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Biology of Reproduction 61, 1513-1520 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Ultraviolet-Irradiated Spermatozoa Activate Oocytes but Arrest Preimplantation Development After Fertilization and Nuclear Transplantation in Cattle1

Vilceu Bordignona, and Lawrence C. Smith2,a

a Centre de recherche en reproduction animale (CRRA), Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Canada J2S 7C6


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Artificial means of parthenogenetically activating mammalian oocytes are believed to lack an essential sperm epigenetic component required for normal development. The main goal of this study was to examine the potential of ultraviolet (UV)-irradiated sperm as a means of functionally eliminating the chromatin component of spermatozoa without affecting the ability to induce activation and support parthenogenetic development in cattle. Spermatozoa were stained with a DNA dye, exposed to various UV irradiation doses, and used to fertilize secondary oocytes. Although the percentage of pronuclei at 18 h postinsemination was similar using treated and control sperm, most oocytes fertilized by UV-irradiated sperm failed to develop beyond the 2-cell stage, suggesting that UV irradiation can functionally destroy the genomic component of spermatozoa with limited effects on the ability to induce oocyte activation. However, when oocytes activated with UV-irradiated sperm were used as hosts for nuclear transfer, developmental rates to cleavage and to blastocyst improved only marginally and remained lower than in the controls, indicating that UV-treated spermatozoa blocked development even in the presence of a diploid donor nucleus. Although DNA replication was not inhibited by UV irradiation treatment, abnormal chromatin morphology after cleavage suggests improper segregation of chromatin to daughter blastomeres during the first mitotic division. Together, these results indicate that although sperm exposed to UV can activate oocytes, a developmental block occurs at or soon after the first mitosis in parthenotes and oocytes reconstructed by nuclear transfer.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A common approach to improving the developmental potential of embryos reconstructed by nuclear transfer is to preactivate host oocytes in order to reduce the levels of the maturation-promoting factor (MPF), the kinase responsible for nuclear envelope breakdown and chromatin condensation in both meiotic and mitotic cells [1]. Host cytoplasts with low MPF prevent the premature condensation and pulverization of blastomere donor chromatin that are normally at the S-phase at the time of transfer [2]. Artificial means of activating oocytes are more effective with use of oocytes after prolonged metaphase II arrest; i.e., aged oocytes are more easily activated than recently matured oocytes [3, 4]. However, development potential after in vitro fertilization (IVF) [5] and nuclear transfer [6] is compromised by oocyte aging.

Artificial agents employed to activate oocytes are mostly unable to mimic the pattern of calcium oscillation induced by the sperm at fertilization [7–9]. It is well established that activation induced by sperm penetration involves a series of oscillations in the intracellular free calcium that persist for several minutes or even hours after fertilization, leading to MPF degradation, meiotic resumption, and passage to interphase [7–10]. In contrast to a single stimulus, repetitive calcium spikes induced by artificial means lead to a prompt and stable degradation of MPF in various species [11–14], indicating that calcium oscillations are required for an effective activation. Furthermore, the calcium-releasing agent introduced by the fertilizing sperm becomes associated with the nucleus up to the 2-cell stage and remains able to induce calcium oscillations, meiotic completion, and pronuclear formation upon transfer back to nonfertilized oocytes [15]. These results suggest that a non-genetic component of sperm plays an important role during the beginning of embryonic development. Moreover, studies indicate that a protein localized at the equatorial region of the spermatozoa is introduced into the oocyte at fertilization and may be the agent responsible for inducing intracellular calcium oscillations [16].

Ablation of spermatozoa DNA using ultraviolet (UV) irradiation has been successfully used for gynogenesis and ploidy manipulation in amphibian [17] and aquatic species [18–21]. In these species, UV irradiation causes the complete inactivation of sperm chromatin without affecting the ability to penetrate or activate the oocyte. UV radiation causes limited DNA fragmentation and is, therefore, preferentially used among other methods to induce sperm chromatin ablation [22, 23]. In fish, UV irradiation has also been used to functionally enucleate eggs in androgenetic studies [24, 25]. In mammals, the effect of UV irradiation has been evaluated in oocytes and eggs [26–28] and has been proposed as a potential mechanism for functional enucleation of recipient oocytes for nuclear transfer [26, 28, 29]. However, use of UV irradiation to destroy DNA as means to functionally remove the genetic contribution of sperm has not been tested to date. If applicable, this procedure could be used not only for activating non-aged oocytes in nuclear transfer but also to assess the genetic and non-genetic contribution of sperm to embryonic development. Therefore, the main goals of this study were, first, to determine whether UV irradiation of sperm leads to chromatin ablation without affecting the ability to activate oocytes at penetration and, second, to verify whether oocytes activated with UV-irradiated sperm can be used as recipient cytoplasts for nuclear transfer. We show that UV-exposed spermatozoa remain capable of penetrating and activating non-aged bovine oocytes without supporting development beyond the 2-cell stage. However, UV-irradiated sperm chromatin appears to participate in the formation of the first mitotic spindle and thereby interferes with the balanced segregation of chromatin to daughter blastomeres.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Oocyte and Embryo Source

Cow ovaries were obtained from a local abattoir, placed in saline at 35°C, and transported to the laboratory within 2 h of slaughter. Cumulus-oocyte complexes (COCs) were aspirated from 2- to 8-mm antral follicles using a 19-gauge needle and selected for the presence of several layers of cumulus cells and oocytes with homogeneous cytoplasm. Selected COCs were washed in Hepes-buffered tissue culture medium (TCM-199; Gibco BRL, Burlington, ON Canada) supplemented with 10% (v:v) fetal calf serum (FCS; Gibco). Groups of 20 COCs were matured for 24 h in 100-µl drops of TCM-199 supplemented with 10% of FCS (Gibco), 1 µg/ml estradiol-17ß (Sigma Chemical Co., St. Louis, MO), 50 µg/ml LH (Ayerst, London, ON, Canada), 0.5 µg/ml FSH (Folltropin-V; Vetrepharm, St. Laurent, PQ, Canada), 22 µg/ml pyruvate (Sigma), and 50 µg/ml gentamicin (Sigma) at 39°C in 5% CO2. IVF was performed using the procedures described previously [30]. Briefly, expanded COCs were placed in 50-µl drops of Tyrode's medium supplemented with 0.6% BSA (fraction V; Sigma), lactate, pyruvate, gentamicin, and 10 µg/ml heparin. Frozen-thawed spermatozoa were washed and centrifuged through a gradient of Percoll (Pharmacia, Bromma, Sweden) and diluted at 106 live spermatozoa/ml. After 4 or 18 h from insemination, oocytes were denuded of cumulus cells by brief shaking and transferred to 50-µl drops of Menezo's B2 medium (MB2; Pharmascience, Paris, France) supplemented with 10% FCS in the presence of bovine oviductal epithelial cells. All the cultures were performed in drops under equilibrated mineral oil at 39°C in a humidified atmosphere of 5% CO2 in air.

UV Sperm Treatment

To amplify the effects of the UV treatment on chromatin, frozen-thawed spermatozoa were incubated for 15 min with 10 µg/ml of Hoechst 33342 (Sigma). After staining, sperm were washed through Percoll, adjusted to a final concentration of 106 live spermatozoa/ml, and placed in 50-µl drops covered with a thin layer (4 ml in a 35-mm-diameter Petri dish) of mineral oil (Sigma). Sperm were exposed to different doses of UV radiation (10, 30, or 50 mJ/cm2) using a UV chamber (GS Gene Linker Ultraviolet Chamber; Bio-Rad, Richmond, CA). An average of 15 in vitro-matured COCs were placed in sperm drops 1 h after UV irradiation and cultured for either 4 or 18 h. Oocytes incubated with sperm during 4 h were denuded of cumulus cells and cultured for a further 12 h in fertilization medium in the absence of sperm, whereas those incubated with sperm for 18 h were denuded of cumulus cells at the end of the insemination period. Both groups were either fixed immediately after removal from IVF drops or cultured for a further 7 days to evaluate their ability to cleave (44 h) and develop to the blastocyst stage (Day 7) in vitro.

DNA Synthesis Assay

Inseminated oocytes were incubated in the presence of 100 µM bromodeoxyuridine (BrdU; 5-bromo-2'-deoxyuridine 5'-triphosphate; Sigma) to determine whether the UV-irradiated sperm affected the pattern of DNA synthesis during pronuclear formation. After IVF using control (nontreated) or UV-irradiated (10 or 30 mJ/cm2) spermatozoa, oocytes were denuded of cumulus cells and incubated in BrdU for various periods (4–18 h, 4–8 h, 8–16 h, or 18–24 h). At the end of each incubation period, oocytes were fixed in 10% formalin for 10 min, permeabilized using 0.5% Triton X-100 (Biopharm, Laval, PQ, Canada) in PBS for 2 h, and washed twice in block solution (PBS, 3% BSA, 0.1% Tween 20) for 20 min at room temperature. Oocytes were incubated for 1 h in 10 µl of anti-BrdU monoclonal antibody (Amersham, Oakville, ON, Canada) containing 1 µg/ml DNase, washed in block solution, and incubated with the secondary antibody (fluorescein-conjugated goat anti-mouse IgG; Sigma) at 1:100 dilution. Finally, oocytes were washed in block solution, mounted onto slides in Mowiol (Aldrich, Milwaukee, WI) containing 5 µg/ml Hoechst 33342, and examined by epifluorescence using a filter block at 380-nm excitation and 420-nm emission (UV-2A; Nikon, Tokyo, Japan). Pronuclei of control and UV-irradiated groups were ranked according to the intensity of the emitted fluorescence.

Chromatin and Spindle Morphology Assay

At 24 h after IVF, inseminated oocytes were fixed in formalin, permeabilized as described above, and exposed to a primary antibody raised against mouse {alpha}-tubulin (Sigma) diluted 1:1000. The secondary antibody was similar to that used for the BrdU assay. Chromatin morphology was evaluated at the pronuclear (18 h) and 2-cell stage (30 h) in both IVF and reconstructed embryos. To determine chromatin morphology and the number of nuclei in blastocysts, embryos were mounted onto a glass slide using Mowiol containing 5 µg/ml of Hoechst 33342 and examined by epifluorescence microscopy.

Oocyte Activation and Reconstruction

Sperm-mediated oocyte activation was assessed at 4 h after insemination with either control or UV-irradiated (10 mJ/cm2) sperm; oocytes presenting a second polar body were selected for micromanipulation. Parthenogenetic activation was obtained by exposure of in vitro-matured oocytes to 5 µM ionomycin (Sigma) for 4 min in TCM-199 Hepes-buffered medium supplemented with 2 mg/ml BSA, washed, and cultured for an additional 2 h for second polar body extrusion. Activated oocytes were enucleated at the telophase II stage by removing a small portion of cytoplasm surrounding the position of the second polar body as described previously [6]. A single blastomere derived from an in vitro-produced morula at Day 5 after IVF was injected into the perivitelline space of the enucleated oocyte. The resulting couplet was placed in a 0.3 M mannitol (Sigma) solution containing 0.1 mM MgSO4 and 0.05 mM CaCl2 and exposed to a 1.5-Kv electrical pulse lasting 70 µsec. After electrical stimulation, oocytes were washed in PBS and cultured in MB2 in the presence of bovine oviductal epithelial cells. Fusion between nuclear donor blastomeres and enucleated oocytes was verified 1 h later, and development to cleavage and blastocyst was observed at 30 h and Day 7, respectively. Androgenetic embryos were produced by fertilizing metaphase-stage enucleated oocytes. After 24 h of in vitro maturation, oocytes were denuded of cumulus cells and enucleated by removing approximately 30% of the cytoplasm surrounding the first polar body. After microsurgery, oocytes were placed in medium containing 5 µg/ml of Hoechst 33342 for 15 min and exposed briefly to UV irradiation to verify the absence of chromatin. Enucleated oocytes were placed in the presence of UV-irradiated (10 mJ/cm2) or control sperm for 4 h, fixed after 18 h (before cleavage) or 30 h (after cleavage), stained, and examined by epifluorescence to assess chromatin morphology.

Statistical Analysis

Percentage development to pronuclear, cleavage, and blastocyst stages were arcsine and square-root transformed to normalize the data. All data were examined by ANOVA, and means were compared using Tukey-Kramer test at 5% level of significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1: Effect of Sperm UV Irradiation on Oocyte Activation and Pronuclear Formation

The first objective of this study was to determine whether UV-irradiated sperm have the ability to activate oocytes and, if so, to determine whether the irradiation of paternal chromatin influences the developmental outcome after fertilization. Pronuclear formation, cleavage, and blastocyst development percentages were assessed in oocytes cultured for 18 h in the presence of control and UV-treated sperm exposed to three different doses of irradiation (Table 1). Sperm exposed to 10 mJ/cm2 of UV irradiation enabled pronuclear formation as often as non-irradiated controls (P > 0.05). However, sperm exposed to higher doses (30 and 50 mJ/cm2) showed a reduced fertilization potential (P < 0.05) as evidenced by a low percentage of pronuclear-stage zygotes at the end of 18-h culture. The percentage of oocytes that cleaved to the 2-cell stage at 48 h postinsemination was significantly reduced (P < 0.05) at all UV doses examined, indicating partial blockage of the first cleavage division regardless of apparently normal activation and pronuclear formation. Development to the blastocyst stage was completely inhibited with sperm exposed to 30 and 50 mJ/cm2 of UV, and only a small percentage of oocytes reached the blastocyst stage when fertilized with sperm exposed to 10 mJ/cm2.


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  		TABLE 1. Percentage pronuclear formation, cleavage, and blastocyst development of oocytes fertilized with UV-irradiated spermatozoa (IVF for 18 h)

To determine with more precision the time of fertilization, a second group of oocytes was placed for a shorter period (4 h) in insemination drops containing either control or irradiated sperm (Table 2). At this shorter insemination period, non-irradiated sperm led to significantly higher pronuclear formation than the irradiated groups (P < 0.05). Nonetheless, although cleavage to the 2-cell stage was severely compromised and the development to blastocyst stage almost completely inhibited, pronuclear formation after 10 mJ/cm2 irradiation was observed in over half of the inseminated oocytes. These results indicate that one can achieve a reasonable percentage of activation in more precisely timed UV-exposed sperm-activated oocytes using low levels of UV irradiation. However, levels of pronuclear formation after fertilization with sperm exposed to 30 mJ/cm2 of UV was remarkably low, precluding possible use for the reconstruction of oocytes activated with UV-treated sperm.


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  		TABLE 2. Percentage pronuclear formation, cleavage, and blastocyst development of oocytes fertilized with UV-irradiated spermatozoa (IVF for 4 h)

Experiment 2: Embryo Reconstructions Using Oocytes Activated with UV-Irradiated Sperm

Our objective in this study was to verify whether oocytes activated with UV-irradiated spermatozoa can be used as host cytoplasts in the reconstruction of oocytes by nuclear transplantation. Host oocytes in control groups were activated either artificially (ionomycin) or through use of non-irradiated sperm (control sperm) and then enucleated at telophase (Table 3). The developmental rate both to cleavage and blastocyst stage was significantly (P < 0.05) reduced with use of recipient oocytes activated with UV-irradiated sperm, indicating that UV-irradiated sperm chromatin was detrimental to development regardless of the presence of a normal diploid donor nucleus. High yields of embryos were obtained with reconstructions using host oocytes activated with non-irradiated sperm, indicating that triploidy did not affect their developmental outcome. The number of nuclei per blastocyst at Day 7 of culture was very similar among treated and control groups.


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  		TABLE 3. Effect of activation of host cytoplasts with UV irradiated spermatozoa (10 mJ/cm2) and the development of nuclear transfer embryos

Experiment 3: DNA Synthesis and Chromatin Morphology Assay

Our final goal was to identify the possible causes for the cleavage and developmental blockage observed in oocytes after activation with UV-irradiated spermatozoa. Initially, DNA synthesis in the resulting male pronucleus was measured using a BrdU uptake assay. Assays were performed at different periods throughout the first cell cycle, i.e., from 4 to 8, 8 to 16, 18 to 24, and 4 to 18 h after insemination with control and 10 or 30 mJ UV-irradiated sperm (Fig. 1). Although a weaker BrdU staining was observed during the period from 4 to 18 h postinsemination in UV-irradiated groups (P < 0.05), all male pronuclei stained positively for BrdU, indicating that DNA replication occurred regardless of the irradiation of spermatozoa. Compared to that in UV-irradiated groups, stronger BrdU staining was seen in the control group during the first few hours after insemination (from 4 to 8 h), suggesting that fertilization occurred later in the UV-irradiated sperm. However, no significant difference in BrdU incorporation was seen during the period of major DNA replication (8–16 h from IVF) or at the end of the first cell cycle from 18 to 24 h postinsemination (P > 0.05).



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  		FIG. 1. Patterns of BrdU incorporation in pronuclei of embryos fertilized with UV-irradiated sperm. Oocytes were placed into fertilization drops in the presence of control (lighter gray bar) and 10 mJ/cm2 (darker gray bar) and 30 mJ/cm2 (black bar) UV-irradiated sperm for 4 h and incubated in the presence of BrdU for various periods after insemination (0 h). The intensity of BrdU staining was assessed subjectively in a total of 227 pronuclear-stage zygotes (average 19 per group) and ranked into four categories: none (0), weak (1), medium (2), and strong (3). Vertical bars represent SE of the mean rankings for each group, and different letters represent differences within periods (P <= 0.05)

To evaluate the morphology of pronuclei at the end of the first cell cycle, chromatin was observed by epifluorescence at 18 h postinsemination. Most androgenetic embryos produced by fertilization of enucleated oocytes using UV-irradiated spermatozoa showed smaller pronuclei with condensed and pulverized chromatin (Fig. 2). None of these abnormalities were observed in androgenetic embryos produced with non-irradiated sperm. However, pronuclear morphology of both inseminated and reconstructed embryos produced with UV-irradiated sperm was apparently not affected (Fig. 3). Most embryos in the latter groups possessed two well-developed pronuclei with decondensed chromatin. Conversely, in relation to fertilized and nuclear transfer embryos, androgenetic groups derived from UV-irradiated spermatozoa showed abnormal chromatin configuration when analyzed as 2-cells at 30 h after insemination. The abnormalities seen included chromatin fragmentation, consisting of threads of chromatin bridging sister blastomeres (Fig. 3, e and f) and pulverized and pyknotic condensed chromatin in nuclear transfer and IVF groups, and total chromatin degradation in the androgenetic group (Fig. 2, c and d). Whereas all 2-cell-stage androgenetic embryos contained abnormal chromatin, nonenucleated oocytes fertilized with 10 and 30 mJ/cm2 UV-irradiated sperm showed 67% and 76% abnormal chromatin, respectively. Most oocytes fertilized with UV-treated sperm stopped development at the 2-cell stage, since 57.1% (n = 28) of oocytes fertilized with UV-irradiated sperm remained as 2-cells at 50 h post-IVF compared to only 7.7% (n = 52) in the control group. Moreover, only 10.7% developed to the 8-cell stage compared with 71.1% in the control group. Together, these results indicate that the chromatin of UV-irradiated sperm is decondensed after penetrating the oocyte and develops a DNA-synthesizing pronucleus that participates in syngamy at the end of the first cell cycle. Nonetheless, most embryos arrested development either before or soon after first cleavage. Although only a small number of oocytes were fixed at mitosis, no apparent abnormality was seen in the alignment of chromatin onto the metaphase plate, suggesting that spindle formation is not directly involved in the abnormalities observed at the 2-cell stage.



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  		FIG. 2. Chromatin morphology of androgenetic embryos inseminated with control and UV-irradiated sperm. Oocytes were enucleated at metaphase II; they were inseminated with either non-irradiated sperm and fixed at the pronuclear (a) and 2-cell (b) stages, or with UV-irradiated sperm (10 mJ/cm2) and fixed at the pronuclear (c) and 2-cell (d) stages. x500 (published at 86%)



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  		FIG. 3. Chromatin morphology of embryos inseminated with control and UV-irradiated sperm. Oocytes inseminated with non-irradiated sperm at the pronuclear (a), 2-cell (b), and 8-cell (c) stages. Oocytes inseminated with UV-irradiated sperm (10 mJ/cm2) at the pronuclear (d), 2-cell (e, f), and 4- to 8-cell (g, h) stages. x500 (published at 86%)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The main goal of the present study was to establish a procedure to induce the functional ablation of sperm chromatin without inhibiting the capacity to penetrate and activate metaphase-arrested oocytes. We show that the capacity of UV-irradiated spermatozoa to penetrate and activate oocytes is dependent on the UV dose applied. Whereas lower doses do not reduce the fertilizing ability of sperm, development to the blastocyst stage was almost completely blocked by UV irradiation, suggesting functional destruction of the chromatin. Transplantation of morula-derived blastomere nuclei into oocytes fertilized with UV-irradiated sperm led to only a partial recovery of potential to develop to the blastocyst stage. Morphological evaluations by DNA staining suggest that the inhibition of development may be caused by the detrimental participation of UV-irradiated sperm chromatin in the first mitotic spindle.

Effect of Sperm UV Irradiation on Oocyte Activation

Results from experiment 1 show that the fertilizing capability of bovine spermatozoa exposed to UV irradiation is dose dependent. Although UV irradiation of sperm had no apparent effect on motility immediately after exposure, only sperm exposed to lower doses of irradiation were able to penetrate and activate in vitro-matured oocytes at a rate similar to that for non-irradiated sperm. Moreover, high doses of radiation (30 and 50 mJ/cm2) led to significantly lower fertilization rates. The chromatic component of sperm is not considered to be involved in oocyte activation [16, 31], suggesting that UV effects on chromatin are unlikely to affect fertilization rates. However, apart from effects on chromatin, exposure to UV may affect other sperm components, such as mitochondria or membrane integrity as evidenced after the irradiation of bovine oocytes [27] and fish spermatozoa [21].

Although lower UV doses did not affect the activation and penetration rates of spermatozoa, development to the 2-cell (cleavage) and blastocyst stages was seriously compromised at low and high levels of UV irradiation. These results contrast with those described in fish and amphibians showing that intermediate UV doses led to low embryonic survival whereas a recovery of development potential was observed with the use of higher doses. It was concluded that intermediate doses of irradiation cause an incomplete destruction of the chromatin, allowing fragments of sperm chromatin to be incorporated into the maternal chromatin, leading to improper expression of the paternal genes, and interfering with the embryonic development [19, 21, 23, 32]. Conversely, while low doses do not cause significant damage to chromatin, high doses render sperm completely nonfunctional and thereby allow for the development of gynogenetic embryos. Surprisingly, complete nonfunctionality was not observed in the present study since higher UV doses drastically reduced the fertilization rate, and no improvement in embryonic development was observed. In contrast to the rapid development of fish and amphibian early embryos, the first cell cycle is much longer in bovine embryos, suggesting that the damage caused to chromatin may be partially repaired by nucleotide excision repair mechanism in oocytes [33–35]. Therefore, partially repaired chromatin would reverse the effect of UV irradiation and thereby have a negative influence on subsequent development. When the time of exposure of irradiated sperm was reduced to 4 h, the overall effect of irradiation was similar to that for the 18-h period. However, a significant reduction in fertilization rate was observed when a lower dose of radiation was applied, indicating that UV-irradiated sperm require a longer period to penetrate and activate oocytes than non-irradiated sperm.

Nuclear Transfer

Our objective in this study was to determine whether oocytes activated with UV-irradiated sperm would develop in vitro after fusion to a diploid nucleus. Results indicate that the development of reconstructed embryos derived from host cytoplast activated with UV-irradiated (10 mJ/cm2) spermatozoa was significantly lower than that of reconstructed oocytes activated with non-irradiated sperm or by ionomycin (Table 3). This suggests that the detrimental effect of UV-irradiated sperm on the development of activated oocytes beyond the 2-cell stage persisted regardless of the presence of a diploid nucleus. However, compared with results obtained after fertilization using sperm exposed to the same UV dose (Tables 1 and 2), more embryos cleaved and developed to the blastocyst stage after nuclear transplantation, suggesting that the transfer of a diploid nucleus at least partially removed the inhibitory effect of UV-irradiated sperm. The transfer of a viable centrosomal pair with the diploid nucleus may have enabled a better developmental outcome. In cattle, establishment of the centrosomal component required for normal spindle assembly seems to have paternal and maternal origins, and either one or two centrosomes are carried into the host oocyte after the transfer of a morula-derived blastomere [36]. Therefore, since UV irradiation may affect the spermatic centrosome and lead to low cleavage, the introduction of normal centrosomes with the transferred blastomere would improve cleavage rates in reconstructed embryos. On the other hand, high rates of cleavage and blastocyst development have been reported in parthenogenetic embryos in cattle [37, 38], suggesting that the paternal centrosome may not be absolutely required for cleavage.

Effect of UV Irradiation on Chromatin

The objective here was to determine the possible causes for the development blockage that occurred after fertilization with UV-irradiated spermatozoa. Patterns of DNA synthesis during the first cell cycle were assessed by BrdU incorporation to determine whether DNA replication was affected by UV irradiation. Our results showed that, independently of UV irradiation, actively replicating male and female pronuclei were observed in all zygotes analyzed, indicating that sperm chromatin had not been completely destroyed by UV irradiation. However, the intensity of BrdU immunostaining was significantly reduced in pronuclei from treated groups, suggesting a lower amount of DNA synthesis after UV irradiation. Reduction of BrdU uptake has been previously reported using cultured human fibroblasts [39]—a UV-induced effect that may be due to a physical blockage of the replication fork movement by lesions on the chromatin [40]. In addition to the general reduction in the BrdU uptake seen during the period of 4–18 h postfertilization, we have shown that oocytes fertilized with irradiated sperm have a reduced synthesis of DNA during the first 8 h postfertilization. A delay in the fertilization time or a slower remodeling of the sperm chromatin may have accounted for this effect. Indeed, as observed in hamster ovary cells synchronized at the G1-phase, UV irradiation causes a dose-dependent delay in entry into S-phase [41].

Concerning the effect of UV irradiation on chromatin morphology, the most remarkable anomaly observed was the different patterns of chromatin fragmentation seen after the first cell division. A high proportion of the embryos showed dispersed fragments of chromatin forming small nuclei or threads of chromatin linking the nuclei of sister blastomeres, indicating that, despite damage caused by UV irradiation, sperm chromatin participated in the formation of the mitotic spindle. Surprisingly, no apparent morphological effect of UV irradiation was observed in the pronuclei during the first cell cycle after fertilization. However, androgenetic embryos derived from enucleated oocytes fertilized with UV-irradiated sperm showed abnormal pronuclear morphology, suggesting that the presence and formation of a female pronucleus in controls had a beneficial effect on the remodeling of irradiated sperm chromatin.

It is known that the main injury induced by UV irradiation on the chromatin is the formation of cyclobutane dimers, a covalent link between adjacent pyrimidine bases, which affect the chromatin activity, including DNA replication and transcription [10, 42–44]. Moreover, in response to the chromatic damage induced by radiation, eukaryotic cells can activate regulatory pathways known as cell cycle checkpoints that control the order and timing of cell cycle transitions to ensure that critical events such as DNA replication and chromosome segregation are completed with high fidelity [45]. Although all cells have a nucleotide excision repair mechanism that is used to remove and replace affected bases and correct the effect of radiation [43], spermatozoa are not provided with this mechanism and are unable to repair damage induced by radiation or chemicals [46, 47]. On the other hand, eggs have an excision-repair capacity [33] that can also act on sperm DNA that has been damaged before fertilization [34, 35]. This mechanism, combined with the long period required to complete the first cell cycle, may have enabled the recovery of UV-induced damaged chromatin, which could account for the inability to induce a complete DNA ablation after UV irradiation. Moreover, our results suggest that the inability of many embryos to cleave when fertilized with UV-irradiated sperm may have been caused by blockage at the G2-phase, since both pronuclei were shown to incorporate BrdU throughout the first cell cycle. Indeed, although DNA-damaged checkpoints can block cells at different phases of cell cycle, including G1/S, S, and G2/M [48], UV-irradiated cells seem to block more frequently at G2 [41, 49]. The G2/M arrest checkpoint is probably caused by breaks in the chromatin, and consists of a period required for repairing DNA damage [48]. Nevertheless, most cleaved embryos showed abnormal chromatin morphology, indicating that they were able to cleave regardless of damaged chromatin and suggesting that G2 cell cycle checkpoints may not be fully operational during cleavage in cattle. Whenever the G2 arrest checkpoint fails, a broken chromosome can be partitioned into separated nuclei, precluding the possibility of their undergoing end-to-end fusion and leading to a variety of outcomes, including degradation or formation of truncated chromosomes [48].

Although the exact molecular mechanism implicated in the arrest of the cell cycle in response to DNA damage remains unclear, the signal seems to depend on the p53 tumor repressor protein [50, 51] that can induce the transcription of several genes, some of which can arrest the cell cycle or even induce cell apoptosis [45]. However, the arrest of cells at G2 stage seems not to be dependent on p53 [52] and possibly involves a block in the cdc25-dependent activation of cyclin/cdc2 kinase [49, 53]. Taking into account that some embryos were arrested before the first cleavage and others after, we may infer that different pathways are involved in the blockage of embryonic development after fertilization with UV-irradiated sperm.

Finally, we demonstrate that bovine oocytes activated with UV-irradiated sperm can be used as hosts in embryo reconstruction by nuclear transfer. Although the development rate to blastocyst was inferior to that obtained with artificially activated oocytes, this procedure could be of particular interest for species in which artificial activation protocols are not effective. Nonetheless, further experiments will be required to determine the ability of these embryos to support normal development to term.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge the technical support of Carmen Léveillée, CIAQ, for providing frozen semen for the IVF protocol and the help of Alan Goff for critical reading of the manuscript and helpful comments.


   FOOTNOTES
 
1 This work was supported by the National Science and Engineering Research Council (NSERC) of Canada. V.B. was supported by a scholarship from CNPq, Brazil. Back

2 Correspondence: Lawrence C. Smith, Centre de recherche en reproduction animale (CRRA), Faculté de médecine vétérinaire, Université de Montréal, C.P. 5000, Saint-Hyacinthe, Canada J2S 7C6. FAX: 450 778 8103; smithl{at}medvet.umontreal.ca Back

Accepted: August 2, 1999.

Received: June 7, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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