Reproduction in mice: Spermatozoa as factors in the development and implantation of embryos

The effects on mouse embryo development in vivo of varying the numbers of spermatozoa used in artificial inseminations was studied. The two criteria used in the evaluation of the progress of embryo development were 1) ability to reach the two-cell stage and 2) success of development from the two-cell stage through implantation. A 44% reduction in the yield of two-cell embryos and a 67% reduction in the number of implants was observed when C3HeB/FeJ females were inseminated with one-twentieth the number of spermatozoa estimated to be present in a typical ejaculate. The reduction in the yield of two-cell embryos was substantially reversed by a second insemination of a large number of heat-inactivated spermatozoa 12 hr after the first insemination. The sperm-dependent reduction in development from the two-cell stage through implantation was prevented only by normal viable (unheated) spermatozoa. These results were rationalized by the hypothesis that in female C3HeB/FeJ mice spermatozoa serve physiological functions beyond the fertilization of ova and that spermatozoa may act to foster early embryo development through modulation of the environments embryos experience as they move through the reproductive tract.     

Development of mouse embryos in vitro is affected by strain and culture medium

One-cell and two-cell embryos from three random-bred strains of mice–CF1, Dub:(ICR), and CFW (Swiss-Webster)–were cultured to the blastocyst stage in Spindle's, Earle's, Ham's F10, Whittingham's T6, or Hoppe and Pitts' medium. CFW embryos were more successful than CF1 and Dub:(ICR) embryos in developing to the blastocyst stage in all five media. Dub:(ICR) and CFW two-cell embryos showed the best development in Spindle's, Whittingham's T6, and Hoppe and Pitts', whereas CF1 two-cell embryos were most successful in developing in Hoppe and Pitts' medium. Similar results were obtained with one-cell embryos, although fewer developed to the blastocyst stage, and T6 rather than Hoppe and Pitts' medium sustained the best development of CF1 one-cell embryos. For all strains, the least successful development was in Ham's F10, but CFW embryos did show good development in this medium. In addition to the effects of various media on mouse embryo development, our results indicate that the strain of mouse used for the bioassay of media is of critical importance. Random-bred CFW (Swiss-Webster) mice are as suitable as a hybrid strain for this purpose.     

A mouse embryo culture system for quality control testing of human in vitro fertilization and embryo transfer media and fetal cord sera

Swiss white mice were superovulated, mated, and sacrificed to recover two-cell embryos that were cultured in Ham's F-10 supplemented with 15% fetal serum. In 16 experiments, media enriched with fetal bovine serum (FBS) supported blastocyst development from 80% ± 19% (mean ± S.D.) of two-cell embryos. Culture media + FBS was the positive control when 74 batches of heat-inactivated human fetal cord serum (hFCS) were tested. Statistical analyses indicated two distinct populations: 49 hFCS promoted blastocyst formation and 25 hFCS grew fewer blastocysts. In five studies, 35/47 two-cell embryos recovered from mice oviducts in media + FBS and immediately incubated formed blastocysts (75% ± 10%). In six comparison studies where the recovered embryos stood at room temperature for 30 minutes before incubation, only 18/57 (29% ± 21%) became blastocysts. When the colony was housed for 1 week in rooms with Shell No Pest Strips as treatment for mites, only 11/125 two-cell embryos became blastocysts (9%). In contrast, animals housed in quarters decontaminated with chlorine bleach had reduced breeding efficiency and produced fewer two-cell embryos. We conclude that (1) Ham's F-10 + FBS is an excellent positive control to test new batches of hFCS; (2) hFCS that supports blastocyst formation from ≥75% of two-cell embryos is adequate for human use; (3) pesticide treatment of breeding colonies and cooling of murine embryos during harvest both impaired in vitro blastocyst development; and (4) chlorine bleach cleansing of animal quarters reduced the number of successful matings.     

Collection of two-cell pig embryos and their culture in media containing different protein components

Eggs were flushed from the oviducts of slaughtered gilts that had been inseminated after synchronization of estrus and ovulation with pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG). Of 347 eggs collected at a recovery rate of 73.8%, 41.8% had not cleaved by 32 h after expected ovulation time. Of those cleaved, 86.9% were at the two-cell stage.Two-cell embryos were cultured in Dulbecco's medium containing either no protein or 20% of one of the following: lyophilized bovine serum albumin, lyophilized fetal calf serum or heat-inactivated serum from slaughter heifers, rabbits or barrows. Dulbecco's medium without protein did not support further development of embryos. Addition of heat-inactivated blood serum from slaughter heifers, rabbits or barrows resulted in development rates similar to those obtained by using commercially available products. Optimal embryonic development rates of 54.5% were obtained with addition of heat-inactivated bovine serum. Of the two-cell embryos only three (2.1%) developed past the four-cell stage in these culture media.     

The effects of U.V.-light, ionizing radiation and the carcinogen N-acetoxy-2-fluorenylacetamide on the development in vitro of one- and two-cell mouse embryos.

The development of one- and two-cell mouse embryos to morula-blastula stages was followed in vitro after treatment with low doses of U.V.-light, ionizing radiation or N-acetoxy-2-fluorenylacetamide. Exposure of one-cell embryos to either radiation source 18 and 24 hours after human chorionic gonadotropin injections prevented maturation, most embryos being arrested at the one-cell stage and a few at the two-cell stage. Two-cell embryos, however, were not sensitive to low doses of either U.V. or X-irradiation and developed normally. Treatment of early one-cell embryos with the carcinogen, N-acetoxy-2-fluorenyl-acetamide (0-7 muM), also arrested development, whereas exposure of late one-cell embryos did not completely prevent maturation to morula-blastula stages. Exposure of two-cell embryos to the same concentration of carcinogen had no effect on their development to blastulas. Results with all three agents showed that mouse embryos at the one-cell stage are more sensitive than those at the two-cell stage, as judged by their ability to develop in vitro.     

Development of in vivo derived diploid and tetraploid pig embryos in a modified medium NCSU 37

The aim of this study was to assess development of diploid and tetraploid in vivo derived pig embryos cultured in a modified medium NCSU 37 in an atmosphere with reduced concentration of oxygen. The tetraploid embryos were produced by electrofusion of two-cell embryos that had been cultured in vitro from the one-cell stage before fusion (cultured two-cell embryos) or by fusion of freshly recovered two-cell embryos. Development to blastocyst stage of tetraploid embryos, generated from the cultured two-cell embryos was significantly inferior to the development of control one-cell embryos (29.1 +/- 9.7% versus 66.8 +/- 9.7%; P < 0.05). However, development of tetraploid embryos produced from the freshly recovered two-cell embryos and control two-cell embryos was very similar (89.9 +/- 6.1% versus 81.3 +/- 3.4%). Detection of chromosomes 1 and 10 by in situ hybridization showed that more than 85% of the cultured control embryos were diploid while 15% of the embryos were mosaic. Among the fused embryos 50% were tetraploid, 29% mosaic and 21% diploid. These data indicate that the modified medium NCSU 37 provides optimum environment for pre-implantation development of pig diploid and tetraploid embryos.     

Treatment of sperm with extracellular adenosine 5'-triphosphate improves the in vitro fertility rate of inbred and genetically modified mice with low fertility

In vitro fertilization (IVF) is one of the most important techniques used for assisted reproduction in mouse colony management. As with natural mating, where mice have varying fertility indices, fertility rates of genetically modified (GM) [transgenic (Tg), knock out (KO) and congenic (Cg)] mice are influenced by their genetic background. Lines of GM mice that have poor fertility have a concomitant poor IVF outcome. Treatment of mouse sperm with extracellular adenosine 5'-triphosphate (ATPe) enhanced in vitro fertilization rates in outbred and hybrid mice. The objective of this study was to analyze the effects of using extracellular adenosine 5'-triphosphate-treated sperm for IVF of inbred wild type, and genetically modified mouse lines, for which standard IVF did not work well. The IVF was performed using the GM mice on C57BL/10SnJ, C57BL/6J, BALB/cJ and NFS/N background strains and wild type (WT) mice such as C57BL/6N, BALB/cAnN, and B6129SF1 strains. Oocytes from superovulated females were fertilized in vitro with sperm from the same background strain, and either treated or not treated with ATPe. The ATPe treatment enhanced IVF outcome in most of the GM and some WT strains, as indicated by the percentage of embryos that progressed to the two-cell stage. There was no marked difference between ATPe treated and control groups for the development rate of two-cell embryos to blastocysts in culture, or in the number of pups born after transfer of two-cell embryos into recipient females. The observed improvement of the IVF results following ATPe treatment of transgenic and KO mouse sperm were a potential solution for improving the outcome of assisted reproduction techniques used for rederivation or for gamete banking.     

In vitro formation of tetraploid rat blastocysts after fusion of two-cell embryos

Gene targeting technology is not available in the rat which is an animal model of major importance, e.g., in cardiovascular research. This is due to the fact that the rat embryonic stem cell (ESC)-like cells established by several groups do not form germ-line chimeras when injected into blastocysts. In the mouse, the aggregation of ESC with tetraploid embryos has allowed the generation of animals completely derived from these cells. However, aggregation of rat ESC-like cells with tetraploid rat embryos has not yet been attempted to evaluate their developmental capacity. Therefore, we established a method to produce tetraploid rat embryos by fusion at the two-cell stage. Chemical fusion by polyethylene glycol (PEG) was shown to be less efficient (56.3% fused embryos) than electrofusion (96.1% fused embryos). The rate of development of fused embryos to blastocysts was independent of the fusion method and similar to the rate of control embryos. However, this rate was lower when the embryos had been cultured from the zygote state before fusion (14-20%) compared to freshly isolated two-cell embryos (41-63%). Alike for the mouse, blastocysts derived from fused two-cell rat embryos contained about half the number of cells as control blastocysts and were homogeneously tetraploid with no evidence of mosaicism. This method may be useful for the establishment of gene-targeting technology in the rat.     

A new vitrification device that absorbs excess vitrification solution adaptable to a closed system for the cryopreservation of mouse embryos

Several closed vitrification devices that avoid contact with liquid nitrogen have been reported. Recently, based on the Kitasato Vitrification System (KVS), we developed the Closed-KVS, which is a closed vitrification device. The KVS is an open vitrification device that can absorb excess vitrification solution. In this study, we performed two experiments to evaluate the efficacy of the Closed-KVS as a vitrification device for the cryopreservation of mouse embryos at the blastocyst and two-cell stage. In the first experiment, the blastocysts were vitrified using either the Closed-KVS or the KVS (control device). The survival, re-expansion, and hatching rates were not significantly different between embryos vitrified using the Closed-KVS and those vitrified using the KVS. In the second experiment, we evaluated the embryonic development of the two-cell stage embryos vitrified using the Closed-KVS. There were no significant differences in the survival, blastocyst formation, or hatching rates between vitrified or non-vitrified embryos. Additionally, we evaluated the cooling and warming rates of these devices using a numerical simulation method. The cooling rates of the Closed-KVS were similar regardless of whether the outer cap was pre-cooled and were lower than those of the KVS. However, the warming rates of the Closed-KVS (irrespective of cap pre-cooling) were the same as those of the KVS (612,000 °C/min). In summary, the Closed-KVS is a novel closed vitrification device for the cryopreservation of mouse embryos at the blastocyst and two-cell stage.     

Birth of mice after intracytoplasmic injection of single purified sperm nuclei and detection of messenger RNAs and MicroRNAs in the sperm nuclei

We have developed a method that effectively removes all of the perinuclear materials of a mouse sperm head, including the acrosome, plasma membrane, perinuclear theca, and nuclear envelope. By injection of a single purified sperm head into a metaphase II mouse oocyte followed by activation with strontium chloride, 93% of the zygotes developed into two-cell embryos. Although only approximately 17% of the transferred two-cell embryos were born alive, all live pups developed into adults, and they appeared to be normal in reproduction and behavior. We detected RNA species, including mRNAs and miRNAs from the purified sperm heads. Our data demonstrate that pure membrane-free sperm heads are sufficient to produce normal offspring through intracytoplasmic sperm injection and that at least part of the RNA molecules are deeply embedded in the sperm nucleus.     

Development of hamster two-cell embryos in the isolated mouse oviduct in organ culture system

Hamster early two-cell embryos developed to the expanded blastocyst stage within the isolated mouse ampulla maintained in organ culture system. Mouse ampullae isolated at different times after treating the mice with human chorionic gonadotropin (hCG) (0–72 h) or pregnant mare's serum gonadotropin (PMSG) (30–32 h) were flushed with culture medium, and hamster early two-cell embryos were introduced into these ampullae. Mouse ampullae isolated at 14–32 h after hCG injection were more favorable for the development of the embryos than those isolated at 70–72 h. When mouse ampullae were isolated 30–32 h after hCG or PMSG treatment, 39% of the cultured eggs developed, some of them to the expanded blastocyst stage after additional culture for 65–70 h. These results indicate that unknown oviductal factors stimulate the development of hamster early two-cell embryos, and these factors are under the control of hCG or PMSG. In addition, these factors are common to the mouse and hamster.     

Comparison of conditioned medium and direct co-culture of human granulosa cells on mouse embryo development

Although numerous investigations have demonstrated the beneficial effects of co-culture system of different somatic cells on in vitro development of embryos, the effects of conditioned-media of co-culture cells have not been well documented. The objective of this study was to compare the effects of human granulosa cells co-culture system and its conditioned medium on the developmental rate of mouse embryos in vitro. Two sets of experiments were undertaken: in the first one 317 mouse one-cell embryos were cultured in human granulosa cell co-culture system (GC). Ham's F10 medium conditioned with granulosa cells (CM) and non-conditioned Ham's F10 for 120 h. In the second experiment. 391 late two-cell embryos were cultured in the 3 fore-mentioned culture treatments for 72 h. Embryos were obtained from NMRI mice. Granulosa cells were collected from patients undergoing an IVF program during oocyte pickup. In the first set of experiments, 23.6, 14.5 and 11.1% of one-cell embryos passed two-cell block and continued growing to 4-cell in GC, CM and HF, respectively. This index in GC was significantly different from two other treatments. Also significantly more embryos reached blastocyst stage in GC compared with two other treatments. The blastocyst rate was not significantly different between CM and HF. In the second set of experiments the proportion of blastocyst stage was significantly higher in CM than that in HF and lower than that in GC. In conclusion, although human granulosa cell-conditioned medium has beneficial effects on mouse embryo development, it was not as effective as co-culture of these cells.     

Hypoxanthine-maintained two-cell block in mouse embryos: dependence on glucose and effect of hypoxanthine phosphoribosyltransferase inhibitors

The culture conditions under which hypoxanthine maintains a two-cell block in preimplantation mouse embryos were assessed. Hypoxanthine prevented embryo development past the two-cell stage at concentrations as low as 30 nM, and this inhibitory activity required the presence of D-glucose. The action of hypoxanthine plus D-glucose was reversed by glutamine and higher lactate. D-mannose substituted for D-glucose in supporting the inhibitory action of hypoxanthine, but L-glucose, D-fructose, and 2-deoxyglucose were much less effective. Other purine derivatives such as inosine and adenosine, but not xanthosine or uric acid, also blocked development at the two-cell stage at a concentration of 30 microM, and guanosine was inhibitory at higher doses. Assays of hypoxanthine phosphoribosyltransferase (HPRT) activity in lysates of four-cell embryos determined that the drugs 6-mercapto-9-(tetrahydro-2-furyl)-purine (MPTF) and 6-mercaptopurine (6-MP), but not 6-azauridine (6-AzaU), prevented salvage of hypoxanthine. In addition, MPTF and 6-MP produced a significant two-cell block, which did not depend upon the presence of hypoxanthine or D-glucose; whereas 6-AzaU was without effect. When embryos were cultured 2 days in the presence or absence of D-glucose, hypoxanthine salvage was significantly reduced in lysates of four-cell embryos exposed to D-glucose. D-glucose had no effect when added directly to the assay mixture. These data demonstrate that the ability of hypoxanthine to block embryo development at the two-cell stage depends on the presence of D-glucose or other glycolyzable sugars and suggest that inhibition of the purine salvage pathway promotes the two-cell block.     

Modifications in major satellite methylation in the nucleus of a two-cell mouse embryo with respect to developmental conditions

A study was conducted of the degree of DNA methylation in the nucleus, in particular, of the major satellite in two-cell mouse embryos developing in the maternal organism, in standard culture medium M16, used for cultivating mouse embryos; and M2 media used for manipulating embryos in air. Two-cell embryo nuclei at 44–46 h after injections of chorionic hormone were investigated. The results are evidence for the dependence of the major satellite’s methylation level on the developmental conditions of embryos. The methylation level of the nuclear DNA was shown to increase with a deterioration of environmental conditions. It was reported that in the case of cultivation in M2 media unsuitable for long cultivation, the DNAís methylation level, the major satellite in particular, was higher compared to other embryo groups. Accordingly, not only a significant number of genes but also sequences of satellite DNA are involved in epigenetic regulation.     

Optimal equilibration conditions for practical vitrification of two-cell mouse embryos

The objective of the study reported here was to elucidate the optimal equilibration conditions for carrying out vitrification of two-cell mouse embryos, using a solution containing 2M dimethyl sulfoxide, 1M acetamide, and 3M propylene glycol (DAP213) as a cryoprotectant. Embryos were subjected to an equilibration process under 20 conditions of a combination of different temperatures (10 to 37 degrees C) and times (5 to 90 sec), and viability of the embryos was assessed by the rate of development into blastocysts and into live fetuses. As a result, these rates of development into blastocysts did not differ from those for unfrozen embryos. The rate of development of frozen-thawed embryos into live fetuses under conditions of 30 sec. at 20 degrees C, which was selected as having by highest operability, was 55.2%, comparable to the value (65.0%) for unfrozen embryos. Thus, the optimal equilibration condition for vitrification of two-cell mouse embryos, using DAP213 solution, was 30 sec at 20 degrees C, under which embryo viability was maximized, and this equilibration process was considered useful as a practical two-cell embryo freezing process in the vitrification method.     

Deficiency in the response to DNA double-strand breaks in mouse early preimplantation embryos

DNA double-strand breaks (DSBs) are caused by various environmental stresses, such as ionizing radiation and DNA-damaging agents. When DSBs occur, cell cycle checkpoint mechanisms function to stop the cell cycle until all DSBs are repaired; the phosphorylation of H2AX plays an important role in this process. Mouse preimplantation-stage embryos are hypersensitive to ionizing radiation, and X-irradiated mouse zygotes are arrested at the G2 phase of the first cell cycle. To investigate the mechanisms responding to DNA damage at G2 in mouse preimplantation embryos, we examined G2/M checkpoint and DNA repair mechanisms in these embryos. Most of the one- and two-cell embryos in which DSBs had been induced by gamma-irradiation underwent a delay in cleavage and ceased development before the blastocyst stage. In these embryos, phosphorylated H2AX (gamma-H2AX) was not detected in the one- or two-cell stages by immunocytochemistry, although it was detected after the two-cell stage during preimplantation development. These results suggest that the G2/M checkpoint and DNA repair mechanisms have insufficient function in one- and two-cell embryos, causing hypersensitivity to gamma-irradiation. In addition, phosphorylated ataxia telangiectasia mutated protein and DNA protein kinase catalytic subunits, which phosphorylate H2AX, were detected in the embryos at one- and two-cell stages, as well as at other preimplantation stages, suggesting that the absence of gamma-H2AX in one- and two-cell embryos depends on some factor(s) other than these kinases.     

小鼠植入前胚胎GCN5和HDAC1表达模式及体外培养对其表达的影响

为探讨小鼠植入前胚胎组蛋白乙酰化酶GCN5(general control of nucleotide synthesis,GCN5) 和组蛋白去乙酰化酶1(histone deacetyluse1,HDAC1)的表达模式及常规体外培养对它们表达的影响,应用荧光免疫细胞化学技术,检测了体内和体外培养的小鼠2、4、8细胞期卵裂胚胎、桑葚胚和囊胚GCN5和HDAC1的表达。结果显示,GCN5在体内组各细胞期卵裂胚胎和桑葚胚的细胞浆内均呈高表达,细胞核内未见明显表达,而囊胚细胞的细胞浆和细胞核内均无表达:HDAC1在体内组小鼠2细胞期胚胎中以细胞浆内表达为主,在其他各期胚胎均以细胞核内表达为主．囊胚期内细胞团部分细胞的细胞核内未见HDAC1表达。GCN5在体外组小鼠植入前各期胚胎均不表达,而 HDAC1的表达强度明显低于体内组的。提示体外培养抑制小鼠植入前胚胎GCN5和明显降低 HDAC1的表达,影响胚胎基因的正确性表达。     

小鼠植入前胚胎GCN5和HDAC1表达模式及体外培养对其表达的影响

为探讨小鼠植入前胚胎组蛋白乙酰化酶GCN5（general control of nucleotide synthesis,GCN5）和组蛋白去乙酰化酶1（histone deacetylasel,HDAC1）的表达模式及常规体外培养对它们表达的影响,应用荧光免疫细胞化学技术,检测了体内和体外培养的小鼠2、4、8细胞期卵裂胚胎、桑葚胚和囊胚GCN5和HDAC1的表达。结果显示,GCN5在体内组各细胞期卵裂胚胎和桑葚胚的细胞浆内均呈高表达,细胞核内未见明显表达,而囊胚细胞的细胞浆和细胞核内均无表达：HDACl在体内组小鼠2细胞期胚胎中以细胞浆内表达为主,在其他各期胚胎均以细胞核内表达为主。囊胚期内细胞团部分细胞的细胞核内未见HDAC1表达。GCN5在体外组小鼠植入前各期胚胎均不表达。而HDAC1的表达强度明显低于体内组的。提示体外培养抑制小鼠植入前胚胎GCN5和明显降低HDAC1的表达,影响胚胎基因的正确性表达。     

Effect of epidermal growth factor on preimplantation development and its receptor expression in porcine embryos.

The present study aimed to determine the influence of exogenous epidermal growth factor (EGF) on in vitro preimplantation porcine embryo development and its mRNA expression for EGF receptor (EGFR). Oocytes were aspirated from abattoir ovaries, selected and cultured in defined, protein-free media for 44 hr before in vitro fertilization (IVF). Thirty-six hours after IVF, two-cell stage embryos were selected and treated or cultured until embryo treatment. In experiment 1, compact morulae were selected on day 4 after IVF and randomly allocated into 5 groups: NCSU 23 with PVA as group 1; NCSU 23 with PVA and 0.1 ng/ml, 1.0 ng/ml, 10.0 ng/ml EGF as group 2, 3, 4, respectively; NSCU 23 with 0.4% BSA as group 5. In experiment 2, treatment groups were the same as in experiment 1 except that 0.1% crystallized BSA was added to both washing media and all treatment groups instead of PVA. In experiments 3 and 4, two-cell stage embryos were treated and cultured in the same experimental design as experiments 1 and 2, respectively. RT-PCR was used to detect the mRNA expression of EGF receptor in compact morulae and blastocysts. The PCR products were subjected to direct DNA sequencing. There was no significant improvement in the development rate of embryos from compact morulae to blastocysts in the presence of various EGF concentrations (0.1, 1.0, 10.0 ng/ml) versus without EGF addition. They were all significantly lower than those embryos cultured in the continuous presence of 0.4% BSA. However, when a reduced concentration (0.1%) of crystallized BSA was added to all the treatment groups, a significantly lower rate of embryo development was observed in control media (NCSU23 with 0.1% crystallized BSA) compared with those developed in culture media with 0.4% BSA. With the addition of EGF at 10 ng/ml (with 0.1% BSA), embryo development rates were significantly improved over the control group (P < 0.05) and were as good as those rates in 0.4% BSA culture group. When embryos were selected and treated from the 2-cell stage, they did not develop to blastocyst stages after five more days' culture without any protein (BSA) or growth factor addition. When 0.1% BSA was included in the media, blastocyst formation rates were significantly improved by EGF addition at the concentration of both 1.0 or 10 ng/ml (P < 0.05) as compared to 0.0 or 0.1 ng/ml. EGFR mRNA was detected in both compact morulae and blastocyst stages of porcine embryos and confirmed by direct DNA sequencing. Our results indicate that IVM-IVF porcine embryo developmental rates could be improved by the addition of EGF in the culture media with the presence of a reduced amount of defined BSA (>97% albumin). However, EGF alone was not able to elicit any stimulatory effects on embryo development in the absence of protein supplementation. Further studies are needed to investigate the potential synergistic factors in embryo culture media to eventually define the porcine embryo culture media.     

Nuclear transfer of mouse follicular epithelial cells pretreated with spermine, protamine, or putrescine

The in vitro and in vivo developmental potential of nuclear transferred embryos receiving follicular epithelial cells pretreated with spermine (5 and 20 mM), protamine (0.25 and 25 mg/ml), or putrescine (1 and 100 microg/ml) at room and reduced temperatures was examined in the mouse. The pretreated donor cells were first fused with enucleated oocytes, and then nuclei from reconstituted eggs at the two-cell stage were fused with the enucleated fertilized two-cell embryos. The proportion of reconstituted embryos that developed into blastocysts was not significantly different among groups. After transfer to recipients, implantation rates were not different between groups and fetuses were obtained in protamine- and spermine-treated groups as well as in control groups. These results demonstrate that pretreatment of nuclear donor cells with spermine, protamine, or putrescine does not enhance the developmental potential in vitro or in vivo in the mouse. J. Exp. Zool. 289:208-212, 2001.