Unité de Maturation Gamétique et Fécondation, INSERM and Institut Fédératif de Recherche sur les Cytokines, Clamart, France
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Abstract |
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Key words: pre-implantation/round spermatids/spermatozoa
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Introduction |
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Spermatogenesis involves many profound morphological and functional changes. One unique aspect of the process is that gene expression continues in the haploid stage of spermatogenesis. Among the genes expressed in spermatids some, such as transition proteins 1 and 2 (Kleene, 1989) and protamines 1 and 2 (Kleene et al., 1984
), are unique to the testis, while others are testis-specific transcripts of unusual size or differently spliced variants of somatically expressed genes (Means et al., 1991
; Bolger et al., 1996
; Walensky et al., 1998
).
Mature spermatozoa, on the other hand, have lost most of their cytoplasm and it is generally acknowledged that they do not perform transcription (Bellvé et al., 1988). Despite this apparently inert state, DNA polymerase activity has been described within the nucleus of mature sperm (Hecht, 1974
), and specific RNA transcripts including c myc (Kumar et al., 1993
), HLA class I genes (Chiang et al., 1994
), zfp59 (Passananti et al. 1995
), ß1 integrin (Rohwedder et al., 1996
), protamines 1 and 2 and transition protein 2 (Wykes et al., 1997
) have been detected. Until now it has been unclear whether these mRNA transcripts are the product of active synthesis in mature spermatozoa or, more likely, synthesized during the final burst of transcription prior to the replacement of histones by protamines. However, the quantity of spermatozoa required for RTPCR analysis of these transcripts suggest that their numbers per spermatozoon must be extremely small.
The injection of a spermatid into an oocyte represents the mass arrival of mRNA from various genes, some of which may be deleterious to the development of the embryo if translated. This is a situation that the oocyte has not been programmed to deal with. Available data do, in fact, indicate that inappropriate transcription of some genes during the period of nuclear reprogramming can have long-term detrimental effects on the embryo (Latham, 1999). The pattern of mRNA expression at various times following spermatid injection is therefore a key element in understanding how the oocyte reacts to restore the conditions for normal development.
We have developed a nested reverse transcription/polymerase chain reaction (RTPCR) assay for the detection of mRNA from six genes at various times following intracytoplasmic injection of a spermatozoon or spermatid into a mouse oocyte. These are: Hprt which is normally expressed continuously in the embryo (Kratzer, 1983); protamine 2 (Prm2), the expression of which is strictly restricted to the spermatid stage (Kleene et al., 1984
); Hsp70.1 which is expressed early in the 2-cell stage (Christians et al., 1995
); and one X- and two Y-located genes which are expressed post-meiotically. These are: Ube1Y, which is not expressed before post-implantatory development of the embryo (Odorisio et al., 1996
); its X homologue, Ube1X, which is expressed in both round spermatids and oocytes; and Smcy, which shows early zygotic expression (Agulnik et al., 1994
). This paper is the first report on gene expression in the early mouse embryo following intracytoplasmic injection of round spermatids.
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Materials and methods |
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Preparation of oocytes
C57BLxCBA F1 female mice (610 weeks old; INRA, Jouy-en-Josas, France) were injected with 10 IU pregnant mare's serum gonadotrophin followed by 10 IU human chorionic gonadotrophin (HCG) 48 h later. Oocytes were collected from oviducts about 15 h after HCG injection. They were freed from the cumulus cells by 3 min incubation at 37°C with 0.1% bovine testicular hyaluronidase (type VI S; Sigma) in M16 medium. The oocytes were rinsed and kept in M16 medium at 37°C for 3 h under 5% CO2 in air. Prior to round spermatid (RS) injection (or spermatozoa injection in preliminary experiments only), the oocytes were activated by 8% ethanol in M16 medium for 3 min at 37°C, then rinsed twice in M16 medium.
Preparation of spermatozoa and spermatids
Spermatogenetic cells were obtained from 813 week hybrid F1 males (C57BLxCBA; INRA). Spermatozoa were collected from the vas deferens. They were expressed into M16/BSA medium and incubated for 1.5 h at 37°C under 5% CO2 in air to allow capacitation. They were finally resuspended in M16/BSA medium containing PVP to a final concentration of 8%.
Round spermatids were isolated from the testes by flow cytometry coupled to cell sorting as previously described (Ziyyat et al., 1999) except that the discontinuous Percoll gradient step was omitted. The sorted spermatids were washed in M16/BSA and resuspended in M16/BSA containing PVP to a final concentration of 8%.
Injection of spermatozoa and spermatids into oocytes
The injection was performed in a cell chamber (POC chamber; Helmut Saur, Reutlingen, Germany) following the method of Kimura and Yanagimachi (1995a,b) (Kimura and Yanagimachi, 1995a, b
), except that no piezo-assisted manipulator was used to facilitate zona pellucida penetration and that the entire procedure was performed at room temperature. The chamber was placed on the stage of an inverted microscope (Nikon Diaphot) equipped with Nomarski differential interference optics. Single spermatozoa were aspirated by the middle of their tails into the pipette, so forming them into a hairpin shape and minimizing the volume of medium (5 µm ID; Humagen, Charlottesville, VA, USA) introduced into the oocyte during the injection. Whole RS, each with its disintegrating plasma membrane, were injected into the oocytes.
Culture and examination of oocytes
After injection with either spermatozoa or RS, oocytes were stored at 80°C in batches of 40, or incubated in M16 medium at 37°C under mineral oil in a plastic dish under 5% CO2 in air for 5 h, then examined using an inverted microscope with a X20 objective. Oocytes with two large pronuclei and one second polar body were considered `fertilized'. They were cultured continuously in M16 medium for up to 24 h, corresponding to the 2-cell stage. The 2-cell stage embryos were transferred into CZB medium for a further 24 h culture taking them to the 4-cell stage. Embryos were cultured at a concentration of 10 embryos per 10 µl volume of medium. The entire culture plate was overlaid with heavy paraffin oil. At the end of the culture period, pools of 20 2-cell stage and 10 4-cell stage embryos were stored at 80°C in a minimal volume of medium.
Nested RTPCR
Messenger RNA for Hprt, Smcy, Ube1y, Ube1X, Prm2 and Hsp70.1 was detected in whole oocyte/embryo homogenates using nested RTPCR. To evaluate the dynamic of the genome and not only the carry-over of inherited transcripts, and considering that all cells are equivalent in a 2- and 4-cell embryo, the results were expressed per cell (i.e. per genome equivalent) rather than per embryo. Pools of 40 oocytes, 20 2-cell or 10 4-cell stage embryos in about 2 µl of medium were heated at 99°C for 1 min and then placed on ice. A mixture of 1 µl (10 IU) DNase I RNase-free (Boehringer Mannheim, Meylan, France), 1 µl 10xGold Taq buffer (Perkin Elmer, Foster City, CA, USA) and 0.5 µl (20 IU) RNase inhibitor (Boehringer Mannheim) was added to the lysate in a final volume of 10 µl. Samples were incubated at 37°C for 10 min followed by 15 min at 70°C to destroy DNase I.
Whole samples containing embryonic RNA were reversetranscribed by incubating at 42°C for 1 h with 2 units MMLV reverse transcriptase (Life Technologies, Cergy Pontoise, France), 3 µmol/l random hexamer, 1 mmol/l each dNTP, 1 µl 10xGold Taq buffer in a final volume of 20 µl. The reaction was terminated by heating at 95°C for 10 min and cooling to 5°C. A total of 5 µl of room temperature reaction volume was used for 35 cycles of first round PCR in a final PCR reaction volume of 50 µl containing 2.5 IU of Ampli Taq gold (Perkin Elmer), 1 mmol/l each dNTP and 10 pmol of each of the sense and antisense primers. A 2.5 µl aliquot of cDNA obtained from the first amplification served as template for a second DNA amplification reaction, using inner nested or semi-nested primers (Table I) (Melton et al., 1984
; Johnson et al., 1988
; Hunt and Calderwood, 1990
; Mitchell et al., 1991
; Agulnik et al., 1994
). This second round PCR was run for 40 cycles. The PCR products (20 µl of each) were analysed on 2% agarose gels stained with ethidium bromide and molecular sizes were determined with the molecular weight marker
X 174 Hae Digest (Sigma).
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Southern blot analysis
The cDNA probes used in this study were as follows: Smcy, a 251 bp PCR fragment (921 bp1171 bp) (Mitchell et al., 1991); Hsp70.1, a 223 bp PCR fragment (2528 bp-2750 bp) (Hunt and Calderwood, 1990
). Fragments were verified by size and restriction enzyme mapping.
For Southern blot analyses, 20 µl of nested PCR products were run on 1.5% agarose gel and blotted to hybond N+ membranes (Amersham, Üppsala, Sweden). Hybridization was carried out overnight using 32P labelled probes and autoradiographic exposure was for 30 min at 80°C.
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Results |
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Discussion |
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In the mouse embryo, a major reprogramming of the pattern of gene expression occurs during the 2-cell stage. While some of these changes may be due to maternal mRNA, many are attributable to the transcription of zygotically expressed genes (Latham et al., 1991). This reprogramming of gene expression is likely to be critical for further embryonic development. The effects on early embryonic development of the presence in the oocyte of mRNA from genes expressed post-meiotically in RS are a matter of speculation. Does the activation of the zygotic genome occur normally in this new context, and what happens to the expression of round spermatid specific genes? To answer the first question, we looked for the expression of Hsp70.1 and Smcy, both known to be activated during the 2-cell stage. To answer the second, we followed the expression of Prm2 and Ube1Y, both of which are expressed specifically in round spermatids. Christians et al. (1995) have shown that the onset of zygotic genome activity is marked by the constitutive expression of Hsp70.1, the major inducible heat shock gene. Our results, which show the expression of Hsp70.1 early in the 2-cell stage, are consistent with those of Christians et al. (1995). Hsp70.1 followed the same temporal pattern of expression whether the embryo came from the injection of RS or spermatozoa; however, the level of expression obtained in embryos from spermatozoa was several orders of magnitude higher than that obtained in embryos from RS.
Smcy codes for an epitope of the male specific transplantation antigen H-Y; in mouse development, Smcy expression has been detected as early as the 2-cell stage (Agulnik et al., 1994). In our work, Smcy expression followed the same temporal pattern as that of HSP70.1; it was expressed during the 2-cell stage but repressed by the 4-cell stage. Smcy was expressed in spermatids, but only at a relatively low level because mRNA from 500 round spermatids was necessary to get a signal following a nested RTPCR assay. This low expression in RS probably explained the absence of a signal following injection in embryos from RS, and it argues for zygotic expression of Smcy in 2-cell embryos. Embryos from spermatozoa expressed Smcy with the same temporal pattern of expression than that observed for Hsp70.1, while RS embryos showed less intense expression at 2-cell and more extended expression at 4-cell. Considering the expression patterns of Hsp70.1 and Smcy in embryos from RS, it would appear that immature male gametes are less efficient at activating the embryonic genome. The expression status of the round spermatid greatly differs from that of the spermatozoon, and this might influence the onset of the zygotic genome activation in different ways: it might delay the first wave of activation; this would explain the expression profile of Smcy in RS embryos, with a retarded peak beyond the 2-cell; or/and it might affect the intensity of the activation leading to low expression of Hsp70.1.
Thompson et al. (1995) demonstrated that the regulation of Hsp70.1 during early mouse development is dependent on the maturation of chromatin structure (Thompson et al. 1995). In fact, the most important event determining the nuclear status of sperm cells is the replacement of histones by protamines, the basic nuclear proteins of mature spermatozoa. To accommodate the cessation of transcription several days before the completion of spermiogenesis, mRNA encoding the protamines is synthesized in round spermatids (steps 79), stored as cytoplasmic ribonucleoprotein particles for up to a week (steps 712), and finally translated in elongated spermatids (steps 1216) (Kleene et al., 1984
). Therefore, the round spermatids that were injected into the ooplasm were able to express their protamine genes. Because paternal pronuclei are more effective than maternal pronuclei in transcribing genes, it has been suggested that important early events in fertilized embryos may be predominantly under paternal control (Henery et al., 1995
). Within the first 8 h following fertilization, the paternal nucleus normally undergoes profound changes; in particular, histones replace protamines during pronucleus formation (McClay and Clark, 1997
). For the normal development of RS embryos it therefore appears crucial to prevent protamine expression from occurring. Our data clearly demonstrate that Prm2 expression was down-regulated in embryos from RS: if Prm2 mRNA was present in oocytes just after RS injection, it was undetectable in 1-cell embryos as early as 5 h following fertilization. As expected, at none of the stages tested were transcripts detected in embryos from spermatozoa.
By contrast, Ube1Y, which is not normally transcribed during pre-implantation development (Odorisio et al., 1996), was expressed in embryos from round spermatids from fertilization through to the 2-cell stage. Even though there is no direct evidence of continued transcription, the level of the message is high and apparently equivalent in 1-cell and 2-cell embryos, independently of the fact that the number of embryos (but not of cells) was halved. Therefore, the absence of detectable Ube1Y message in the 4-cell is rather the expression of down-regulation than dilution of the paternal mRNA. No transcripts were detected in embryos from spermatozoa at any of the stages tested. Ube1Y, a Y-linked gene, is transcribed in the testis together with its ubiquitously expressed homologue on the X chromosome (Ube1X). It encodes ubiquitin-activating enzyme E1, an enzyme essential for eukaryotic cell proliferation. X and Y transcripts are present in spermatogonia A, almost undetectable in pachytene spermatocytes, and then return to high levels in round spermatids (Odorisio et al., 1996
). Ube1X was expressed in embryos from both RS and spermatozoa up to the 2-cell stage. Ube1X is not only maternally-inherited, but also actively transcribed in the spermatid, while it is not expressed in the preimplantation embryo. If maternally inherited transcripts are diluted from 1-cell to 4-cell, because we are halving the number of embryos (but not of cells) at each stage, the absence of detectable signal in 4-cell embryos must also reflect the arrest of transcription from the paternal copy of the gene. The complete amino acid sequence of Y- and X-encoded proteins indicates that they probably have the same function; this could explain why Ube1Y is not rapidly repressed following fertilization with RS.
Immediately after micro-injection, the expression status of the embryo is obviously the picture of both paternal and maternal contributions: spermatids expressed Ube1Y and Prm2 while spermatozoa which are transcriptionally quiescent have undetectable levels of Ube1Y and Prm2 mRNA in extracts from 100 spermatozoa (A.Z. and A.F., personal observation) and, similarly, embryos from RS expressed both Ybe1Y and Prm2 while embryos from spermatozoa did not. Besides, Ube1X followed the same pattern of expression in embryos from RS or spermatozoa, whether the oocytes are activated with alcohol or not. Therefore, the differences between sperm-derived and spermatid-derived embryos are likely to reflect the differences in the expression status observed between spermatozoa and spermatid.
In summary, our results suggest that, in the mouse, regulatory mechanisms are activated soon after fertilization with RS to inhibit the inappropriate transcription of male post-meiotically expressed genes. The kinetics of this inhibition was dependent on the gene to be switch off: the protamine 2 gene was rapidly repressed following fertilization, while Ube1Y expression persisted up to the 2-cell stage. On the other hand, mRNA species of RS origin that persist in the early embryo permit the expression of Hsp70.1 and Smcy in a narrow window during the first phase of the activation of the zygotic genome. Even though embryos from RS developed up to the 4-cell stage more or less normally after a short period for reprogramming of the male genome, it does not foresee their future. More work will be necessary to explain the failure of RS embryos, particularly in humans, to support full embryonic development. This should include following-up embryos beyond the 4-cell stage, and an analysis of gene expression at later stages. In interpreting the results we have to bear in mind that the embryos we analysed were from RS of normally fertile mice, and that the findings are not necessarily applicable to embryos from the RS of infertile men. Particularly, RS from patients with complete spermiogenesis failure had significantly higher frequencies of apoptosis-specific DNA damage in comparison with patients with incomplete spermiogenesis failure (Tesarik et al., 1998a). They also failed to support the special form of Ca2+ oscillations that is the sign of normal activation of the oocyte (Tesarik et al., 1998b
). These observations might explain the low success rates of spermatid conception in these cases.
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Acknowledgements |
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Notes |
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References |
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Submitted on October 9, 2000; accepted on March 14, 2001.