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a Wisconsin Regional Primate Research Center and
b Department of Animal Health and <<016>>Biomedical Sciences, University of Wisconsin, Madison, Wisconsin 53715
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-amanitin prevented incorporation of label in 2-cell embryos, and treatment with ribonuclease reduced the signal to background levels, indicating that [3H]uridine was incorporated into mRNA and not rRNA or DNA. Nucleolar incorporation of [3H]uridine was not evident in pronucleate-stage or 2- to 5-cell embryos, but it was detected in one 6-cell embryo and in all 8-cell to blastocyst-stage embryos. Fibrillarin was first expressed in some 6- to 7-cell embryos, but it was consistently expressed in all 8-cell embryos. Fibrillarin was localized to the perimeter of the nucleolar precursor bodies, forming a ring that completely encapsulated these structures. Fibrillarin was not expressed in 8- to 16-cell embryos cultured with -amanitin, indicating that it is transcribed, rather than recruited, at the 8-cell stage. In conclusion, in in vitro-fertilized macaque embryos developing in vitro, extranucleolar synthesis of mRNA is initiated at the 2-cell stage while the onset of nucleolar transcription occurs at the 6- to 8-cell stage, coincident with expression of fibrillarin.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-amanitin-sensitive protein synthesis [5–10] and transformation of the nucleolus precursor into an active fibrillo-granular nucleolus with onset of rRNA synthesis [11–19]. This process has been commonly referred to as "genome activation," and it is coincident with this critical stage when preimplantation embryos experience species-specific blocks to development in the presence of -amanitin and under various culture conditions in vitro. Although a major burst in protein synthesis at the time of genome activation has been described for many species, lower levels of mRNA synthesis have been detected before synthesis of -amanitin-sensitive proteins in mouse [20, 21] and cow [22–24] embryos. It is unknown whether these early messages are translated into proteins at this time or stored for future translation. Nevertheless, these studies indicate that activation of the embryonic genome may not be a rapid one-step process but may begin before the species-specific stage at which preimplantation development becomes dependent upon genomic transcription. Additionally, while the developmental importance of the onset of mRNA synthesis has been the focus of many studies, relatively few studies have addressed the importance of nucleolar transcription.
In human embryos, the major genome activation event is thought to occur between the 4- and 8-cell stages [5, 6, 14–17]. Rhesus monkey embryos cultured in the presence of -amanitin do not progress beyond the 16-cell stage, and 50% are developmentally arrested at the 10-cell stage [25], indicating that the major onset of embryonic transcription in rhesus monkeys most likely occurs during the fourth cell cycle, as in humans. However, characterization of the onset of embryonic transcription during MET has not been described for any nonhuman primate species. Specific aims of this study were to determine the time of the onset of nucleolar and extranucleolar transcription and the expression of the nucleolar protein fibrillarin in macaque preimplantation embryos developing in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rhesus monkeys (Macaca mulatta; n = 22) received twice-daily i.m. injections of 30 IU recombinant human FSH (Organon Inc., West Orange, NJ) for 8 days, beginning on Days 1–3 of the menstrual cycle (Day 1 = first day of menstruation). Ovaries were examined on Day 8 via ultrasonography of sedated (ketamine HCl) monkeys to evaluate the follicular response to gonadotropin stimulation. If several 4- to 5-mm follicles were present, either urinary (Sigma Chemical Co., St. Louis, MO) or recombinant (Ares Advanced Technology, Randolph, MA) hCG (1000 IU) was injected (i.m.) on treatment Day 9 for induction of oocyte maturation. Oocytes were aspirated laparoscopically 27–30 h after injection of hCG from follicles 3 mm or greater in diameter, using a modified Renou Device with a 20-gauge aspiration needle, into Tyrode's lactate-Hepes medium (37°C) containing 10 IU/ml heparin [26].
Oocyte Culture/In Vitro Fertilization (IVF)
Oocytes were retrieved from aspirates using an EM Con filter (Veterinary Concepts, Spring Valley, WI). In some cases, cumulus masses were treated with 0.1% hyaluronidase to facilitate recovery of oocytes. Oocytes that appeared normal (nonvesiculated, round, and medium to lightly pigmented) and did not contain an intact germinal vesicle were cultured for 4–10 h post-aspiration in 50-µl drops of modified CMRL-1066 medium (CMRL; [27]) containing 20% bovine calf serum (BCS) under 3.5 ml of mineral oil at 37°C in a humidified atmosphere of 5% CO2 in air [28]. Before insemination, oocytes were examined for evidence of nuclear maturation and were inseminated between 31 and 38 h post-hCG.
Semen was collected by penile electroejaculation, and sperm capacitation and IVF were done as described previously [26]. Briefly, 20 x 106 washed motile sperm/ml were resuspended in 2 ml Tyrode's albumin lactate pyruvate (TALP) medium overlaid with 2 ml mineral oil, incubated at 37°C in 5% CO2 in air for 1–10 h, and then incubated for an additional 1 h in the presence of 1.0 mM each of caffeine and dibutyryl cAMP to induce hyperactivation [29]. Hyperactivated sperm were then diluted (1 x 105-2 x 105) into 100-µl drops of TALP medium containing 2% BCS and were coincubated with oocytes for 12–16 h at 37°C in a humidified atmosphere of 5% CO2 in air. Sperm and remaining cumulus cells were then removed manually by pipetting through a pulled glass pipette, and oocytes were examined for evidence of fertilization.
Embryo Culture
Embryos having 2 pronuclei were placed into 50-µl drops (1–5/drop) of modified CMRL-BCS and cultured for 19–24 h (2-cell), 30–35 h (4-cell), 41–56 h (8-cell), 88 h (16- to 32-cell), or 184 h (blastocyst) postinsemination at 37°C in a humidified atmosphere of 5% CO2:5% O2:90% N2 [28]. Embryos were placed into fresh culture medium every other day and were examined daily using Nomarski optics (x200–400) on a Nikon (Garden City, NY) Diaphot TMD microscope with a heated (37°C) environmental control chamber and continuous flow of 5% CO2 in air [30].
Analysis of RNA Synthesis
A total of 66 embryos from 11 rhesus macaques were incubated for either 6 h (pronucleate stage, n = 14), 6–10 h (2-cell, n = 18), 5 h (4- to 5-cell, n = 12), 4 h (7- to 8-cell, n = 14), 1 h (16-cell, n = 4), or 10 min (blastocyst, n = 4) in 25-µl drops of CMRL-BCS medium containing 200 µCi/ml of [5,6-3H]uridine (Amersham, Oakville, ON, Canada), as described by Plante et al. [31]. Earlier-stage embryos were cultured for longer periods of time to insure detection of label in embryos demonstrating low levels of incorporation. Negative controls for background labeling were cultured as described above but supplemented with 5.0 mg/ml of unlabeled uridine. Some 2-cell embryos were cultured with [3H]uridine in the presence of -amanitin (25 µg/ml; inhibitor of mRNA synthesis; n = 8) and processed for autoradiography. After culture with [3H]uridine, embryos were washed in cold PBS containing 10% BCS (PBS-BCS) and then held for 30 min in PBS-BCS supplemented with 2.5 mg/ml uridine. Whole embryos were then spread on slides with 1:1 methanol:glacial acetic acid as described by King et al. [32], fixed overnight in 3:1 methanol:glacial acetic acid, and stored at 4°C until processed for autoradiography. Some slides with spread 2-cell embryos (n = 4) were treated with ribonuclease (RNase)-A (100 µg/ml; [24]) before processing for autoradiography.
Autoradiography was performed in total darkness as described by Plante et al. [31]. Briefly, slides were dipped into NTB2 photographic emulsion (Eastman Kodak Co., Rochester, NY) diluted 1:1 with water and dried at room temperature. Spread embryos were exposed for 9 days at 4°C, developed in D19 (Eastman Kodak Co.), fixed in Kodak fixer (Eastman Kodak Co.), and then dried overnight at room temperature. Embryos were then mounted with Vectashield mountant (Vector Laboratories, Burlingame, CA) and evaluated by lightfield microscopy.
Immunocytochemistry
Immunocytochemistry was done as described previously by Collas et al. [33] and modified for macaque embryos, as described below. A total of 57 embryos (pronucleate stage, n = 5; 2-cell, n = 4; 4-cell, n = 6; 5-cell, n = 4; 6-cell, n = 3; 7-cell, n = 4; 8-cell, n = 12; morula, n = 17; blastocyst, n = 3) from 11 rhesus macaques were attached to poly-L-lysine-coated slides, fixed in methanol-free formaldehyde for 1 h, and then permeabilized at 37°C in 0.1 M PBS containing 1.0% Triton X-100 (PBS-Triton). After a glycine rinse for reducing free aldehydes, embryos were blocked in PBS-Triton containing 3 mg/ml nonfat dry milk (NFDM) and then incubated for 45 min at 37°C with the anti-fibrillarin antibody purchased as the nucleolar pattern component of the Anti-Nuclear Antibody kit (ANA-N; Sigma). After being rinsed in PBS-Triton-NFDM, embryos were incubated as above in fluorescein isothiocyanate (FITC)-conjugated goat anti-human immunoglobulin diluted in PBS with Evans blue dye (Sigma). After a rinse in PBS-Triton containing 20 µg/ml Hoechst 33342 (Sigma), specimens were mounted in Vectashield mountant and stored at 4°C in the dark. Negative controls for background fluorescence were treated as described above, but the primary antibody (ANA-N) was omitted. Some 8- to 16-cell embryos were cultured for 3 h in the presence of -amanitin (25 µg/ml; inhibitor of mRNA synthesis) and then processed for immunocytochemistry to determine whether fibrillarin is transcribed or recruited at the stage at which it is initially expressed. Several arrested embryos (
8-cells) were also probed with ANA-N. Whole mounts were examined at 100–200x using a Nikon Optiphot-2 epifluorescence microscope. UV and FITC signals were detected using appropriate filter combinations and were photographed with a Nikon 2000 camera mounted on the microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Representative autoradiographs of rhesus preimplantation embryos cultured for various lengths of time with [3H]uridine are illustrated in Figure 1. Incorporation of label was not detected in any pronucleate-stage embryos cultured from 12 to 18 h postinsemination (6 h) with [3H]uridine (Fig. 1, a and b). Culture periods of 8–10 h were attempted, but with these longer intervals, embryos had undergone syngamy and/or cleavage. Extranucleolar incorporation of [3H]uridine was first detected in 2-cell embryos (Fig. 1, c and d) cultured 6 h with [3H]uridine, and it was incorporated at all subsequent stages through the blastocyst stage (Fig. 1, e–l). Culture with -amanitin, an inhibitor of mRNA synthesis, prevented incorporation of label into 2-cell embryos (Fig. 2b), and treatment with RNase reduced the signal to background levels (Fig. 2c), indicating that [3H]uridine was incorporated into mRNA and not rRNA or DNA. The presence of label over the nucleolus, indicative of nucleolar incorporation of [3H]uridine into rRNA, was not evident in 2- to 5-cell embryos, but it was evident in some blastomeres of one 6-cell embryo (not shown), and was consistently present in all blastomeres of 8-cell embryos (Fig. 1, g and h) and at all subsequent stages through the blastocyst stage (Fig. 1, i–l), indicating that transformation of the nucleolus precursor into an active fibrillo-granular nucleolus with onset rRNA synthesis is probably initiated at the 6- to 8-cell stage in macaque embryos developing in vitro.
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Expression of Fibrillarin
Representative images of rhesus preimplantation embryos probed with ANA-N and counterstained with Hoechst 33342 are shown in Figure 3. In no case was fibrillarin expressed in any blastomeres of pronucleate-stage (Fig. 3, a and b) or 2- to 5-cell embryos (Fig. 3, c–h). Expression of fibrillarin was first detected in two 6-cell embryos (Fig. 3, i and j) but was consistently expressed in all 8-cell embryos (Fig. 3, k and l) and at all subsequent stages through the blastocyst stage (Fig. 3, m–p). The presence of label in some 6-cell embryos may imply that expression of fibrillarin is initiated during the third cleavage division, coincident with the onset of nucleolar transcription. In all cases, the FITC signals were localized to the perimeter of the nucleolus, appearing in most cases as two distinct fluorescent circles encapsulating each nucleolus. In some cases, only one nucleolus within a blastomere was labeled. Morula-stage embryos probed with secondary antibody alone exhibited only background fluorescence, confirming the specificity of the antibody (not shown). Expression of fibrillarin was absent in 8- to 16-cell embryos cultured for 3 h with -amanitin (Fig. 4), indicating that fibrillarin is transcribed, rather than recruited, at the 8-cell stage, coincident with the onset of nucleolar transcription. In 4 of 27 embryos having 8 cells, expression of fibrillarin was absent in 2–40% of the morphologically normal-appearing blastomeres (not shown). No signal was detected in metaphase nuclei or in any cleavage arrested embryos ( 8-cells).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-amanitin-sensitive protein synthesis [3, 5–10] and transformation of the nucleolus precursor into a fibrillo-granular nucleolus actively engaged in rRNA synthesis [11–19]. During this critical period, embryos experience species-specific blocks to development in the presence of -amanitin and under various culture conditions in vitro, indicating that MET may be one of the most important events for successful development during preimplantation embryogenesis. The timing of various aspects of MET have been characterized in several species (mouse [20, 34]; hamster [35]; sheep [36]; pig [10, 18]; cow [7, 8, 11, 12, 22–24, 37, 38]; horse [13, 39]; goat [40]), including humans [5, 6, 14–17, 41]. In the present study, we have used sensitive autoradiographic and immunocytochemical techniques to characterize the timing of the onset of nucleolar and extranucleolar transcription and expression of the nucleolar protein fibrillarin during MET in rhesus monkey embryos developing in vitro.
In rhesus monkey embryos, the onset of extranucleolar (mRNA) transcription was initially detected by autoradiography at the 2-cell stage. In no case was [3H]uridine incorporation detected in pronucleate-stage embryos. Although the possibility of very low levels of mRNA synthesis by pronucleate-stage embryos cannot be ruled out, the whole embryo spreading technique used in this study for autoradiography is considerably more sensitive than autoradiography of embedded/sectioned embryos [31], which has commonly been used to assess the onset of embryonic transcription. Although it could be argued that [3H]uridine might have been converted into DNA in 2-cell embryos during the 6-h culture period, treatment with -amanitin, an inhibitor of RNA polymerase II, prevented incorporation of label into 2-cell embryos, and treatment with RNase reduced labeling to background levels, clearly demonstrating its incorporation into mRNA. Thus, in macaque embryos developing in vitro, activation of embryonic transcription is initiated by the 2-cell stage. In contrast, extranucleolar incorporation of [3H]uridine was first detected in human embryos at the 4-cell stage [14, 15]. However, in those studies, embryos were embedded and sectioned for autoradiography, which may not have been sensitive enough to detect RNA synthesis occurring at earlier stages [31].
Although extranucleolar RNA synthesis was observed in 2- and 4-cell embryos, nucleolar RNA synthesis was not apparent until the 6- to 8-cell stage. Although only one 6-cell embryo exhibited heavy label over the nucleolus of 2 blastomeres (not shown), label was consistently present over the nucleoli of 8-cell embryos. Although it cannot be unequivocally proven in whole embryo spreads that label present over the nucleolus actually reflects nucleolar incorporation of the label, it is unlikely that label would have consistently concentrated so heavily directly over the nucleolus of each blastomere, beginning at the 6- to 8-cell stage, if it were not actually nucleolar label. It is possible that very low levels of nucleolar RNA synthesis may have been occurring at earlier stages. However, in similar autoradiographic studies in sectioned human embryos, nucleolar label was not detected until the 6- to 8-cell stage, along with a corresponding increase in extranucleolar RNA synthesis [14–17]. Because cleavage is often asynchronous in rhesus embryos, the presence of nucleolar label at the 6-cell stage may imply that nucleolar transcription is initiated during or after the third cleavage division. The onset of nucleolar RNA synthesis in human embryos at the 6- to 8-cell stage was also associated with DNA penetration into the nucleolus from adjacent chromatin [15, 16] and inhibition of stage-specific changes in the qualitative pattern of polypeptide synthesis after treatment with the -amanitin [5, 6]. Likewise, in bovine embryos, extranucleolar RNA synthesis was initially detected in 2-cell embryos [22–24], while nucleolar RNA transcription was not detected until the 8-cell stage [12], in association with major qualitative changes in -amanitin-sensitive protein synthesis [7, 8, 37, 38]. These studies as well as similar studies in sheep [36], goats [40], pigs [18], and horses [13, 39] confirm that the onset of nucleolar RNA synthesis is associated with ultrastructural features of nucleologenesis consistent with the major activation of the embryonic genome. Nevertheless, it is also apparent that genome activation is not a rapid, one-step process, but rather that a gradual activation of embryonic genes occurs during the course of preimplantation development, beginning as early as the 2-cell stage in bovine and primate embryos. However, the importance of this early mRNA transcription is not clear, as cleavage is not inhibited by -amanitin at the 2- and 4-cell stages in human [6], rhesus monkey [25], or bovine [7] embryos. There is some evidence that this initial transcription, or `first wave" of transcription, may be important for synthesis of transcription factors required for the major burst of embryonic transcription [42], but this remains to be substantiated.
In the present study, the onset of fibrillarin expression mirrored that of [3H]uridine incorporation into the nucleolus, first appearing in 6- to 8-cell embryos. In no case was fibrillarin detected in 4- or 5-cell embryos. In similar studies in mice [43, 44], rabbits [45, 46], and cows (unpublished results), expression of fibrillarin at the periphery of the nucleus was initially detected at the species-specific time of genome activation, coincident with expression of nucleolin [43], protein B23 [47], and RNA polymerase I [44]. Following genome activation, the fluorescent pattern progressively changed into either a continuous layer of label in the cortex (mouse [43, 44]) or into branch-shaped speckles in reticulated nucleoli (rabbit [45]; cow, unpublished results). In the present study, fibrillarin was also localized to the perimeter of the nucleolar precursor bodies from the 6- to 8-cell through the morula stage, forming a ring that completely encapsulated these structures, before changing into a reticulated-like pattern at the blastocyst stage. Although the specific functions of fibrillarin in embryonic transcription have not been completely defined, its expression at the periphery of the nucleolar precursor body along with nucleolin, protein B23, and RNA polymerase I [43–45, 47], which coincides with the onset of nucleolar transcription in mouse [43, 44], cattle [47], and rabbit [46] embryos, suggests a role of this protein in rRNA gene transcriptional activity. Moreover, lymphocytes, which have transcriptionally inactive nucleoli, exhibit weak labeling with fibrillarin antibodies [48]. In somatic cells, fibrillarin is a major component of U3 small nuclear ribonucleoprotein [49], which binds to pre-rRNA, and is involved in the earliest cleavage step of pre-rRNA processing [50–53]. Fibrillarin is also indicated in methylation of pre-rRNA [54], as well as ribosome assembly [52, 53] and transport of preribosomes to the cytoplasm [55].
In the present study, fibrillarin was nondetectable in embryos cultured with -amanitin, indicating that fibrillarin is transcribed by the embryonic genome, rather than recruited or translated from maternal messages, at the 8-cell stage, coincident with the onset of nucleolar transcription. Therefore, fibrillarin may be considered an early marker of genome activation or of the transcriptional status of the nucleolar precursor body among individual blastomeres. Pinto-Correia et al. [46] used fluorescent antibodies to fibrillarin to evaluate the reprogramming of transcriptional activity in rabbit embryos reconstructed by nuclear transfer, demonstrating that transcription in blastomeres from 16-cell rabbit embryos was turned off when transferred to enucleated oocytes, indicative of nuclear reprogramming.
The time of cleavage arrest for the majority of macaque embryos is during, or after, the third cleavage division [27, 28, 56], similar to that reported for human embryos [5, 57–60]. Previous studies indicate that rhesus monkey embryos cultured in the presence of -amanitin do not progress beyond the 16-cell stage, and 50% are developmentally arrested at the 10-cell stage [25]. On the basis of these findings and results of the present study, developmental arrest during this time period may result from failure of timely onset of embryonic genome activation [6, 14, 15, 41, 61, 62]. Autoradiographic studies of [3H]uridine incorporation into IVF human embryos obtained from gonadotropin-stimulated women have shown that transcription failure is not uncommon in blastomeres of human 8-cell and morula-stage embryos [14]. These blastomeres express very low levels of extranucleolar RNA synthesis and a complete absence of nucleolar RNA synthesis, typical of 4-cell embryos, with up to 30% of blastomeres in human IVF 8-cell and morula-stage embryos exhibiting this impairment. Failure in timely onset of rRNA synthesis may have pronounced effects on development, since embryos must support their demand for protein synthesis using maternally inherited ribosomes, which are rapidly exhausted during the first three cleavages [14]. Some of these embryos in which the switch from maternal to embryonic gene activity has failed in a large proportion of blastomeres can progress to the morula stage [16, 41, 63, 64] but fail to develop into blastocysts [61, 63]. Similar autoradiographic studies in bovine embryos [65] demonstrated that, unlike 8-cell embryos derived from oocytes from medium to large antral follicles, those derived from oocytes from small (1–2 mm) antral follicles exhibited very low levels of extranucleolar RNA synthesis and the absence of nucleolar RNA synthesis, indicative of a delayed onset of gene transcription. This was observed not only among embryos but also among blastomeres within the same embryo [65], and was associated with a high incidence of developmental failure [66]. In the present study, based upon expression of fibrillarin, transcription failure was detected in blastomeres of only 4 of 27 nonarrested embryos ( 8-cell). Although no conclusions can be drawn from such a limited number of embryos, the relatively high incidence of transcription failure reported for IVF human embryos [14, 16, 41, 61] may reflect the selection of incompetent "spare" embryos for experimental purposes or may be associated with suboptimal culture conditions or particular ovarian stimulation protocols used in those studies. Under the culture conditions used in the present studies, approximately 50% of embryos develop into blastocysts. Fibrillarin was not detected in 8-cell embryos that had undergone cleavage arrest, although it is unknown whether this was a cause or an effect of developmental failure. Preliminary studies in rhesus monkeys suggest that failure in the timely onset of nucleolar transcription may be more prevalent in embryos derived from in vitro-matured oocytes (unpublished results). This may not be surprising since genome activation is thought to be under control of maternally inherited molecules, which may be deficient in oocytes developing or maturing in a suboptimal environment.
In conclusion, in IVF macaque embryos developing in vitro, extranucleolar transcription of mRNA is initiated at the 2-cell stage, while the onset of nucleolar transcription of rRNA occurs at the 6- to 8-cell stage, coincident with the expression of fibrillarin. Evidence suggests that the timing of MET is similar in human and nonhuman primates. Results of the present study have important implications in assessing the role of transcriptional impairments in the relatively high incidence of developmental failure that occurs during the transition from maternal to embryonic control of development in primate preimplantation embryos in vitro.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: R. Dee Schramm, Wisconsin Regional Primate Research Center, 1223 Capitol Court, Madison, WI 53715. FAX: 608 263 3524; schramm{at}saimiri.primate.wisc.edu
Accepted: October 9, 1998.
Received: July 7, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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