To examine the effects of diethylstilbestrol (DES) on male pubertal development and thyroid function, juvenile male Sprague-Dawley rats were given DES daily by oral intubation at doses of 10, 20 and 40 μg/kg/day from postnatal day 33 for 20 days. Prepuce separation was significantly delayed at the dose of 20 μg/kg/day and above in the DES-treated rats. DES treatment induced a significant reduction in the weights of testes, epididymides, the ventral prostate, seminal vesicles plus coagulating glands and fluid, levator ani bulbocavernosus muscles, Cowper's glands and the glans penis. The weights of the liver and adrenals increased in the DES-treated animals. DES caused a dose-dependent reduction in germ cells; in particular the spermatids were mainly affected. The serum levels of testosterone and luteinizing hormone were significantly reduced in the DES-treated groups, but that of estradiol decreased. No differences were observed in the serum thyroxine levels of the control and DES-treated groups. In microscopic observation of the DES-treated animals, degeneration of germ cells and tubular atrophy in the testis were noted, but there were no microscopic changes in the thyroid. These results indicate that DES affected the pubertal development of juvenile male rats and that its mode of action may be related to alterations in hormone levels.

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The precise role of Dickkopf-1 (Dkk-1) during early bovine trophoblast development and subsequent placentation is not fully understood. Using somatic cell nuclear transfer (SCNT) generated pregnancies as a model of poor placentation we have found that mean levels of Dkk-1 mRNA were 1.5 fold lower in SCNT fetal cotyledon tissue at Day 50 of gestation than those resulting from artificial insemination (AI) and 2 fold lower at Days 100 and 150 (P<0.004). Dkk-1 expression in cotyledon tissue was localized by in situ hybridization to fetal binucleate cells (BNCs). Examining conceptuses from blastocyst stage we show that Dkk-1 mRNA was first evident between Days 15-20 of gestation in trophoblast tissue (when BNCs first appear) prior to the initial expression of the BNC specific bovine placental lactogen (bPL) on Day 20. Dkk-1 mRNA levels were higher than bPL in trophoblast tissue throughout the pre-attachment period (Days 24-31), however, this reversed during cotyledon development with only a subset of the bPL immunoreactive BNCs also containing Dkk-1 protein, suggesting a specific role for Dkk-1 during early placentation. One function of Dkk-1 is as an antagonist of the Wnt signaling pathway and, although Wnt5A and Wnt7A mRNAs were expressed in Day 50 bovine cotyledons, their expression levels were similar between AI and SCNT. In addition, the nuclear localization of β-catenin, which is an indicator of activation of the Wnt pathway, was also similar between AI and SCNT cotyledon tissue. Transcriptional control of Dkk-1 was not due to changes in DNA methylation levels in the promoter region as methylation levels were no different when comparing AI and SCNT tissues. The decreased expression of Dkk-1 in SCNT cotyledons that are prone to abnormal placentation suggests a role in cotyledon formation but the mechanism and regulatory control is yet to be revealed.

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When intracytoplasmic sperm injection (ICSI) is applied in the rat, sperm chromatin is introduced into the oocyte together with the acrosome, which does not enter the cytoplasm of the oocyte during normal fertilization, resulting in the rat giving birth to pups. Since successful ICSI was reported in rats, but with low efficiency, it has been observed that the acrosome of the sperm head seems to have detrimental effects on the embryonic development of ICSI oocytes. To improve ICSI in rats, the effects of removal of the acrosomal membrane from rat sperm on the development of ICSI oocytes were examined. While most control (non-treated) sperm had an intact acrosomal membrane, the Triton X-100 (TX)- and lysolecithin (LL)-treated groups showed high percentages of sperm with a removed acrosomal membrane. The timing of pronuclear formation in ICSI-oocytes using TX- or LL-treated sperm was significantly accelerated compared with that of the control sperm (P<0.05). However, neither TX nor LL treatment affected amounts of PLCζ in rat sperm. The rates of offspring derived from TX- (20.3 ± 4.4%) and LL-treated sperm (19.0 ± 2.8%) were also significantly higher than that of the control group (7.6 ± 2.3%; P<0.05). Our data clearly indicate that removal of acrosomal membranes from sperm by reagents is effective for generation of offspring via ICSI in rats.

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The purpose of this study was to investigate fertilization ability and embryo development to the blastocyst stage after reciprocal in vitro fertilization (IVF) between yak and cattle in an attempt to clarify the problem of low conception rate after mating yak females with cattle bulls. In vitro-matured (IVM) cattle and yak oocytes were inseminated with either Holstein or yak spermatozoa, and after an 18-h of coincubation period, a proportion of the oocytes was fixed and examined for sperm penetration, polyspermy and male pronuclear formation. The remaining oocytes were cultured in vitro and evaluated for cleavage and blastocyst formation rates. The percentage of IVM oocytes penetrated by spermatozoa ranged from 78.5 to 90.5%, and the formation of one or two pronuclei and the incidence of polyspermy did not differ among the different combinations. The cleavage and blastocyst rates were not affected by the species of the sperm, but they were affected by the species of the oocytes (P<0.05), with cattle oocytes having a higher (P<0.05) cleavage and blastocyst rates (69.9 and 31.3%) than yak oocytes (62.7 and 11.5%). The blastocyst formation rate was calculated from the cleaved zygotes. The interaction between sire and oocytes species (P<0.05) influenced blastocyst formation rate, with the highest blastocyst rate occurring in cattle oocytes fertilized with yak spermatozoa (36.5%) and the lowest rate occurring in yak oocytes fertilized with yak spermatozoa (9.4%). The effect of heterosis was apparent at the blastocyst stage, but there was a large reciprocal difference in blastocyst production between crosses. It was concluded that the low conception rate that results from crossing yaks with cattle is not due to either a species-specific block of fertilization or the developmental competence of the early stage embryo.

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Von Willebrand factor (vWF), a large multimeric glycoprotein present in blood plasma, is a blood protein of the coagulation system. It is defective in von Willebrand disease and is involved in a large number of other diseases, including thrombotic thrombocytopenic purpura-hemolytic uremic syndrome and heyde's syndrome. We have developed a line of transgenic swine harboring recombinant human von Willebrand factor (rhvWF) cDNA through microinjection of fertilized one-cell pig zygotes. Expression of rhvWF in the mammary gland and secretion of rhvWF into the milk of the transgenic swine were confirmed by immunohistochemical and western blot analyses, respectively, and rhvWF proteins were detected in milk from all lactating founder females at concentrations that were 28- to 56-folds greater than that in circulating human plasma. The amino acid sequence of rhvWF protein in the transgenic pig milk matched that of vWF produced from human blood plasma. This study provides evidence that production of rhvWF from transgenic pig milk is a potentially valuable technology and can be used as a cost-effective alternative in clinical applications.

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Myelocytomatosis oncogene (c-myc) is a major transcriptional regulator that controls various biological processes, and its deregulated expression causes carcinogenesis. To investigate the involvement of c-myc in oogenesis and preimplantation development, the expression of c-MYC during these stages was examined by immunocytochemistry. A strong c-MYC signal was detected in the nucleus of growing and fully grown oocytes as well as in preimplantation embryos before the morula stage. The signal intensity decreased slightly at the morula stage, and no signal was detected in blastocysts. Close observation of the nucleus revealed that c-MYC was localized in small granules that appeared to be nuclear speckles controlling pre-mRNA splicing. Although the number of granules decreased during oocyte growth, their size increased. After fertilization, the granules of c-MYC disappeared from the pronuclei, and c-MYC was evenly distributed in the nucleoplasm at the 1-cell stage, but the granules reappeared at the 2-cell stage. These results suggest that c-myc is involved in oocyte growth and preimplantation development and that its role changes during these stages.

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Production of hyaluronan (hyaluronic acid: HA) was demonstrated in denuded mouse oocytes (DOs) by the enzyme-linked immunosorbent assay, and the role of HA in enlargement of the perivitelline space in the oocytes was examined. The incidence of polyspermy following insemination was also observed in DOs in which HA synthesis was inhibited. HA was not detected in culture medium containing DOs immediately after collection. After culture for 7 h, 4.75 pg of HA per DO was detected in the medium, and the mean amount of HA significantly increased to 20.78 pg 14 h after culture. When DOs were cultured in medium containing 0.25 mM 4-methylumbelliferone (MU), an inhibitor of HA synthase, the mean amount of HA in the culture medium with DOs was 8.61 pg, which was significantly smaller than the amount in the control medium with non-treated DOs (21.59 pg). The mean size of the perivitelline space in oocytes cultured with cumulus cells (5.40 μm) did not differ from that (5.08 μm) of DOs. The mean size of the perivitelline space was significantly smaller in the MU-treated DOs (3.58 μm) than in the control DOs (4.65 μm). The fertilization rate did not differ between the MU-treated DOs (84.9%) and control DOs (81.0%), whereas the incidence of polyspermy was significantly higher in the MU-treated DOs (13.3%) compared with the control DOs (2.1%). These findings clarified that the HA involved in enlargement of the perivitelline space in oocytes is synthesized and secreted by the oocytes themselves. They also suggest that there is a close relationship between the size of perivitelline space and the incidence of polyspermy in mouse oocytes.

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This study aimed to identify protein(s) that bind(s) to the highly AT-rich sequence of porcine Fshb promoter region -852/-746 (named Fd2) by the Yeast One-Hybrid Cloning System and finally a paired related homeodomain transcription factor, Prx2, known as a key factor for skeletogenesis was cloned. RT-PCR analysis of fetal and postnatal porcine pituitaries demonstrated that Prx2 starts to be expressed at around fetal days 40-50 just before the beginning of Lhb-expression and that the level of Prx2 increases after birth. Immunohistochemical analysis of the prepubertal porcine pituitary revealed that some Prx2-positive cells overlap some Lh β-positive cells. Transient transfection assay using non-pituitary CHO cells and pituitary tumor-derived LβT2 cells revealed that Prx2 plays a cell-type dependent role in modulation of the Fshb promoter, showing stimulation in CHO cells and repression in LβT2 cells via the regions of Fd2 and -596/-239. The binding ability of Prx2 to the regions of Fd2 and -596/-239 was confirmed by electrophoretic mobility shift assay. DNase I footprinting revealed that broad regions of Fd2 were bound by Prx2 and that -596/-239 contained seven Prx2-binding sites. The SELEX method using a random N15-mer oligonucleotide pool demonstrated that Prx2 monomer binds to a TAATT motif, which is present in Fd2 and -596/-239. However, the binding of Prx2 to TAATT with a single molecule and its inverted repeat with two molecules could not induce transcriptional activation, indicating that the Prx2-dependent transcriptional modulation demonstrated in cultured cells is not introduced by Prx2 alone. Thus, this study demonstrated for the first time that Prx2 is expressed in the pituitary gland and at least in a part of gonadotropes in which Prx2 may play a role in repression of the Fshb gene.

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To investigate the possible role of the vasculature in the local regulation of corpus luteum (CL) function, we determined the densities of capillaries and large blood vessels in the center of the bovine CL during the estrous cycle and following prostaglandin (PG) F2α-induced luteolysis. The CLs at the early (Days 2-3 post-ovulation), developing (Days 5-7), mid (Days 8-12), late (Days 15-17) and regressed (Days 19-21) stages were collected. In addition, the CLs were collected by transvaginal ovariectomy from 12 cows (Day 10 after ovulation), i.e., non-treated (n=3, 0 h, control), at 0.5 (n=3), 2 (n=3) and 12 h (n=3) after injection of a luteolytic dose of PGF2α. Immunohistochemical staining with von Willebrand Factor (specific for endothelial cells that are found in both types of blood vessels) revealed that the density of the luteal blood vessels was significantly higher at the developing and late luteal stages (P<0.05) than at the other stages, whereas the number of larger blood vessels (those stained with α-smooth muscle actin) was higher at the late and regressed luteal stages (P<0.05) than at the other stages. Furthermore, both the density of blood vessels and the number of blood vessels with smooth muscle were significantly higher in the CLs obtained at 2 h and 12 h after PGF2α administration (P<0.05) than in those without PGF2α treatment. These results suggest that the number of blood vessels with smooth muscle per unit area in the regressing CL increased as a result of losing steroidogenic cells and capillaries. The overall results demonstrate that the capillaries disappeared earlier than the large blood vessels during structural luteolysis and suggest that the loss of capillaries in the CL results in a reduced supply of nutrients and oxygen to luteal cells followed by cell death.

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The mammalian brain exhibits sex differences with respect to structure and function. In our previous report, we found that progranulin (PGRN)-deficient (pgrn^-/-) mice displayed an alteration in male-type behaviors, including reduced frequency of ejaculation and elevated levels of aggression and anxiety. The aim of the present study was to elucidate the role of PGRN in sex differences in anxiety. In the elevated plus maze, wild-type (pgrn^+/+) female mice spent more time in the closed arms than the pgrn^+/+ males, suggesting that the level of anxiety was higher in females than males. On the other hand, no sex difference was observed in the pgrn^-/- mice, and their anxiety levels were almost the same as those of the pgrn^+/+ females. To elucidate the effect of testosterone on male anxiety, male mice were castrated at 5 weeks of age and silastic tubes filled with either testosterone or cholesterol were then implanted into them for one week. These treatments did not affect anxiety in the open field in either genotypes, although the pgrn^-/- males exhibited higher anxiety than pgrn^+/+ males. Next, we measured the volume of the paraventricular nucleus (PVN) and the locus ceruleus (LC), as these are anxiety/stress-related nuclei that are known to have sex differences in their structures. In the pgrn^+/+ mice, there was a tendency for the volume of the LC to be larger in males than females. In addition, the pgrn^-/- mice had a larger volume of LC than the pgrn^+/+ mice, although no sexual differences were observed. The number of cells in the LC was also larger in the pgrn^-/- than in the pgrn^+/+ mice. No significant differences in the volumes of the PVN were observed between genotypes or sexes. These results suggest that PGRN plays a role in organization of the LC, which eventually modulates anxiety in novel environments.

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The present study was undertaken to compare the changes in circulating levels of inhibin-B, prolactin, follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol-17β, progesterone and testosterone during the different reproductive states of turkey hens. Blood samples were collected during different reproductive states, at laying, incubating and out of lay. Inhibin-B was measured by ELISA, while other hormones were determined by Chemiluminescent Microparticle Immunoassay (CMIA). The results revealed highly significant differences among the hen's states for all serum hormone concentrations. The highest levels of inhibin-B and prolactin were observed in incubating hens, while the lowest values were observed in laying hens. In contrast, the highest levels of FSH, LH, estradiol-17β, progesterone and testosterone were found in the laying group, while the lowest values were found in the incubating group. The progesterone level was higher in the laying group compared with the other groups. These results clearly demonstrate that negative correlation was found between both the inhibin-B and prolactin levels and the gonadotropin and steroid hormone concentrations during the different reproductive states of the turkey hens. In addition, the results suggest that inhibin-B may be involved in control of FSH and LH secretion.

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The sex ratio of mammals has previously been shown to be affected by maternal stress. In our previous study, the proportion of female embryos collected from superovulated and artificially inseminated Holstein heifers that were frequently placed in stanchions and subjected to transrectal examinations of the ovaries during the follicular phase tended to be higher than the expected 50%. The goal of the present study was to test the validity of this observation using a greater number of heifers. Superovulated heifers were artificially inseminated at 56 and 72 h after PGF_2α treatment using a single batch of frozen semen. Frequent capture (FC), transrectal examination and/or blood sampling were performed at 4-h intervals from 36 to 76 h after PGF_2α treatment (n=13). Nine heifers were used as the Control (non-treatment). Seven-day-embryos were recovered by uterine flushing. Male and female embryos were separated using the loop-mediated isothermal amplification procedure. The proportion of female transferable embryos in the FC group (67.8%, 78/115) was significantly higher than that in the Control group (51.2%, 43/84, P<0.05). The peak concentration of plasma cortisol during the follicular phase following superovulatory treatment was 20.6 ng/ml in the FC group. These results suggest that subjecting heifers to stress during the follicular phase following superovulatory treatment may increase the female sex ratio of embryos.

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The objective of this research was to examine the effects of high concentrations of glucose on mouse embryos developing in vitro by studying embryo viability, mitochondrial content and expression of glucose transporters. Addition of 55 mM glucose to the culture medium of two-cell stage embryos significantly reduced the formation of morulae and blastocysts, resulting in fewer cells in the blastocyst stage embryos and increased levels of apoptosis. Quantitative reverse transcriptase (RT) PCR analysis revealed that the expression levels of the pro-apoptotic genes Bax and Casp3 at the blastocyst stage were increased significantly by the addition of either 25 or 55 mM glucose to the culture medium. However, addition of 25 or 55 mM glucose to the culture medium did not change the copy numbers of the apoptosis-related miRNAs mmu-mir-15a, mmu-mir-16 and mmu-mir-21. MitoTracker Green fluorescence revealed a decrease in the mitochondrial mass. The expression levels of the mitochondrial DNA-encoded genes Cox1 and Cox2 decreased sharply with the addition of 25 or 55 mM glucose to the culture medium. Both transcripts and protein synthesis of the glucose transporters Glut1 and Glut3 were reduced in blastocysts cultured in the presence of either 25 or 55 mM glucose. These results suggest that hyperglycemia reduces both mitochondrial content and expression levels of glucose transporters in mouse embryos developing in vitro and that this may result in apoptosis in these embryos.

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To improve bovine somatic cell nuclear transfer (SCNT) efficiency, we studied various aspects to optimize the experimental procedures. Firstly, donor cells were treated with pronase, which resulted in a higher fusion rate than that of cells without the pronase treatment (78.3 vs. 53.9%). Secondly, when fused embryos were activated either by chemical (ionomycin + cyclohemixide (CHX)) or electrical + CHX stimulation, the cleavage and blastocyst formation rates were comparable amongst these treatment groups (P>0.05); however, mortality following electrical + CHX activation was significantly higher than that observed with the chemical activation, regardless of the pronase treatment (P<0.05). Finally, we compared the culture conditions of the reconstructed embryos using ACM medium plus mouse embryonic fibroblasts (MEF) vs. B2 medium plus granulose cells (GC), and the results clearly demonstrated that the former culture conditions led to a higher blastocyst rate, 90-day pregnancy rate, and newborn rate, than that observed for culture in B2 medium plus GC (46.7 vs. 34.7%, 36.1 vs. 9.6% and 25.9 vs. 5.8% for the blastocyst, pregnancy and newborn rates, respectively). In summary, the efficiency of bovine SCNT can be greatly improved using optimized operational procedures, including treating the donor cells with pronase, activation of fused embryos by ionomycin + CHX and the culture of the reconstructed embryos in ACM + MEF media.

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Use of sperm separation methods such as density gradient centrifugation for selecting the best spermatozoa for animal breeding is constrained by the problem of dealing with the large volumes of ejaculate produced by the males of some species, such as boars. The purpose of this study was to compare density gradient centrifugation (DGC) with centrifugation on a single layer of colloid (SLC) for the preparation of ejaculated boar spermatozoa using Androcoll^TM-P. There was no difference between the two techniques in terms of sperm motility or duration of motility after selection, and sperm motility was retained for at least 24 h longer in the centrifuged sperm preparations than in controls (uncentrifuged aliquots). Sperm motility was significantly better (P<0.001) in the centrifuged sperm preparations (means ± sd: SLC 79.6 ± 8.1% and DGC 74.2 ± 12.0%) than in the uncentrifuged controls (62.9 ± 12.7%). The mean yield of motile spermatozoa for SLC was 67.5 ± 25.6%, and for DGC it was 59.6% ± 22.3% (not significant, ns). Sperm survival was significantly increased by colloid centrifugation (control 3.1 ± 0.3 days, SLC 5.5 ± 0.79 days, DGC 5.75 ± 0.62 days; P<0.001 for uncentrifuged versus centrifuged; SLC vs. DGC, ns). Moreover, boar spermatozoa could be stored for 24 h before centrifugation without any detrimental effect on sperm motility or duration of motility. In a further experiment, larger volumes of ejaculate were processed easily on a modified SLC, indicating that this method may be practical for processing large volumes of boar ejaculates.

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In mammals, growing oocytes with a diameter less than 80% of that of full-grown oocytes cannot start meiotic maturation, and their maturation promoting factor (MPF) cannot be activated by hormonal stimulation or isolation from follicles. The aim of the present study was to identify the key molecules responsible for meiotic failure of these growing oocytes (referred to as "small oocytes" in the present study). To this end, we altered the expression of the molecules involved in MPF activation in the small oocytes of pigs by injecting them with mRNA or antisense RNA (asRNA) and examined the effects on the meiotic ability of the small oocytes. Immunoblotting analyses revealed three defects in small oocytes compared with full-grown oocytes, an inactive mitogen activated protein kinase (MAPK) cascade, a failure of cyclin B synthesis and an insufficient amount of Cdc2. Injection with mRNAs of Mos, the uppermost molecule of the MAPK cascade, cyclin B1, cyclin B2 or Cdc2 into small porcine oocytes indicated directly and for the first time that the cause of meiotic failure of porcine small oocytes is an insufficient amount of Cdc2 rather than MAPK inactivation or failure of cyclin B synthesis. Next, in order to suppress Myt1 and Wee1B, which phosphorylates at inhibitory phosphorylation sites of Cdc2 and inactive MPF, we injected their asRNAs into the porcine small oocytes and found that the Wee1B asRNA significantly increased meiotic ability, whereas the Myt1 asRNA had no effect. When Cdc2 overexpression and suppression of Wee1B expression were simultaneously induced in the small oocytes of pigs, about 70% of the small oocytes resumed meiosis, and this rate was nearly comparable with that of the full-grown oocytes. These results strongly suggest that an insufficient amount of Cdc2 and continuous activation of Wee1 B are the cause of meiotic failure of small oocytes in pigs.

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Technical refinement of boar sperm cryopreservation is indispensable for effective breeding of the rare Okinawan native pig, the Agu. The objective of the present study was to determine whether addition of low-density lipoprotein (LDL) extracted from hen egg yolk to the freezing extender improves the characteristics of cryopreserved Agu spermatozoa. Ejaculated Agu sperm frozen in extender supplemented with 2, 4, 6, 8 or 10% LDL instead of egg yolk was thawed, and the post-thaw sperm characteristics were evaluated. Treatment with 4-8% LDL during cooling and freezing significantly increased the intracellular cholesterol content, as compared to that of sperm frozen in extender containing 20% egg yolk (P<0.05). Higher potential resistance to cell damage from cryoinjury was also observed in sperm frozen in extender supplemented with LDL: the integrities of plasmalemma and DNA, mitochondrial activity and proteolytic activity of the acrosomal content in the post-thaw sperm were superior to those of sperm that were not treated with LDL. Moreover, the percentages of total motile sperm and the extent of rapid progressive motility at 1 and 3 h after incubation were markedly higher in sperm treated with 4 or 6% LDL, and these sperm also had more ATP. However, LDL did not inhibit in vitro sperm penetrability, even though the cholesterol content of post-thaw sperm was higher after treatment with LDL. These findings indicate that addition of 4-6% LDL instead of egg yolk to the freezing extender improves the post-thaw characteristics of Agu sperm by protecting sperm against cold shock damage during cryopreservation.

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Somatic cell nuclear transfer has many potential applications in the fields of basic and applied sciences. However, it has a disadvantage that can never be overcome technically-the inflexibility of the sex of the offspring. Here, we report an accidental birth of a female mouse following nuclear transfer using an immature Sertoli cell. We produced a batch of 27 clones in a nuclear transfer experiment using Sertoli cells collected from neonatal male mice. Among them, one pup was female. This "male-derived female" clone grew into a normal adult and produced offspring by natural mating with a littermate. Chromosomal analysis revealed that the female clone had a 39,X karyotype, indicating that the Y chromosome had been deleted in the donor cell or at some early step during nuclear transfer. This finding suggests the possibility of resuming sexual reproduction after a single male is cloned, which should be especially useful for reviving extinct or endangered species.

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Angiotensin II (Ang II), a vasoactive peptide, is secreted by the bovine oviduct and is involved in modulation of local oviductal contraction. Ang II biosynthesis and release during the normal estrous cycle and the effects of luteinizing hormone (LH) and ovarian steroids on biosynthesis and secretion of Ang II were investigated. During the preovulatory period, increases in mRNA expression for Angiotensin converting enzyme 1 (ACE-1) and release of Ang II peptide were detected. Microdialysis of oviductal segments in vitro showed that LH alone significantly increased Ang II release, and combined infusion of LH+E₂+P₄ caused an increase in Ang II release. In cultured oviductal epithelial cells, LH increased Ang II release and ACE-1 mRNA expression, and E₂+P₄ enhanced stimulatory effect of LH on Ang II release and ACE-1 mRNA expression. Thus, it can be concluded that the oviductal Ang II system is upregulated by LH and ovarian steroids during the periovulatory period and may enhance local oviductal contraction. These events could stimulate transport of gametes to the fertilization site.

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The aim of this study was to test the effect of actinomycin D on the expression of selected genes and to elucidate possible components of its apoptotic pathway in mouse embryos. Selected mRNAs and Trp53 protein were examined in blastocysts cultured for 24 h in vitro with or without the presence of a high concentration of actinomycin D. In all tested genes, the relative quantities of mRNA were significantly lower in treated blastocysts than in controls. The mRNA quantities of H2afz, Actb, Bax, Bad and Bcl2 were reduced at a similar rate, but the decreases in Bcl2l2 and Trp53 mRNA were significantly greater. Treatment with actinomycin D also changed the ratio between the mRNA levels of some pro-apoptotic and anti-apoptotic genes: the Bad/Bcl2l2 and the Bax/Bcl2l2 ratios were on average 4.39 and 2.66 times higher in the treated embryos than in the controls, respectively. Generally, treatment led to developmental arrest and significant increase in the incidence of cells with typical apoptotic features. However, its effect on Trp53 protein expression was not significant. The results suggest that mechanisms beyond the apoptotic effect of actinomycin D might include specific changes in the expression of pro-apoptotic and anti-apoptotic genes, shifting the expression ratio in favor of the pro-apoptotic ones. The results also show that the role of Trp53 is probably not crucial in this apoptotic pathway.

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