Nucleic acid synthesis and development of human male pronucleus J. Tesa\l=r%v\\l=i'\kand V. Kope\l=c%v\n\l=y'\ Centre for Reproductive Medicine, Purkyne University Medical Faculty, CS-65677 Brno, Czechoslovakia; and * Research Institute ofAnimal Production, Prague, Czechoslovakia

Summary. Polyspermically penetrated human zona-free eggs prepared from oocytes that had failed to be fertilized in an in-vitro fertilization programme were used. The pronuclear synthetic activity was evaluated by high-resolution autoradiography and correlated with the development of pronuclear structure. Incorporation of [3H]- thymidine, signalling the occurrence of a DNA synthetic phase, was only detected in structurally fully developed pronuclei previously shown to appear no sooner than 12 h after gamete union. However, [3H]adenosine was incorporated into very early pro- nuclei which had not yet completed the development of their nuclear envelopes and which first appeared about 4 h after sperm\p=n-\eggfusion. In the absence of DNA synthe- sis (shown by the lack of thymidine incorporation), this early adenosine incorporation apparently reflects an early pronuclear RNA synthesis. Taken together, these results indicate that nucleic acid synthesis in human male pronuclei is tightly bound to the development of a corresponding pronuclear structure and that DNA synthesis, beginning about 12 h after fertilization, is preceded by a slight but evident RNA synthesis taking place during an early stage of human male pronuclear formation. Keywords: pronuclear development; DNA synthesis; RNA synthesis; nucleolar development; human; zona-free eggs

Introduction

The transformation of the fertilizing sperm nucleus into a male pronucleus is one of the most complex and least understood processes in early mammalian development. Paternal DNA repli¬ cation, occurring in synchrony with the replication of maternal DNA, is believed to be the first functional task to be exerted by the newly formed male pronucleus. The onset and duration of the DNA synthetic phase (S phase) in 1-cell zygotes have been studied in several mammalian species: rabbit (Oprescu & Thibault, 1965; Szollosi, 1966), mouse (Luthardt & Donahue, 1973; Siracusa et al, 1975; Abramczuk & Sawicki, 1975; Krishna & Generoso, 1977; Howlett & Bolton, 1985), hamster (Naish et al, 1987), and cow (Eyestone & First, 1988). Unlike DNA, the ability of pro- nuclei to synthesize RNA, which, for mammals, has only been examined in mouse zygotes (Clegg & Piko, 1982, 1983; Vasseur et al, 1985), appears to be very limited. Study of the development of male pronuclear structure and function in human zygotes has been made possible by using in-vitro inseminated zona-free human eggs coming from unsuccessful attempts at in-vitro fertilization in clinical programmes for infertility treatment. Most of these unfertilized eggs are penetrated by multiple spermatozoa if inseminated again in the zona-free state and develop morphologically normal male pronuclei (Tesarík et al, 1988a). More important, most of these eggs retain the ability to support male pronuclear development for at least 2 days after tPresent address: INSERM, Unité 187, Hôpital Antoine-Béclère, 92141 Clamart, France.

Downloaded from Bioscientifica.com at 10/09/2021 01:16:57PM via free access the initial fertilization attempt (J. Tesarík & V. Kopecny, unpublished). In this paper we have examined the ability of developing human male pronuclei to incorporate nucleic acid precursors.

Materials and Methods

Preparation ofpolypronuclear zona-free eggs. Human oocytes were obtained from women treated for infertility by in-vitro fertilization. Methods used for monitoring the follicular development, oocyte recovery, sperm preparation and in-vitro insemination were as previously described (Tesarík el ai, 1988a). Oocytes (n = 27) that lacked pronuclei 14-18 h after insemination but had separated the first polar body were used in this study. The zona pellucida of unfertilized mature oocytes, the majority of which were previously shown to have only achieved the second meiotic metaphase in the insemination culture (Tesafik el ai, 1988a), was removed by incubation for 5 min in 01% (w/v) pronase (Sigma, St Louis, MO, USA) dissolved in Dulbecco's phosphate-buffered saline (Serva, Heidelberg, FRG), followed by mechanical removal of zona remnants. Zona-free oocytes were placed in plastic tubes containing 1 ml Ham's FIO medium (Serva) modified as described previously (Tesarík el ai, 1988a) and supplemented with 16% heat-inactivated human serum. Oocytes were then reinseminated in vitro with about 2 IO5 spermatozoa per tube. The spermatozoa used for reinsemination were prepared from fresh sperm samples obtained from healthy normospermic donors. After 1 h of co-culture with spermatozoa, zona-free eggs were washed from loosely attached spermatozoa and transferred into fresh medium for further culture. The duration of this additional culture varied between 1 and 16 h. Only after this culture period, allowing pronuclear development to proceed, were the eggs incubated with labelled nucleic acid precursors.

Nucleic acid labelling. Two different nucleic acid precursors tagged with 3H were used. For DNA labelling, eggs were incubated at 37°C with [methyl-3H]thymidine (UVVVR, Prague, Czechoslovakia; sp. act. 2-6 Ci/mmol). This DNA precursor was diluted in serum-supplemented and gas equilibrated (90% N2, 5% 02 and 5% C02) Ham's FIO medium (as above) to a radioactivity concentration of 2 pCi/ml. The incubation time was 1 h. Other eggs were incubated under the same conditions with 100 pCi [8-3H]adenosine/ml (UVVVR; sp. act. 27 Ci/ mmol). This precursor is incorporated into both DNA and RNA and, unlike the conventionally used RNA precursor, uridine, it is taken up much more efficiently by early (mouse) embryos (Clegg & Pikó, 1982). The incubation time for [8-3H]adenosine was 1-3 h. Autoradiography. After the incubation with nucleic acid precursors, eggs were thoroughly washed in fresh medium to remove unincorporated radioactivity and fixed with a mixture of 2-5% glutaraldehyde and 0-6 paraformaldehyde in 0-1 M-cacodylate buffer (pH 7-2). Specimens were then washed in buffer, post-fixed using the osmium-ferricyanide method (McDonald, 1984), embedded in Epon and sectioned with an Ultrotome III LKB ultramicrotome. Thick sections (~ 0-5 µ ) were coated with Ilford K5 liquid nuclear emulsion (Ilford, Basildon, Essex, UK) and exposed for 3-6 weeks. Thin sections were coated with Ilford L4 nuclear liquid emulsion and exposed for 2-3 months. Thick- and thin-section autoradiographs were developed with D 19. They were then stained with toluidine blue and with uranyl acetate and lead citrate, respectively. Thick section autoradiographs were examined in a light microscope, while thin- section autoradiographs were viewed in a transmission electron microscope. Staging of pronuclear development. Developing male pronuclei were identified by their close association with remnants of the sperm flagellum and were classified into four developmental stages. Briefly, Stages 1 and 2 were characterized by partial and total sperm chromatin decondensation, respectively. Both of these stages lacked a nuclear envelope, but the assembly of a new nuclear envelope around pronuclei began at Stage 2. By contrast, Stage 3 pronuclei were surrounded by a continuous nuclear envelope—although this was morphologically immature (dilated perinuclear space and few nuclear pores). Stage 3 pronuclei also showed a profound structural reorganization of chromatin, marked by the loss of its homogeneous distribution and the development of chromatin clusters of various sizes. Nucleolar precursors began their development at this stage. Stage 4 was characterized by the presence of a fully developed nuclear envelope. The typical feature of the nuclear envelope at this stage was the presence of numerous vesicles located in dilated segments of the perinuclear space. The development of nucleolar precursors had also been completed by this stage.

Results

Incorporation of [3 ]thymidine An overview of pronuclear development and labelling in eggs incubated with [3H]thymidine is shown in Table 1. No incorporation of [3H]thymidine was observed during Stages 1, 2 and 3 of pronuclear development. The first incorporation of this DNA precursor could only be detected in morphologically fully developed pronuclei that had reached Stage 4, as shown by the typical

Downloaded from Bioscientifica.com at 10/09/2021 01:16:57PM via free access Table 1. Quantitation of pronuclear development in the cohort of eggs incubated with [3H]thymidine Time after No. of pronuclei* Egg insemination no. (h) Total Stage 1 Stage 2 Stage 3 Stage 4

1 1 2 1 3 1 4 6 5 6 6 6 7 12 8 12 9 12 10 16 11 16 12 16 13 16

* Labelled pronuclei in bold type.

Fig. 1. Electron-microscope autoradiographs of fully developed male pronuclei (Stage 4) labelled with [3H]thymidine. (a) Heavily labelled chromatin concentrated at the periphery of a pronucleus (pn) and contrasting with the unlabelled cytoplasm. Note the double-membrane- bounded vesicles (v) apparently detaching from the nuclear envelope to the nearby cytoplasm. 25 000. (b) Homogeneous nucleolar precursor (np) unlabelled in its interior but surrounded by strongly labelled chromatin. A nuclear-envelope-derived cytoplasmic vesicle ( ), 30 000. Downloaded from Bioscientifica.com at 10/09/2021 01:16:57PM via free access Table 2. Quantitation of pronuclear development in the cohort of eggs incubated with [3H]adenosine Time after No. of pronuclei* Egg insemination no. (h) Total Stage 1 Stage 2 Stage 3 Stage 4 1 1 2 3 1 4 1 5 6 6 6 7 6 8 12 9 12 10 12 11 12 12 16 13 16 14 16

*Labelled pronuclei in bold type.

Table 3. Summary of developmental changes in the ability of human male pronuclei to incorporate nucleic acid precursors Stage of Time after pronuclear gamete fusion Thymidine Adenosine development (h)* incorporation! incorporationf 1 1 4 + 12 + + + +

*Time after in-vitro insemination of zona-free eggs when a particular stage first appears. tScoring of labelled nucleoside incorporation: -, absent; +, low; -t- +, high. features of the nuclear envelope and nucleolar precursors (Fig. 1). The presence of numerous out- pocketings of the perinuclear space containing vesicles filled with material of medium electron density (Fig. la) was one of these characteristic features. Nucleolar precursors had completed their development at this stage and appeared as homogeneous filamentous intranuclear bodies (Fig. lb). All pronuclei that had reached this developmental stage (as assessed by fine-structural mor¬ phology) were labelled (Table 1). The label was accumulated in the same regions as condensed chromatin, preferentially near the nuclear envelope (Fig. la). The interior of the nucleolar pre¬ cursors was free of label, whereas labelled DNA was concentrated around their periphery (Fig. lb). The absence of DNA within the fully developed nucleolar precursors was expected, based on the disappearance of typical chromatin structures from these bodies.

Incorporation of [3HJadenosine In contrast to [3H]thymidine, which was only incorporated into morphologically fully developed male pronuclei (Stage 4), incorporation of [3H]adenosine (Table 2) was evident as early as Stage 3 of pronuclear development (Fig. 2a). The label was preferentially located in regions occupied by dense chromatin clusters occurring either individually or as parts of developing nucleolar precursors (Fig. 2b). In the latter case, it was often associated with aggregates of chromatin intermingled with Downloaded from Bioscientifica.com at 10/09/2021 01:16:57PM via free access Fig. 2. Electron-microscope autoradiographs of developing male pronuclei (Stage 3) labelled with [3H]adenosine. (a) Low-magnification view showing the overall labelling intensity of a Stage 3 pronucleus. Note the immaturity of the nuclear envelope with dilated perinuclear space and only rare nuclear pores, 14000. (b) High-magnification view showing labelling in chromatin clusters (arrows) some of which participate in the formation of nucleolar precursors (double arrows), 32 000. Downloaded from Bioscientifica.com at 10/09/2021 01:16:57PM via free access interchromatin granules, whereas the fusing dense bodies, representing another building element of nascent nucleolar precursors (J. Tesarík & V. Kopecny, unpublished), were unlabelled. The label could be detected within newly formed nucleolar precursors as long as the complexes of condensed chromatin and interchromatin granules could be morphologically recognized in them (Fig. 3a). At Stage 4, when the granules and chromatin disappeared from the nucleolar precursors, their interior was no longer labelled and the label only remained associated with the chromatin, occasionally persisting for some time in their very marginal regions (Fig. 3b). Fully developed homogeneous nucleolar precursors were, as for [3H]thymidine autoradiography (see Fig. lb), free of label.

Structural and temporal correlations of developmental changes in [3Hj'thymidine and [3 H¡adenosine incorporation into pronuclei The changes in the ability of developing male pronuclei to incorporate the two nucleic acid precursors used in this study are summarized in Table 3. From the developmental pattern of [3H]thymidine incorporation it is evident that S phase does not begin until the pronuclei have fully accomplished their ultrastructural development (Stage 4), at least 12 h after gamete fusion. The same intensity and distribution of labelling in Stage 4 pronuclei were observed for [3H]adenosine, a precursor that can also be incorporated into replicating DNA. A weak but evident labelling with [3H]adenosine was detected as early as Stage 3, when S phase had not yet begun, as indicated by the absence of [3H]thymidine incorporation (Table 3). This adenosine incorporation, occurring before the onset of DNA replication, can only be ascribed to an early pronuclear RNA synthesis.

Discussion

This study was performed with eggs that had failed to be fertilized in a clinical in-vitro fertilization programme. Accordingly, the normality of these eggs with regard to the phenomena described in this study must be considered before further conclusions and interpretations are made. We have demonstrated that, if spermatozoa are normal, the majority of failed fertilization attempts are due to functional immaturity of the zona pellucida and are caused by premature insemination of oocytes that have not yet reached metaphase II at the moment of the first contact with spermatozoa (Tesarík et al, 1988a). Tesarík et al. (1988a) also showed that such unfertilized oocytes, if stripped of the zona pellucida and reinseminated, become penetrated and develop apparently normal pronuclei. The ability of these oocytes to support pronuclear development remains virtually unchanged when they are cultured for up to 48 h (J. Tesarík & V. Kopecny, unpublished). While it is clear that such eggs cannot be considered normal, because they are polyspermic, they represent a good and easily reproducible model for the study of pronuclear events, particularly suitable for the analysis of structure—function correlations. In this study we have shown that DNA synthesis in the developing human male pronucleus does not begin until 12 h after gamete fusion. This relatively late onset of S phase conforms with the findings obtained in other mammalian species (e.g. Howlett & Bolton, 1985; Naish et al, 1987) and is apparently related to the complexity of transformations which the sperm nuclear template must undergo before it becomes available to enzymes required for DNA synthesis. Even though the reduction of sperm chromatin disulphide bonds (Perreault et al, 1984) and removal of protamines from the sperm DNA (Kopecny & Pavlok, 1975; Ecklund & Levine, 1975) are thought to be prerequisites for the start of DNA synthesis, additional steps are obviously required (Naish et al, 1987). Our experiments have confirmed the view that the availability of the male pronuclear template for DNA synthesis is strictly correlated with the development of a corresponding pronuclear structure (see below). In addition to nuclear template availability, the actual DNA replicating capacity of male pro- nuclei is presumably dependent on specific actions of egg cytoplasmic factors. This may explain Downloaded from Bioscientifica.com at 10/09/2021 01:16:57PM via free access Fig. 3. Electron-microscope autoradiographs showing developing nucleolar precursors in Stage 3 (a) and Stage 4 (b) pronuclei labelled with [3H]adenosine. (a) Labelling of chromatin struc¬ tures at the periphery (arrows) and in the interior of a developing nucleolar precursor (np). 50 000. (b) A more advanced stage of nucleolar precursor development in a Stage 4 pro- nucleus showing a quite different labelling pattern as compared with Stage 3 (see Fig. 3a). Abundant labelling is present in dense chromatin regions restricted to the periphery of the nucleolar precursor (np), whereas its interior is free of label, v,DownloadedDouble-membrane-bounded from Bioscientifica.com at 10/09/2021 01:16:57PM vesicle detaching from the nuclear envelope, 45 000. via free access why DNA synthesis in human male pronuclei can start much sooner during pronuclear develop¬ ment in some heterologous systems (Gordon et al, 1985; Ohsumi et al, 1986), as compared with our findings. These comparisons suggest marked interspecies differences in the mode of action of cytoplasmic factors controlling the development of pronuclear structure and function. The finding of [3H]adenosine incorporation into Stage 3 pronuclei, which have not yet started DNA synthesis, implies the occurrence of RNA synthesis in this intermediate state of pronuclear development. Adenosine or adenine tagged with 3H- or 14C-radioisotopes were used in different studies of nucleic acid synthesis. Differentiation between the incorporation of these precursors into DNA or RNA was obtained either by extraction/enzymic digestion of one of the nucleic acids or by an autoradiographic analysis of the time course and topology of the precursor incorporation into cells with regard to the patterns characteristic for each of the nucleic acids (Walker & Leblond, 1958; Harris, 1959). There is no incorporation of [3H]adenosine into nuclear proteins (Harris, 1959; L. Piko, personal communication). Since our data, based on detailed fine-structural evidence, exclude labelling in DNA, the most probable candidate for this early pronuclear labelling with [3H]adenosine is RNA. Recent results obtained with a similar model of pronuclear development (hamster/hamster, boar/hamster) (V. Kopecny, A. Pavlok & M. Tománek, unpublished data) revealed a difference in the incorporation pattern of [3H]adenosine into early versus late pronuclei, similar to that described in this study (compare Figs 3a and 3 b). Whether or not RNA synthesis continues at Stage 4 of pronuclear development cannot be determined with the methodology employed in this study because, after the onset of S phase, the eventual labelling of newly synthesized RNA would be masked by concomitant incorporation of [3H]adenosine into DNA. There is little information about the transcriptional activity of mammalian pronuclei in the literature. While a major outburst of RNA synthesis in the mouse embryo only occurs at the 2-cell stage (Clegg & Pikó, 1982; Pikó & Clegg, 1982), a limited incorporation of [3H]adenosine into RNA has been detected biochemically in 1-cell mouse zygotes (Clegg & Pikó, 1982). Labelling of very early hamster and ram pronuclei with [3H]adenosine has also been observed (Kopecny et al, 1986). In the human embryo, RNA synthesis could first be observed at the 4-cell stage using auto¬ radiography and [3H]uridine as precursor (Tesarík et al., 1986a, b). However, by analogy with mouse embryos (Clegg & Pikó, 1977), it is presumed that adenosine is taken up much more efficiently into early human embryos as compared with uridine. Moreover, the direct cytoautoradiographic method used in the present study is more sensitive than methods detecting label incorporated into RNA after its isolation from the cells by at least two orders of magnitude. Accordingly, the present observations do not imply a major activation of the embryonic genome in 1-cell human zygotes and they are therefore not in conflict with the previous findings that have allocated the most relevant enhancement of human embryonic gene transcription (Tesarík et al, 1986a, b) and the first embryonic gene expression (Tesarík, 1987; Braude et al, 1988; Tesarík et al, 1988b) between the 4- cell and 8-cell stages of preimplantation development. The slight incorporation of [3H]adenosine into developing male pronuclei obviously reflects an extremely low level of RNA synthesis. This very early transcriptional activity in the 1-cell zygote may be necessary for the basic constitutive pronuclear events (see below). Considerable evidence is available to suggest that the DNA synthetic activity of pronuclei is tightly bound to the development of an appropriate pronuclear structure (Naish et al, 1987; Sheehan et al, 1988). Our findings corroborate these data and extend them to pronuclear RNA synthesis. From an opposite viewpoint, the early pronuclear RNA synthesis may play a permissive role for the occurrence of some structural developmental changes. For instance, the limited trans¬ criptional activity of the chromatin taking part in the assembly of nucleolar precursor bodies may be necessary for the accomplishment of this developmental process. This requirement would not be surprising as there is a great deal of similarity between the ultrastructural picture of the assembly of homogeneous nucleolar precursors in human male pronuclei (J. Tesafik & V. Kopecny, unpublished) and nucleolar reformation in mitotic animal cells (for review see De la Torre &

Downloaded from Bioscientifica.com at 10/09/2021 01:16:57PM via free access Gimenez-Martin, 1982), a process which can be inhibited by selective impairment ofthe transcription of ribosomal genes (Benavente et al, 1987). In conclusion, these observations have clearly established the relationship between the develop¬ ment of human male pronuclear structure and the ability of pronuclei to synthesize nucleic acids. Experiments aimed at determining the causality of the relationships between individual phenomena of these processes and examining the effects of cytoplasmic factors are underway.

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