Molecular Human Reproduction, Vol. 5, No. 9, 845-850,
September 1999
© 1999 European Society of Human Reproduction and Embryology
Regulation of embryo development |
Alternative splicing of the telomerase catalytic subunit in human oocytes and embryos
1 The Institute for Reproductive Medicine and Science of Saint Barnabas Medical Center, Gamete and Embryology Laboratory, 101 Old Short Hills Road, Suite 501, West Orange, NJ 07052 USA
Abstract
The human telomerase catalytic subunit (hTCS) is a ribonucleoprotein which synthesizes telomere repeats on the ends of chromosomes. Telomerase activity is thought to be essential in maintaining normal telomere length in immortal (cancer) and germ cells. The objective of this study was to determine the gene expression of telomerase mRNA in human oocytes at different meiotic stages and in embryos. Normal and abnormal human oocytes, preimplantation embryos, and blastocysts were analysed for the presence and expression of the hTCS transcripts. Multiple telomerase mRNA products were identified by reverse transcription–polymerase chain reaction (RT–PCR) using primers within the reverse transcriptase domain. DNA sequencing of these amplicons suggest that there are alternative splicing variants which align to other telomerase reverse transcriptase (RT) consensus domains. Surprisingly, in unfertilized and immature gametes, as well as preimplantation embryos, hTCS expression revealed three different PCR product sizes, 457, 421 and 275 bp. The frequency of the 275 bp DNA product was 6.6% in oocytes (two out of 30) compared with 56.6% (17 out of 30) in poorly developing human preimplantation embryos (P < 0.005). The presence of alternately spliced mRNA variants in human preimplantation embryos may suggest a lack of telomerase activity and thus chromosomes associated with shortened telomeres.
human oocytes/human preimplantation embryos/RT–PCR/telomerase/telomeres
Introduction
It is now believed that telomere shortening is a key to biological ageing (Lange et al., 1998). Telomeres are repeats of DNA protecting the stability of chromosomes and are essential for chromosome end maintenance. In humans, the DNA sequence consists of the TTAGGG repeats. Telomerase is the ribonucleoprotein enzyme that adds these DNA repeats to the tails of the chromosome. The human telomerase catalytic subunit (hTCS), or human telomerase reverse transcriptase, a recently cloned enzyme, is structurally related to reverse transcriptase and thus represents the first member of this family with essential cellular function (Meyerson et al., 1997
; Nakamura et al., 1997). Interestingly, telomerase activity is high in germ, embryonic and cancer cells. All of these are considered to be telomerase positive (Kim et al., 1994; Wright et al., 1996). The enzyme is missing in telomerase-negative cells, e.g. most somatic cells. Immortal cancer cells divide uncontrollably and are telomerase positive. The mechanism which converts somatic cells to cancer cells is unknown. A possible new partner for telomerase regulation is a human protein called tankyrase, which controls whether telomerase can do its job by removing proteins that otherwise block the access of telomerase to the chromosome ends (Smith et al., 1998).
The oocyte appears to be the most likely place for the initial setting of a telomeric clock, since telomeres should be added to the chromosomes prior to fertilization and before the onset of zygotic transcription and blastulation in the early embryo. Logically, both spermatozoa and oocytes should have the longest telomeres to ensure the transmission of full-length chromosomes to the progeny. Very little is known about telomerase activity in germline and embryonic cells, especially in the human. Telomerase activity was first detected in Xenopus oocytes and embryos (Mantell et al., 1994). Telomerase activity has been detected in human testes (Fujisawa et al., 1998) and ovaries at all stages: fetal, newborn and adult. It has also been found in human blastocysts, but not in mature spermatozoa or oocytes (Wright et al., 1996). Telomerase activity was also found in rat oocytes from the early antral and pre-ovulatory follicles, as well as in ovulated oocytes and 4-cell embryos (Eisenhauer et al., 1997).
It has been recently shown that various tumours, cell lines and even normal tissues such as testes, show considerable differences in telomerase mRNA splicing patterns (Killian et al., 1997). Alternate splicing of hTCS may be important for regulation of telomerase activity. Alternately, spliced variants expressed in germline cells may indicate jeopardized growth potential and serve as markers for embryonic health. With the recent cloning of the hTCS gene, it is now possible to detect telomerase-positive cells by a more sensitive reverse transcription–polymerase chain reaction (RT–PCR), rather than the telomerase enzymatic assay termed telomeric repeat amplification protocol (TRAP) (Kim et al., 1994; Meyerson et al., 1997; Nakamura et al., 1997). The objective of this study was to determine the expression of the hTCS mRNA in different meiotic stage human oocytes and preimplantation embryos. We have examined the hTCS expression and mRNA splicing variants in compromised and potentially normal human embryos and oocytes at different stages of meiosis.
Materials and methods
Collection of human oocytes and embryos
A total of 20 immature human oocytes, at either the germinal vesicle (GV) stage (O-GV-C) or the metaphase I (MI) stage (O-MI-C), 10 mature metaphase II (MII) oocytes (O-MII-C), 30 day 3 cleaved embryos (E-CLV-C) and five day 5 blastocysts (E-BL-C) were donated by couples attending the in-vitro fertilization (IVF) programme at the Institute of Reproductive Medicine and Science at Saint Barnabas Medical Center (Table I). This material can be considered to be compromised since the oocytes did not reach MII. The embryos were not transferred or cryopreserved since they were considered sub-optimal. Normal MII oocytes (O-MII-N) were donated anonymously from patients attending another IVF Clinic (Hamburg, Germany). Normal embryos were supplied by a 38 year old patient who previously delivered triplets after IVF at The Institute for Assisted Reproduction at The Presbyterian Hospital, Charlotte, NC, USA. All the material obtained which was classified as normal MII oocytes (n = 2), and 8-cell embryos (n = 2) were selected from same cohorts which had established pregnancies. These embryos were of comparable quality by morphological criteria and developmental rate.
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The protocol for research was approved by the Internal Review Board in 1995. Ovarian stimulation was carried out using down-regulation and gonadotrophin substitution. Embryos on day 2 and 3 of development were scored for morphological characteristics including percentage fragmentation, type of fragmentation, slow development, arrested development and multinucleation (Munné et al., 1995
; Warner et al., 1998). Data was stored, retrieved and analysed using an online database (EggCyte; ART Inst. of NY & NJ, USA). All oocytes and embryos were washed three times in sterile phosphate-buffered saline (PBS) in plastic dishes and then transferred to 0.2 ml tubes in as small a volume as possible. If necessary, the zona pellucida was removed prior to sample lysis using acidified Tyrode's solution (pH 2.5). A 100 µl sample of denaturing solution (Micro-RNA Isolation Kit; Strategene Inc, La Jolla, CA, USA) was added immediately after transfer and the tube was briefly vortexed. Some samples were frozen and stored at –70°C prior to RNA purification.
RNA purification
Previously frozen samples were thawed and RNA isolation was performed using the Micro-RNA Isolation Kit (Stratagene Inc, La Jolla, CA, USA) according to the manufacturer's protocol. Copies (106) of the plasmid pAW109 mRNA (Perkin Elmer Inc, Foster City, CA, USA) were added as a positive control, along with 20 µg of ultra pure glycogen carrier (Boehringer Mannheim Inc, Indianapolis, IN, USA), prior to isopropanol precipitation. Each dried RNA sample had an 8.5 µl aliquot of a master mix solution consisting of 7.25 µl Molecular Biology Grade Water (5 Prime-3 Prime Inc, Boulder, CO, USA), 0.2 µl 0.1 mol/l dithiothreitol (DTT), 1 µl 50 µmol/l random hexamers (Perkin Elmer Inc), and 0.05 µl 40 IU/µl RNAse inhibitor (Perkin Elmer Inc). The annealing of random hexamers to RNA was performed by incubating the sample at 70°C for 6 min followed by cooling at 25°C (Brenner et al., 1997).
Reverse transcription
The reverse transcription reaction was performed by incubating the mRNA with 11.5 µl master mix solution consisting of 4 µl of 25 mM MgCl2, 2.0 µl 10x PCR buffer (Perkin Elmer Inc), 4.0 µl 100 mM dNTP, 1.0 µl RNase inhibitor (Perkin Elmer Inc), 0.5 µl 50 IU/µl Maloney murine leukaemia virus (MMLV) reverse transcriptase (Perkin Elmer Inc), and 0.5 µl random hexamers. The mRNA was reverse transcribed at 37°C for 1 h, followed by inactivation of the MMLV enzyme by incubation at 95°C for 5 min (Brenner et al., 1997).
hTCS PCR amplification
An aliquot of cDNA was used as a template for PCR amplification using a 9600 Perkin Elmer system, in a total reaction volume of 50 µl containing 1.5 mmol/l MgCl2, 1 mmol/l dNTP mix, 1x Q Solution, 0.5 µmol/l of each gene-specific primer, and 1.25 IU Taq DNA polymerase (Quiagen Inc, Chatsworth, CA, USA). The following amplification profile was applied: 1 cycle 96°C for 3 min; 40 cycles of 96°C for 20 s, 55° for 20 s, 72°C for 20 s; 1 cycle, 72°C for 7 min; hold at 4°C. Gene-specific PCR primers were synthesized by Genosys Inc (The Woodlands, TX, USA). As an external positive control, plasmid pAW109 mRNA (106 copies) was amplified using primers DM151F and DM152R for interleukin (IL)-1
. The amplification resulted in a PCR product size of 308 bp from pAW109 and 427 bp from the sample being tested. To amplify hTCS genes which contain complex splicing patterns in human oocytes and embryos, a nested PCR strategy was employed using the following PCR primers: HT1875F and HT2781R for the first PCR amplification; and HT2026F and HT 2482R for the nested PCR amplification (Table II; Kilian et al., 1997). To obtain better resolution, PCR products were separated by 3% gel electrophoresis using NuSieve GTG agarose (FMG Inc, Rockland, ME, USA), stained with ethidium bromide and visualized under UV illumination.
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DNA sequencing analysis
In order to confirm the identity of the amplified hTCS PCR products and their splicing variants, the amplicons were separated on 2% NuSieve GTG agarose and gel purified using a QIAquick Gel Extraction Kit (Qiagen Inc, Chatsworth, CA, USA). A 100–200 ng sample of gel purified DNA was subjected to sequence analysis using an ABI 377 automated DNA sequencer (Retrogen Inc, San Diego, CA, USA).
Results
Expression of telomerase in normal oocytes and embryos.
Potentially normal human oocytes (n = 4) and embryos (n = 2) were analysed for the mRNA expression of hTCS. All samples analysed, irrespective of the source, showed the same pattern of expression (Table III). In Figure 1, both normal human oocytes (O-MII-N; lanes 2–5) and day 3 human embryos (E-CLV-N: lanes 6 and 7) expressed the 457 bp amplified product as opposed to the 457, 421 and 275 bp amplicons in the testis control (lane 8). There was considerable difference in hTCS mRNA abundance in lanes 2 and 4 compared with lanes 3 and 5 in normal human MII oocytes. Even though nested PCR was not quantitative, these differences suggest that different human oocytes have varying hTCS mRNA copy numbers. When the 457 bp product was DNA sequenced, it was found to correspond to the predicted hTCS DNA sequence (Meyerson et al., 1997; Nakamura et al., 1997).
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Expression patterns of hTCS in compromised oocytes and embryos
For these experiments (Table I
), 30 compromised oocytes at different stages of oocyte maturation (10 O-GV-C, 10 O-MI-C, 10 O-MII-C), 30 compromised preimplantation embryos (E-CLV-C) and five blastocysts that developed from sub-optimal day 3 embryos (E-BL-C) were evaluated for the presence of hTCS mRNA. In all experiments, a positive control using Burkitt's lymphoma mRNA or testis RNA was used since these tissues are known to express alternately spliced variants of hTCS (Kilian et al., 1997; Meyerson et al., 1998). Additionally, lung mRNA was used as a negative control since this is a telomerase-negative tissue (Kilian et al., 1997). For these experiments, cDNA was synthesized from the test sample or control and amplified by RT–PCR using specific hTCS primers (Table II). In Figure 2, the RT–PCR results of alternative splicing of hTCS mRNA in O-MII-C human oocytes (lanes 2–4) and E-CLV-C embryos (lanes 5–7) are shown. Nested PCR was performed using primers HT1875/HT2781 and HT2026/HT2482 and resulted in different splicing variants. In three lanes, (lane 1, Burkitt's lymphoma RNA; lane 2, O-MII-C; and lane 5, E-CLV-C), the PCR amplification of hTCS revealed the presence of three distinct sized bands, the 457, 421 and the 275 bp products. In four lanes (lanes 3 and 4, O-MII-C; and lanes 6 and 7, E-CLV-C), only the the 457 bp product was present. The summary of hTCS expression patterns in all oocytes analysed and embryos is shown in Table III. In 30 discarded human oocytes, the 457 bp band was found in compromised oocytes (O-GV-C, O-MI-C, O-MII-C), 90, 100 and 100% respectively; the 421 bp band was found in compromised oocytes (O-GV-C, O-MI-C, O-MII-C), 60, 50 and 20% respectively; whereas the 275 bp was found in compromised oocytes (O-GV-C, O-MI-C, O-MII-C), 10, 0, and 10%; and the 247 bp band was never found in compromised oocytes. The common hTCS splicing pattern detected in compromised preimplantation human embryos showed the presence of three splicing variants (457, 421 and 275 bp). Among 30 discarded E-CLV-C embryos, the 457 bp band was found in 93% of the embryos, the 421 bp band was found in 63% of the embryos, and the 275 bp product was found in 56.6% of the embryos. The frequency of the 275 bp DNA product was 6.7% in oocytes (two out of 30) compared with 56.6% (17/30) in compromised preimplantation embryos (P < 0.005).
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In order to evaluate the expression of hTCS at later developmental stages when the embryonic genome was activated, five compromised blastocysts (E-BL-C) were simultaneously analysed by RT–PCR using external positive RNA control pAW109, interleukin-1
(IL-1) primers, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping genes and the hTCS primers (Table II). Interestingly, three out of five of the compromised blastocysts were negative for the presence for hTCS transcripts, one blastocyst revealed three different PCR products (457, 421 and 275 bp), and the other two embryos displayed two bands (457 and 275 bp), but at much lower intensity than other PCR products generated from other oocytes and preimplantation embryos (Table III). To prove these results were correct, and not an artefact of different mRNA concentration, external (DM151, DM152) and internal (GAPDH) controls were amplified, and the intensity of the PCR products of both controls, reflecting the amount of mRNA isolated from each individual blastocysts, was the same in each sample. Normal blastocysts were not analysed since the compromised blastocyst data confirmed the hTCS message was absent or down-regulated.
DNA sequencing and alignment with other telomerases of hTCS
Multiple hTCS RNA variants in human oocytes and embryos suggests alternative splicing. In order to confirm the identity of amplified PCR products and to determine the molecular basis of alternative splicing in oocytes and embryos, DNA sequence analysis was performed. All three PCR products 457, 421 and 275 bp were analysed from two different oocyte and embryo samples. Multiple amino acid alignment confirmed that the 421 bp PCR fragments had a deletion of 36 bp, while there was a deletion of 182 bp explaining the presence of the smaller 275 bp band (Figure 3). The structure of telomerase reverse transcriptase contains eight conserved motifs: motif T, motif 1, motif 2, motif A, motif B', motif C, motif D and motif E (Nakamura et al., 1997). In some embryos and oocytes, motif A of the hTCS contains the 36 bp deletion, resulting in the 421 bp band, while the 275 bp PCR product contains a 182 bp deletion between motif A and motif B of telomerase (Figure 3).
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Discussion
All human oocytes of various maturity and preimplantation embryos express hTCS mRNA, but expression varies dramatically. With the recent cloning of the hTCS gene, it is now possible to detect telomerase-positive cells by the more sensitive RT–PCR rather than the TRAP assay (Kim et al., 1994; Meyerson et al., 1997; Nakamura et al., 1997). Normal human oocytes and preimplantation embryos showed only the 457 bp PCR product. When these PCR products were DNA sequenced and compared with the GenBank sequence, the DNA sequence was identical to the first identified hTCS gene. Surprisingly, in some compromised gametes and embryos, hTCS expression revealed three different PCR product sizes: 457, 421 and 275 bp. The 421, 275 and 247 bp DNA products were significantly less frequent in compromised oocytes than compromised embryos.
Our data is consistent with the understanding that telomerase activity and telomeric timing is necessary and present in human germline and embryonic tissues. However, little has been known about telomerase expression in early human development and, until recently, there has been confusion regarding whether telomerase activity was present in human oocytes at all (Edwards et al., 1997). Telomerase activity has been detected during Xenopus oogenesis and embryogenesis via the TRAP assay (Mantell et al., 1994). Telomerase activity has been detected in human testes, ovaries, and blastocyst stage embryos, but not in individual unfertilized eggs (Wright et al., 1996). But Hsueh's group (Eisenhauer et al., 1997), using a similar TRAP enzymatic assay, found that telomerase was present in rat oocytes from the early antral and preovulatory follicles as well as in ovulated eggs. Interestingly, the level of activity was significantly lower (50-fold) in ovulated oocytes than in those from immature follicles. To ensure maintenance of telomere length stability, and thus normal cellular senescence, it follows that telomerase must be active during meiosis.
There are several explanations for the discrepancies between the literature and our current findings. We have found that in order to detect telomerase activity in individual human oocytes the nested hTCS RT–PCR method is more sensitive for analysis of individual human oocytes and embryos. Also, we have found differences in hTCS mRNA expression in individual GV, MI, MII human oocytes, day 3 embryos, and blastocysts. Comparing the human data, the developmental stages of the human oocytes were not determined (Wright et al., 1996) and lower levels of activity may be associated with a mature or ovulated oocyte. The data presented here suggests that telomerase is actively transcribed, as a maternal mRNA, in the oocyte. It becomes down-regulated when the zygotic genome is activated at the late 8-cell stage in human embryos. Finally, it is barely detectable at the blastocyst stage (Wright et al., 1994; Edwards et al., 1997). Our data also suggests that telomerase may be important during meiosis since there are different alternate splicing variants present during maturation, although this may be related to the condition of the oocyte.
Using DNA sequencing analysis, the splicing variants detected here were aligned with other telomerase reverse transcriptases (RT) and with members of other RT families. Telomerase proteins contain conserved motifs T (telomerase-specific), 1, 2, A, B', C, D, and E. In many of the compromised human oocytes and embryos there were splicing variants of the eight conserved hTCS motifs: motif A contained a 36 bp deletion, resulting in the 421 bp band, while the 275 bp PCR product contained a 182 bp deletion between motif A and motif B of telomerase RT protein (Figure 3). This was a surprising result, but certain tumours, cell lines, fetal tissues and normal tissues, such as testes, show similar splicing variants (Killian et al., 1997; Ulaner et al., 1998). The 36 bp deletion in the conserved region of motif A may be critical for RT function. The large deletion of 182 bp between motif A and motif B may encode a truncated protein. It is unclear whether alternate splicing variants of hTCS are important for the regulation of telomerase activity or whether they give rise to proteins with different biochemical functions.
Alternate mRNA splicing is a common mechanism for regulating gene expression in specific tissues or developmental cells (Ulaner et al., 1998). Whether alternate splicing patterns in human embryos are associated with specific morphological abnormalities, e.g. fragmentation, multinucleation, arrested or slow embryos, is still under examination (Munné et al., 1995; Warner et al., 1998). Although there is a general correlation between hTCS mRNA values and measurable telomerase activity, it is unclear whether there is less telomerase enzymatic activity in cancer cells, somatic cells or germ cells, all of which exhibit prominent splicing variants resulting in truncated proteins (Killian et al., 1997; Meyerson et al., 1997). Experiments to investigate the correlation of hTCS mRNA values, telomerase enzymatic activity, and telomere length are in progress.
One of the more intriguing questions is whether one can estimate an individual's life span by measuring the length of the telomeres in the unfertilized oocyte or an individual blastomere of a human embryo. Perhaps the lack of telomerase activity in some human oocytes may be associated with shortened telomeres, and thus linked to reproductive senescence and/or chromosomal abnormalities such as translocations or aneuploidy. It is possible that differing telomerase activity in individual oocytes and embryos may serve as a marker for embryonic health. If the oocyte cytoplasm, which contains telomerase, is essential for the maintenance of chromosomal stability and telomere length, injection of the telomerase enzyme or donor oocyte cytoplasm may be able to reset or disturb the `telomeric clock' in abnormal human oocytes (Cohen et al., 1998).
Acknowledgments
The authors gratefully acknowledge the efforts of the team of embryologists at the Institute for Reproductive Medicine and Science of Saint Barnabas Medical Center: Jason A.Barritt, Laurie Ferrara, Nury Steuerwald for editorial assistance; and Doctors Richard T. Scott, Paul Bergh, Patricia Hughes, Michael Drews, Larry Grunselt, and Benjamin Sandler for the clinical support of this study.
Notes
2 To whom correspondence should be addressed 
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Submitted on November 11, 1998; accepted on May 28, 1999.
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