The morphological changes following Sendai virus-enhanced interaction of purified fractions of human spermatozoa with various types of fibroblast lines were examined over a period of several days. The incorporation of spermatozoa was monitored by a variety of immunofluorescent staining techniques, including one for protamin. The initially high incorporation frequency (30–40 %) was accompanied by swelling of the acrosome and nucleus, which, in most instances, was followed by gradual lysis of the sperm components. However, a small proportion of all incorporated spermatozoa (2 %) exhibited chromatin decondensation, accompanied by a shift from a protamin to histone content and induction of RNA and DNA synthesis. These latter cells appear to fit the criteria for true reactivation and electron-microscope studies show that they do not undergo phagocytosis as do the majority of incorporated spermatozoa.

Since the discovery that genetically inactive nuclei, such as those of chicken erythrocytes, can be reactivated following hybridization with a transcriptionally active, cell type (Harris, 1965; Harris & Cook, 1969), several attempts have been made to reactivate either spermatozoa or spermatids by fusion with somatic cells. The conclusionof Sawicki & Koprowski (1971) was that Sendai virus fusion of rabbit spermatozoa with various types of somatic cells produced no reactivation, but that lysolecithin fusion can result in nuclear swelling followed by induction of DNA synthesis in a small number of instances (Gledhill, Sawicki, Croce & Koprowski, 1972). Others have claimed that both mouse and rat spermatozoa can be incorporated spontaneously into somatic cells, resulting in a subsequent transfer of DNA from the sperm to the host cell nucleus and induction of new host cell characteristics (Bendich, Borenfreund & Sternberg, 1974; Higgins, Borenfreund & Bendich, 1975), without evidence for an associated RNA or DNA synthesis of the sperm nucleus. Nyormoi, Coon & Sinclair (1973) claimed that Sendai virus fusion of rat spermatids with thymidine kinase-negative mouse cells, followed by standard HAT selection, results in the isolation of hybrid clones, containing a more or less haploid number of rat chromosomes. However, as demonstrated by Phillips et al. (1976), using human spermatozoa in conjunction with an HPRT-negative mouse line, isolation of hybrid clones is likely to result from fusion with contaminating, diploid, somatic cells present in the sperm cell suspension. These authors also conclude that phagocytosis is the method of entry of the spermatozoa into fibroblasts and that degeneration of the spermatozoa occurs within a matter of days.

This study examines the phenomenon of Sendai virus-enhanced incorporation of human spermatozoa into somatic cells at the heterokaryon level, to see whether lack of reactivation (Sawicki & Koprowski, 1971), followed by breakdown (Phillips et al. 1976), is a general rule for this system, or whether there is also evidence for reactivation of the type described by Elsevier & Ruddle (1976) for mouse spermatids and Gledhill et al. (1972) for rabbit spermatozoa. We make use of fluorescent antibody staining to localize the presence of spermatozoa in the cytoplasm of fibroblasts and to help monitor the structural and chemical changes shown by the spermatozoa over a period of several days.

Preparation of human spermatozoa for cell fusion

Fresh human sperm samples were obtained either from healthy normal donors, or from patients visiting the hospital for routine fertility examination. Only ejaculates with a cell concentration and mobility greater than 2× 107/ml and 20% respectively were accepted for further processing, which involved fractionating the sperm sample in a 10% − 15% − 25% sterile bovine serum albumin (BSA) column at 37 °C (Ericsson, Langevin & Nishino, 1973). After 2-h incubation, the bottom fraction was isolated and the concentration of spermatozoa adjusted with Earle’s solution to 5 × 10 6 /ml. In early experiments these spermatozoa were subsequently swollen by incubation in a 50 rπM Tris Cl buffer with 2 mM dithiothreitol (DTT) for 45 min at room temperature. A fresh trypsin solution (concn./normality) was added to a final concentration of 15 μ g/ml. The reaction was terminated by adding an excess of phenyl-methanesulphonylfluoride (PMSF), when at least 50 % of the sperm heads were swollen.

Cell fusion

Cell fusion between swollen and/or unswollen spermatozoa of the purified sperm fraction was performed using one of the following cell lines: an SV40-transformed human kidney line (NB); 2 HPRT-negative and 1 wild type mouse line (A9, PG19 and 3T3 respectively). The procedure generally followed was to seed 5 × 104 cells onto each of 30 microscope slides and to grow these for 24 h in Earle’s MEM, or HAM’s F10, enriched with 10% newborn calf serum. Following removal of the medium, 5 × 105 spermatozoa were layered onto each slide and permitted to sediment for 15 min. Five hundred HAU (haemagglutinating units) of β-propiolactone-inactivated Sendai virus were then added to each slide, which were stood at 4 °C for 7 min and subsequently for another 30 min at 37 °C. Medium, of composition described above, was then added, changed after 4 days and thereafter every 48 h, depending upon the length of the experiment.

Morphological analysis of heterokaryon

At time intervals varying between 0 · 5 and 144 h following fusion the slides were rinsed twice in phosphate-buffered saline (PBS) and the cells fixed either in acetone at —20 °C, if a subsequent immunofluorescent staining reaction was to be employed, or in ethanol: acetone (1: 1) at room temperature. Evidence for incorporation of sperm nuclei into host cell cytoplasm was based upon the presence of small nuclei with or without associated sperm organelles, such as mid-piece and tail, and which also demonstrated differential staining behaviour to the nuclei or micronuclei of the host cells of the following types:

A positive sperm nucleus and negative cell nucleus staining, based upon an immunofluorescent staining of sperm protamin (Kolk, Samuel & Rümke, 1974), using rabbit antiserum (dilution 1:20) and a tetraethylrhodamineisothiocyanate (TRITC)-coupled goat anti-rabbit conjugate (Nordic, dilution 1:20).

Negative sperm nucleus and positive cell nucleus staining, based upon detection of SV40 antigen in the host cell nuclei, following treatment with hamster antiserum (dilution 1:20) and thereafter a fluoresceinisothiocyanate (FITC)-conjugated pig anti-hamster serum (Nordic, dilution 1:50). Frequently, the 2 treatments were combined. Following fusion with mouse cells, the preparations were stained with Hoechst 33258 as described by Elsevier & Ruddle (1976). In our system, incorporated spermatozoa were in general weakly fluorescent or unstained, as distinct from the intensely fluorescent mouse nuclei.

All examinations were carried out on a Leitz microscope fitted with vertical u.v. illumination for fluorescence and Heini phase-contrast for bright-field work. One thousand host cells from each slide were scored for evidence of sperm cell incorporation following one of the staining techniques described above. The stage coordinates of each heterokaryon were noted and a photograph or small diagram made to help in relocating the cell for subsequent investigations, including autoradiography, Giemsa staining etc. Incorporated spermatozoa were classified according to their level of morphological transformation into the following groups: normal (n), swollen acrosomal region (sa), swollen head (sh), chromatin decondensation (cd), nucleus (nu) and fragmentation (f).

Electron-microscopic investigation of heterokaryons was undertaken at 20 h, 2 and 4 days following fusion. The cells were removed from the culture vessel following trypsin treatment (0 5 % soln., Difco), spun down, washed twice in PBS and fixed in 15 % glutaraldehyde and 1% osmium tetroxide. Embedding was carried out in Epon 812 (Luft, 1961) and ultrathin sections were cut using a diamond knife. Following section staining in 1 % uranyl acetate and 1 % lead hydroxide, examinations were made on a Siemens Elmiskop 1 and photographs of each heterokaryon taken at magnifications of 4000 and 14000 times, respectively, using Ilford SP 332 film.

RNA synthesis

Fused cells were grown in medium containing 1 μCi/ml of [1H]uridine (Radiochemical Centre, Amersham, sp. act. 19 · 7 Ci/mM) for the last 3 h preceding fixation. The cells were fixed at time intervals between 12 h and 6 days following fusion. Individual heterokaryons were identified by one of the methods described above, the coordinates noted and autoradiograms made by coating the slide with Ilford L4 emulsion and exposing at 4 °C for between 2 and 4 weeks. Following development in Kodak D19 and fixation with Snelfix, the autoradiograms were counterstained with Giemsa (2% aq, Gurr) and embedded in malinol. The frequency of nucleoli in nuclei of presumptive sperm origin was carried out by detecting heterokaryons as described above and restaining the cells with one of the following nucleolus-staining reations; methyl green/pyronin (0 5 % w/v, pH 4 8) and acridine orange (1 μg/ml, Gurr) both for detection of concentrations of RNA, or silver nitrate staining, which is specific for ribosomal RNA cistron activity and is localized to nucleoli and the interphase nucleus (Bloom & Goodpasture, 1976).

DNA synthesis

The cells were cultured in HAM’s F10 containing 1· 5 Ci/ml of [3H]thymidine (Radiochemical Centre, Amersham, sp. act. 26 Ci/mM) for 5 h and then fixed immediately. The identification of heterokaryons and preparation of autoradiograms were as desciibed for RNA synthesis.

Histone synthesis

The possibility of transition from protamin to histone in the sperm nuclear component of heterokaryons was investigated by plotting changes in the frequency of positively stained sperm nuclei with the immunofluorescent protamin reaction at various time intervals following fusion and associated changes in the histone-staining reaction given by brilliant sulphaflavin at pH 8·1, following DNA extraction in 5 % TCA for 15 min at 90 °C (Ruch, 1970). Our own pilot experiments show, that the latter reaction can differentiate between histone and protamin containing nuclei by yielding green fluorescence (540 nm) with histone-positive nuclei and yellow fluorescence (590 nm) with protamin-positive nuclei.

Sperm purification

The ejaculates used in this study had an average concentration of 6· 7×107/ml and a motility of 56%. Following separation in a BSA column, 10% of the spermatozoa were found in the bottom fraction and were characterized by an extremely uniform morphology, absence of immature cells and a high motility (95%) when compared with the top fraction, which contained cells in all stages of spermiogenesis. This population exhibited a low motility (25%) and included diploid somatic cells such as leukocytes and epithelial cells. The contamination frequency of diploid cells in the bottom fraction was less than 4 × 10−5.

It was noted, that the presence of immature stages in the top fraction correlated with a positive histone staining reaction with the brilliant sulphaflavin reaction, suggesting an incomplete changeover from histone to protamin in many immature cells (Fig. 1).

Fig. 1.

A. Unpurified human ejaculate, containing diploid leukocytes and early stages from spermiogenesis; phase-contrast. × 310. B. Same field stained with brilliant sulphaflavin, following DNA extraction with hot TCA. Only protamin-containing spermatozoa are brightly fluorescent, c. Purified sperm sample, following BSA fractionation; phase-contrast, ×310. D. Same field stained with brilliant sulphaflavin. Only mature spermatozoa are present.

Fig. 1.

A. Unpurified human ejaculate, containing diploid leukocytes and early stages from spermiogenesis; phase-contrast. × 310. B. Same field stained with brilliant sulphaflavin, following DNA extraction with hot TCA. Only protamin-containing spermatozoa are brightly fluorescent, c. Purified sperm sample, following BSA fractionation; phase-contrast, ×310. D. Same field stained with brilliant sulphaflavin. Only mature spermatozoa are present.

Cell fusion

The mixing of spermatozoa with any of the cell lines used, resulted in a spontaneous uptake of spermatozoa into an average 8 % of cells within 24 h. The frequency of uptake was significantly increased when inactivated Sendai virus was used as a fusion agent in varying concentrations and optimum results were achieved with 500 HAU of virus per slide (Fig. 2), resulting in uptake in 30–40% of cells. In 2% of cells there was evidence of morphological changes suggestive of heterokaryon formation, as considered below, which was almost absent either in control cultures without virus (0 · 1 %), or without spermatozoa (< 0 ·01 %). We also found, that fusion enhancement with polyethylene glycol (Elsevier & Ruddle, 1976) resulted in a lower heterokaryon production according to our criteria (1 · 8%) than Sendai virus.

Fig. 2.

The effect of Sendai virus concentration on the uptake of spermatozoa by NB cells. No. of haemagglutinating units (HAU): ○–○, 250; □ □, 500; × … ×, 1000–3 000; •–•, none. Ordinate, % of cell population showing association. Abscissa, no. of associated spermatozoa/cell.

Fig. 2.

The effect of Sendai virus concentration on the uptake of spermatozoa by NB cells. No. of haemagglutinating units (HAU): ○–○, 250; □ □, 500; × … ×, 1000–3 000; •–•, none. Ordinate, % of cell population showing association. Abscissa, no. of associated spermatozoa/cell.

Variation of other fusion parameters, such as the temperature at which fusion is completed and prior swelling of the spermatozoa as a result of technical procedures (Kolk, Samuel & Rümke, 1974) showed that completion at 37 °C with unswollen spermatozoa resulted in the highest production of heterokaryons and this technique was accordingly standardized for all subsequent experiments.

One of the problems with long-term cultures (more than 5 days), was that the host cells could continue dividing with the possible loss of spermatozoa. Suppression of host cell division by X-irradiation (10 J kg− 1) not only permitted continuance of the slide cultures for up to 3 weeks, but also clearly suppressed the frequency of heterokaryon production (0 · 9%), indicating that sperm cell reactivation is at least partially dependent upon the activity of a viable or undamaged host cell. Long-term cultures were eventually produced by using the mouse cell line 3T3 as host cell. This line exhibits contact inhibition for prolonged periods of time when maintained in preconditioned medium.

Heterokaryon analysis

One of the biggest problems inherent in this work, is to distinguish between micronuclei originating from the host cell nucleus and those from spermatozoa. However, the use of one of the fluorescent staining techniques described in the methods section, permitted the unequivocal identification of sperm nuclear material in the cytoplasm of host cells. In the case of fusions involving NB cells for example, the NB cell micronuclei were almost always distinguished by a positive SV40 T-antigen reaction (Fig. 3). Control cultures without spermatozoa, resulted in a frequency of less than 0 ·01 % cells showing negatively stained micronuclei, as opposed to the 2 · 0% + 0·49 (s.D.) found in experimental cultures 144 h following fusion. Similar differences were also found for other cell lines and staining techniques (Fig. 4).

Fig. 3.

Heterokaryon, containing a NB cell nucleus and a much smaller sperm nucleus (arrow); phase-contrast, × 690. B. Same heterokaryon stained for SV40 T-antigen. Only the NB nucleus shows fluorescence. C. NB cells grown on a microscopic slide, with a micronucleus (arrow) present; phase-contrast, × 690. D. Same cells, stained for SV40 T-antigen. All nuclei, including the micronucleus, show positive fluorescence.

Fig. 3.

Heterokaryon, containing a NB cell nucleus and a much smaller sperm nucleus (arrow); phase-contrast, × 690. B. Same heterokaryon stained for SV40 T-antigen. Only the NB nucleus shows fluorescence. C. NB cells grown on a microscopic slide, with a micronucleus (arrow) present; phase-contrast, × 690. D. Same cells, stained for SV40 T-antigen. All nuclei, including the micronucleus, show positive fluorescence.

Fig. 4.

A. A mouse-sperm heterokaryon stained with Hoechst 33258. The mouse nucleus contains brightly fluorescing aggregates of constitutive heterochromatin, while the sperm nucleus has very weak fluorescence. × 690. c. Same heterokaryon, phase-contrast. B. Mouse fibroblast with a micronucleus, originating from the main nucleus. Hoechst 33258 staining results in an equally intense fluorescence in both nuclei. ±690. D. Same cell, phase-contrast.

Fig. 4.

A. A mouse-sperm heterokaryon stained with Hoechst 33258. The mouse nucleus contains brightly fluorescing aggregates of constitutive heterochromatin, while the sperm nucleus has very weak fluorescence. × 690. c. Same heterokaryon, phase-contrast. B. Mouse fibroblast with a micronucleus, originating from the main nucleus. Hoechst 33258 staining results in an equally intense fluorescence in both nuclei. ±690. D. Same cell, phase-contrast.

The incorporated spermatozoa demonstrated a variety of morphological changes and in isolated examples, the whole sperm structure was lost and an enlarged nucleus appeared within 30 min. More generally, however, this change was initiated only after several hours or even days. Table 1 describes the variation in sperm morphology induced over a period of 6 days. The first alteration observed was the puffing of the acrosomal region, followed by swelling of the entire head to 4 times or more its original size. The majority of swelling occurred during the first 72 h, following which the frequency decreased, either by fragmentation, or changes induced by further reactivation.

Table 1.

Percentage of reactivated fused and attached spermatozoa

Percentage of reactivated fused and attached spermatozoa
Percentage of reactivated fused and attached spermatozoa

Apparently, incorporation within the host cell cytoplasm is not required to induce swelling, since attached spermatozoa also showed enlarged heads as well. This phenomenon was only rarely observed in free-lying sperm cells and the few examples we found, possibly had become detached from cell membranes (Table 1). When swelling of the outer membranes was almost accomplished, decondensation of chromatin led to an increase of the nuclear volume. This disaggregation of chromatin started from the nuclear edge in the region of the acrosome and later was continued more centrally, so that the chromatin looked fragmented (Fig. 3B). Finally, in 2% of the cells, interphase nuclei arose, in which the chromatin was equally distributed (Fig. 5D). The ultimate nuclear size varied from about 12 to 30 μm in diameter and depended upon the preceding enlargement of the sperm head. Chromatin decondensation also occurred in spermatozoa adherent to cell surfaces, but apparently never to the level of a complete interphase nucleus.

Fig. 5.

A. Association of human spermatozoa with NB cells immediately following treatment with Sendai virus. The spermatozoa are not yet incorporated and are moiphologically unchanged, × 380. B. Incorporated, swollen, sperm head showing early chromatin decondensation; large blocks of highly condensed chromatin are still visible. × 380. C. Presumptive heterokaryon, showing a swollen sperm head with almost completely dispersed chromatin. × 380. D. Heterokaryon, containing a NB host nucleus and

Fig. 5.

A. Association of human spermatozoa with NB cells immediately following treatment with Sendai virus. The spermatozoa are not yet incorporated and are moiphologically unchanged, × 380. B. Incorporated, swollen, sperm head showing early chromatin decondensation; large blocks of highly condensed chromatin are still visible. × 380. C. Presumptive heterokaryon, showing a swollen sperm head with almost completely dispersed chromatin. × 380. D. Heterokaryon, containing a NB host nucleus and

In addition to these changes, an increasing number with time of incorporated spermatozoa, usually located inside vacuoles, was broken down, so that only small nuclear and acrosomal remnants remained (Fig. 5E). In consequence of this, the with a nuclear membrane (Fig. 6). These observations will form the basis of a separate communication.

Fig. 6.

EM picture of a sperm nucleus inside mouse 3T3 cytoplasm. The sperm chromatin is highly decondensed and a nuclear membrane is present. × 56000. cm, host cell membrane; sc, sperm chromatin; snm, sperm nuclear membrane.

Fig. 6.

EM picture of a sperm nucleus inside mouse 3T3 cytoplasm. The sperm chromatin is highly decondensed and a nuclear membrane is present. × 56000. cm, host cell membrane; sc, sperm chromatin; snm, sperm nuclear membrane.

Fig. 7.

Membrane fusion between the 3T3 mouse cell membrane and the human sperm acrosome. The mouse fibroblast-develops microvilli, extruding from the cytoplasm during early contact with spermatozoa. × 78000. cm, host cell membrane; tain, inner acrosomal membrane; sc, sperm chromatin.

Fig. 7.

Membrane fusion between the 3T3 mouse cell membrane and the human sperm acrosome. The mouse fibroblast-develops microvilli, extruding from the cytoplasm during early contact with spermatozoa. × 78000. cm, host cell membrane; tain, inner acrosomal membrane; sc, sperm chromatin.

RNA synthesis

The frequency of nucleolus formation in reactivated sperm nuclei identified by immunofluorescent staining, followed by Giemsa staining, is given in Table 2.

Table 2.

Nucleoli in interphase sperm nuclei

Nucleoli in interphase sperm nuclei
Nucleoli in interphase sperm nuclei

This shows an average increase of 5 % in frequency of nucleolus formation with each period of 12 h. The provenance of the nucleoli was confirmed by silver nitrate staining in a parallel experiment (Fig. 8).

Fig. 8.

A. A sperm-mouse heterokaryon stained with Hoechst 33258 for sperm nucleus identification. × 380. B. Silver staining according to Bloom and Goodpasture of the same heterokaryon. Note the silver concentrations over the sperm nucleus (arrow), indicating active rRNA cistrons. × 380.

Fig. 8.

A. A sperm-mouse heterokaryon stained with Hoechst 33258 for sperm nucleus identification. × 380. B. Silver staining according to Bloom and Goodpasture of the same heterokaryon. Note the silver concentrations over the sperm nucleus (arrow), indicating active rRNA cistrons. × 380.

Both acridine orange and methyl fast-green staining, resulted in deep red staining of the sperm nuclei with little or no differentiation, which could possibly indicate the presence of high concentration of RNA throughout the sperm nucleoplasm. The detection of tritiated uridine incorporation by autoradiography showed that silver grains could be detected over both host cell and sperm nuclei with the highest concentrations located above the nucleoli (Table 2). An apparent synchronization of RNA synthesis was observed with approximately equal grain densities over both the host cell and sperm nuclei. Labelling of the sperm nucleus occurred in about 2 · 2% of heterokaryons studied. For this study, we assumed that a positive labelling was present, when the concentration of silver grains per nucleus was at least 3 times higher than a comparable adjacent background area and when the silver grains were concentrated above the nucleoli. The average grain count per sperm nucleus was 25 (range 19-31) and that of an equivalent background area 3.

DNA synthesis

Our SV40-transformed NB cells were active in virus production, so that culturing in medium containing [3 H]thymidine for several hours, resulted in labelling of the viral DNA as well. The autoradiographs showed a resultant high density of silver grains above the cytoplasm and nuclei, which was unrelated to cellular DNA synthesis. We circumvented this difficulty by repeating the experiments with Aq and PG19 cells. Following identification of sperm nuclei with Hoechst 33258 and subsequent preparation of autoradiograms, the labelling distribution shown in Table 3 was found. In most instances of sperm nucleus labelling, an identical labelling was found above the host cell nucleus (Fig. 9), suggesting synchronization of DNA synthesis.

Table 3.

Labelling distributions obtained with [3 H]uridine and [3 H]thymidine

Labelling distributions obtained with [3 H]uridine and [3 H]thymidine
Labelling distributions obtained with [3 H]uridine and [3 H]thymidine
Fig. 9.

A. Mouse-human sperm fusion culture stained with Hoechst 33258 to detect sperm nuclei. × 310. B. Epi-illuminated autoradiograph of the same cells, following a 5-h incubation in medium containing tritiated thymidine. Silver grains arc visible at the same density over the sperm nucleus and mouse nuclei, indicating synchrony of DNA synthesis within the same heterokaryon. × 310.

Fig. 9.

A. Mouse-human sperm fusion culture stained with Hoechst 33258 to detect sperm nuclei. × 310. B. Epi-illuminated autoradiograph of the same cells, following a 5-h incubation in medium containing tritiated thymidine. Silver grains arc visible at the same density over the sperm nucleus and mouse nuclei, indicating synchrony of DNA synthesis within the same heterokaryon. × 310.

Protamin-histone exchange

Fig. 10 shows the relationship between immunofluorescent protamin staining and the protamin-histone staining provided by brilliant sulphaflavin of sperm nuclei following fusion. This latter technique results in mature sperm nuclei, having a yellow fluorescence with a peak at 590 nm (protamin staining) and reactivated sperm nuclei and host cell nuclei with a green fluorescence peak at 540 run (histone staining). It is clear that as the percentage of sperm nuclei showing protamin staining with either staining reaction decreases, so the percentage of histone-positive nuclei increases. Microspectrofluorometric analysis of the spectral shifts involved in the brilliant sulphaflavin reaction, suggest a clear change in the histone-protamin composition in the opposite direction to those found during spermiogenesis which is not attributable to either chromatin concentration, or degeneration effects (own unpublished observations).

Fig. 10.

Percentage of enlarged sperm nuclei showing protamin binding with anti protamin serum and FITC conjugate (◼–◼) and staining with brilliant sulphaflavin for protamin (•––) and histone (○–○).

Fig. 10.

Percentage of enlarged sperm nuclei showing protamin binding with anti protamin serum and FITC conjugate (◼–◼) and staining with brilliant sulphaflavin for protamin (•––) and histone (○–○).

From the results presented in this report, it can be concluded that human spermatozoa can be incorporated into the cytoplasm of various somatic cell lines. Following fusion, more than 30% of the parental cells contained one or more spermatozoa within their cytoplasm. Although the majority of those sperm cells exhibited no morphological alterations and was subsequently broken down within a matter of days, 22% of all incorporated spermatozoa demonstrated reactivation, consisting of nuclear swelling, chromatin decondensation and more rarely, RNA and DNA synthesis.

From electron-microscopic studies we concluded that most spermatozoa enter the host cell cytoplasm by endocytosis, but that fusion between the sperm and host cell membranes also occurs, although less frequently. This latter process finally brings the sperm chromatin, without any surrounding nuclear or cytoplasmic membrane, into direct contact with the cytoplasm as regularly observed in normal fertilization (Austin, 1969; Longo, 1973). It appears that only this category of sperm nuclei exhibit complete chromatin dispersion. In some instances, however, an incomplete chromatin decondensation was observed in phagocytosed sperm nuclei with a subsequent lytic breakdown. The ultrastructural observations confirm our results from the light microscope, but differ from those of Phillips et al. (1976), who found no reactivation and only lytic breakdown of sperm nuclei following the passive incorporation of spermatozoa into fibroblasts. Therefore it seems likely that incorporation leading to reactivation must be mediated via a fusion agent.

The claims over the relative efficiency of various fusion agents for promoting sperm cell fusion and reactivation appear to be inconsistent. Sawicki & Koprowski (1971), for example, found in contrast to our human sperm system, that Sendai virus does not enhance the fusion and reactivation of rabbit spermatozoa with fibroblasts. The limitations of Sendai virus were also commented upon by Elsevier & Ruddle (1976), who concluded that PEG was a much more efficient fusion promotor in their mouse spermatid fibroblast system. However, the frequency of heterokaryon production (2 × 10−2) in our experiments compared to that (1 × 10−4) found by Elsevier & Ruddle, does not lead us to believe that Sendai virus is inferior to PEG. Indeed, we found only small differences in the efficiency of these 2 agents with PEG giving a slightly lower heterokaryon frequency (1 · 8×10− 2). Although we have no experience in the use of lysolecithin, many of the structural alterations of rabbit and bull spermatozoa occurring in lysolecithin-mediated fusions (Gledhill et al. 1972; Koprowski & Croce, 1973; Lucy, 1975) are similar to those found in our experiments.

The mature spermatozoon has a highly differentiated series of membranes, which must first normally undergo the structural changes involved in the acrosome reaction, before the membrane fusion involved in fertilization can occur (Austin, 1965). Although no use was made of an accepted capacitation procedure during our experiments, one might speculate that the BSA or Tyrodes’ buffer used during sperm purification has affected the sperm membrane structure in an analogous fashion to capacitation. Blank, Soo & Britten (1976) have claimed, that mammalian spermatozoa show changes in their membranes, resembling those occurring prior to fertilization, when exposed to albumin solutions. However, our ultrastructure studies gave no indication of a higher frequency of spermatozoa showing an acrosomal reaction following BSA incubation, as compared to control sperm samples. Furthermore, capacitation following BSA incubation would probably result in a reduced Sendai virus binding (Poste, 1972). It is possible, however, that the high Ca2+ ion concentration of the Tyrodes’ buffer-stimulated heterokaryon formation (review, Poste & Allison, 1973).

Swelling of the sperm head is a universal feature of sperm reactivation and we accordingly attempted to increase our heterokaryon frequency by using sperm cells, which had been artificially swollen with DTT and trypsin treatment prior to fusion. This resulted, however, in a lower heterokaryon frequency (0 · 8%) possibly caused by membrane damage, which, following prolonged incubation, can also lead to loss of DNA (Kolk et al. 1974). Similarly, fusion with X-irradiated fibroblasts resulted in a lower heterokaryon formation (o·9%), suggesting the requirement of a fully active, undamaged, host cell cytoplasm and/or membrane to evoke sperm reactivation.

Host cell micronuclei falsely identified as sperm in origin would lead to an increased scoring of heterokaryons. However, control cultures without spermatozoa contained less than 0 ·01 % micronuclei with negative T-antigen staining, which would be scored as false positives. Another possible source of error in heterokaryon scoring is via infection of the sperm nucleus by SV40 virus and subsequent expression of T-antigen, which would diminish the number of identifiable heterokaryons.

Using serological and histological staining techniques, we found a loss of protamin from sperm nuclei, which appeared to correlate with nucleus enlargement. Furthermore we have evidence from histological staining, that protamin is replaced by histone. However, an objective interpretation of the subtle colour differences involved in the brilliant sulphaflavin reaction provided by histone and protamin is difficult and we are currently confirming these results using specific anti-histone sera.

Morphological changes alone are inadequate evidence for nuclear reactivation which also involves gene transcription. Our studies have shown that RNA synthesis, in particular ribosomal RNA synthesis, and DNA synthesis occur in a small proportion of enlarged sperm nuclei, which can be regarded as fitting the criteria for reactivation.

Unfortunately, the low frequency of sperm reactivation implies an impossibility of detecting human enzyme activities from whole cell lysates. The only way to establish transcription of genes other than those for rRNA would be to work on the single cell level, using host cells deficient in a particular enzyme or to make use of antibodies directed against human specific antigens.

The frequency of RNA and DNA synthesizing sperm heterokaryons within a whole fusion population is very low, as compared to frequencies found in fusions between somatic cells, In our laboratory, selection of somatic hybrid cells has an efficiency of 1 × 10− 5 hybrid clones averaged out over many different types of cell selection and fusion systems. Calculating back from observed heterokaryon frequencies for somatic cell and sperm fusions respectively, we estimate a theoretical efficiency for hybrid clone production from sperm fusions of 1 × 10− 10. This low frequency is confirmed by our failure to date to isolate hybrid clones despite extensive efforts. We can also conclude, that our sperm purification procedure has effectively removed contaminating diploid cells which was a possible source of hybrid clone formation in the sperm fusion experiments described by Nyormoi et al. (1973).

These results are disappointing, since we had hoped to use sperm fusions as a method of ultimately visualizing and analysing the chromosome content of individual sperm cells. Premature condensation of sperm chromosomes of the type described by Johnson, Rao & Hughes (1970) for the bull, appears unsuitable for the detailed karyotypic analysis necessary to define meiotic non-disjunction. Similarly the failure to date to produce hybrid clones, also prevents karyotypic analysis at the cell hybrid level.

Moreover, a study of early mitotic divisions in synchronized fusion cultures, revealed no evidence for intact human chromosomes, except for isolated mouse nuclei containing apparent human Y-chromosomes, identified by quinacrine fluorescence. This is not surprising in itself, since in our hands, karyotypic analysis of the much more favourable somatic cell fusions at the first presumptive division following fusion, revealed that the majority of divisions observed were from homokaryons and only a small percentage from heterokaryons. Therefore, the fate of heterokaryons containing reactivated sperm nuclei can only be speculated upon at present. They may disappear from the culture by cell death or, in some instances, disintegrated sperm nuclei may be incorporated into the host cell nucleus by chance (Gledhill et al. 1972; Bendich et al. 1974; Higgins et al. 1975) or they may be extruded from the cytoplasm.

This work was supported by grant no. 13-23-13 of Fungo,’ s-Gravanhage.

Austin
,
C. R.
(
1965
).
Fertilization (Foundations of developmental biology series)
.
Englewood Cliffs, N. J
.:
Prentice Hall
.
Austin
,
C. R.
(
1969
).
Variations and anomalies in fertilization
.
In Fertilization, Comparative Morphology, Biochemistry and Immunology
, vol.
2 (ed
.
C. B.
Metz
&
A.
Monroy
), pp.
437466
.
New York
:
Academic Press
.
Bendich
,
A.
,
Borenfreund
,
E.
&
Sternberg
,
S.
(
1974
).
Penetration of somatic mammalian cells by sperm
.
Science N. Y
.
183
,
857
859
.
Blank
,
M.
,
Soo
,
L.
&
Britten
,
J. S.
(
1976
).
Adsorption of albumin on rabbit sperm membranes
.
J. Membrane Biol
.
29
,
401
409
.
Bloom
,
S. E.
&
Goodpasture
,
C.
(
1976
).
An improved technique for selective silver staining of nucleolar organizer regions in human chromosomes
.
Hum. Genet
.
34
,
199
206
.
Elsevier
,
S. M.
&
Ruddle
,
F. H.
(
1976
).
Haploid genome reactivation and recovery by cell hybridization
.
Chromosoma
56
,
227
241
.
Ericsson
,
R. J.
,
Langevin
,
C. N.
&
Nishino
,
M.
(
1973
).
Isolation of fractions rich in human Y-sperm
.
Nature, Lond
.
246
,
421
424
.
Gledhill
,
B. L.
,
Sawicki
,
W.
,
Croce
,
C. M.
&
Koprowski
,
H.
(
1972
).
DNA synthesis in rabbit spermatozoa after treatment with lysolecithin and fusion with somatic cells
.
Expl Cell Res
.
73
,
33
40
.
Harris
,
H.
(
1965
).
Behaviour of differential nuclei in heterokaryons of animal cells from different species
.
Nature, Lond
.
206
,
583
588
.
Harris
,
H.
&
Cook
,
P. R.
(
1969
).
Synthesis of an enzyme determined by an erythrocyte nucleus in a hybrid cell
.
J. Cell Sci
.
4
,
121
133
.
Higgins
,
P. J.
,
Borenfreund
,
E.
&
Bendich
,
A.
(
1975
).
Appearance of foetal antigens in somatic cells after interaction with heterologous sperm
.
Nature, Lond
.
257
,
488
489
.
Johnson
,
R. T.
,
Rao
,
P. N.
&
Hughes
,
S. D.
(
1970
).
A HeLa cell inducer of premature chromosome condensation active in cells from a variety of animal species
.
J. Cell. Physiol
.
76
,
151
158
.
Kolk
,
A. H. J.
,
Samuel
,
T.
&
Rümke
,
P.
(
1974
).
Auto-antigens of human spermatozoa
.
Clin. exp. Immun
.
16
,
63
76
.
Koprowski
,
H.
&
Croce
,
C. M.
(
1973
).
Fusion of somatic and gametic cells with lysolecithin
.
Meth. Cell Biol
.
7
,
251
260
.
Longo
,
F. J.
(
1973
).
Fertilization: A comparative ultrastructural review
.
Biol. Reprod
.
9
,
149
215
.
Lucy
,
J. A.
(
1975
).
Aspects of the fusion of cells in vitro without viruses
.
J. Reprod. Fert
.
44
,
193
205
.
Luft
,
H. H.
(
1961
).
Improvements in epoxy resin embedding methods
.
J. biophys. biochem. Cytol
.
9
,
409
414
.
Nyormoi
,
O.
,
Coon
,
H. G.
&
Sinclair
,
J. H.
(
1973
).
Proliferating hybrid cells formed between rat spermatids and an established line of mouse fibroblasts
.
J. Cell Sci
.
13
,
863
878
.
Phillips
,
S. G.
,
Phillips
,
D. M.
,
Dev
,
V. G.
,
Miller
,
D. A.
,
Van Diggelen
,
O. P.
&
Miller
,
O. J.
(
1976
).
Spontaneous cell hybridization of somatic cells present in sperm suspensions
.
Expl Cell Res
.
98
,
429
443
.
Poste
,
G.
(
1972
).
Mechanisms of virus induced cell fusion
.
Int. Rev. Cytol
.
33
,
1 57
252
.
Poste
,
G.
&
Allison
,
A. C.
(
1973
).
Membrane fusion
.
Biochim. biophys. Acta
300
,
421
465
.
Ruch
,
F.
(
1970
).
Introduction to Quantitative Chemistry
, vol.
2 (ed
.
L.
Wied
&
G.
Bahr
), p.
446
.
New York and London
:
Academic Press
.
Sawicki
,
W.
&
Koprowski
,
H.
(
1971
).
Fusion of rabbit spermatozoa with somatic cells cultivated in vitro
.
Expl Cell Res
.
66
,
145
151
.