-amanitin for varying lengths of time up to 33 h P4CC after WM culture was chosen as an end point because of findings by our group as well as others [11, 19]. In our experiments, the culture of early 4-cell embryos in the inhibitor blocked progression to the next cell stage in all cases, while extended culture of 4-cell embryos in WM alone resulted in cleavage to the 8-cell stage and beyond (data not shown). More specifically, Schoenbeck et al. [19] showed that placement of embryos in alpha-amanitin at 0, 4, 8, 12, 16, and 20 h P4CC resulted in no cleavage to the 8-cell stage. However, culture in inhibitor starting at 24, 30, and 36 h P4CC resulted in cleavage of 40%, 100%, and 100%, respectively. This implied that porcine zygotic activation occurred by 24 h P4CC. The uptake and incorporation of [35S]methionine as an indirect indicator of zygotic gene activation by the 4-cell-stage porcine embryo has also been employed [11, 19]. A qualitative change in protein profiles at 16 h P4CC with culture in the presence of the inhibitor, alpha-amanitin, was observed [19]. In addition, it was shown that maternally directed protein synthesis decreased from the time of fertilization to 16 h P4CC with a subsequent increase at later time points. Taken together, the alpha-amanitin and [35S]methionine data provided strong evidence that a major shift in embryonic control occurred from approximately 16 to 24 h P4CC. With these findings in mind, we decided to study P4CC time points that occurred earlier than (5, 10 h P4CC), at the time of (18, 25 h P4CC), and later (33 h P4CC) than the suspected 4-cell-stage transitional period.
Problems associated with porcine IVM-IVF may potentially contribute to adverse changes in the molecular mechanisms responsible for early embryo development, ultimately leading to abnormalities in cell cycle control and loss of in vitro embryos. Key protein participants at the G2 to M transition that may be affected by IVM-IVF conditions include cyclin B1, cdc25C, p34cdc2, and others. These problems prompted the study of some of the possible molecular mechanisms, such as transcriptional control over the MZTT, that may contribute to this large in vitro embryonic loss in pigs. The lack of a difference in quantity of the in vivo- and in vitro-derived embryo transcripts as described above was unexpected, but not surprising. Since multiple levels of posttranscriptional control appear to be occurring on the actual protein participants in the cell cycle, our data suggest that translational control of key molecular participants as well as differences in maternal transcript degradation may be potential areas where problems with in vitro-derived embryos occur. Our current studies of cyclin B1 and cdc25C protein expression patterns in both in vivo- and in vitro-derived porcine embryos over the MZTT will help to answer these questions.
Our findings of decreasing cyclin B1 transcript levels over both the in vivo- and in vitro-derived 4-cell porcine embryo, along with an undetectable shift to embryonic control, suggest that some form of posttranscriptional regulation of cyclin B1, as discussed above, is active at the MZTT. Others [8, 32] have pointed out that wide variation in overall cell cycle control is to be expected when specific species are studied using newly emerging technologies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Randall S. Prather, 162 Animal Science Research Center, University of Missouri, Columbia, MO 65211. FAX: 573 882 6827; pratherr{at}missouri.edu
Accepted: July 15, 1999.
Received: April 15, 1999.
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Means bearing different lowercase letters differ significantly (P < 0.03). Asterisk (*) designations below 33-h bars indicate hours P4CC when alpha-amanitin exposure began and continued to 33 h