The quality of porcine oocytes is affected by sexual maturity of the donor gilt

Although differences in the quality of oocytes derived from young gilts and adult sows are well documented, evidence concerning gametes of pre-pubertal and cycling gilts is scarce and inconsistent. The aim of this work was to establish whether sexual maturity of gilts affects the quality of their oocytes with the use of the brilliant cresyl blue (BCB) test, oocyte diameter and apoptosis. Ovarian morphology was evaluated, and gonads with corpus luteum or albicans were recognized as originating form cycling gilts (C) and those with follicles as originating form pre-pubertal females (P). Altogether 952 cumulus-oocyte complexes (COCs; group P: 554; group C: 398) were examined, whereas 149 COCs, not subjected to BCB test, served as a control for TUNEL. COCs of proper morphology were evaluated by the BCB test which differentiated two categories of gametes: more competent, BCB+, and less competent BCB- oocytes. The control group comprised oocytes of proper morphology aspirated from ovaries of P and C gilts not subjected to BCB test. Finally five groups of COCs were matured in vitro: 1/P-BCB+, 2/P-BCB-, 3/C-BCB+, 4/ C-BCB- and 5/ control. Significantly more large oocytes (≥ 120 μm), more BCB+ oocytes and more high quality (both BCB+ and ≥ 120 μm) oocytes originated from ovaries of cycling gilts than pre-pubertal gilts (p<0.001). The rate of mature oocytes at the MII stage differed significantly between C-BCB+ (68.5%) and P-BCB+ (32.9%) oocytes. The incidence of apoptosis among BCB-treated oocytes after in vitro maturation was 21.4% and was similar to that observed in control oocytes (17.4%). BCB+ oocytes from cycling gilts showed significantly higher (28.7%) incidence of apoptosis than that of the group P (16.2%). Interestingly, high quality oocytes displayed a similar level of apoptosis regardless of the donor puberty. We demonstrated that C gilts provided more BCB+ oocytes as well as more large oocytes than P gilts, although C-BCB+ oocytes showed higher apoptotic rate. In conclusion, high incidence of apoptosis and a big variation in the diameter of more competent BCB+ oocytes make the BCB test a less effective selection tool than previously reported.     

Sexual maturity and reproductive phase of oocyte donor influence the developmental ability and apoptosis of cloned and parthenogenetic porcine embryos

This study investigated the influence of the sexual maturity and reproductive phase of oocyte donor on the developmental ability and quality of porcine embryos produced by somatic cell nuclear transfer (SCNT) or parthenogenesis (PA). Blastocyst quality was evaluated in terms of hatching ability, total nuclei number and types of apoptosis. Results revealed that maturation rate was not influenced by the reproductive status of the oocyte donor. However, when subjected to PA or SCNT, embryos derived from sexually mature sow oocytes developed to blastocysts at higher rates and had higher cell number than those derived from immature gilt oocytes (p<0.05). Significant effect of reproductive phase, luteal versus follicular, was also noted with luteal stage oocytes yielding higher (p<0.05) rate of blastocyst formation (PA: 54.3+/-1.3% versus 44.8+/-0.3%; SCNT: 29.4+/-0.2% versus 22.7+/-0.1%). Blastocysts derived from luteal phase oocytes also had higher (p<0.05) hatching ability (PA: 44.2+/-1.1%; SCNT: 39.6+/-4.7%) and cell number (PA: 77.4+/-4.9; SCNT: 54.9+/-2.4) than those derived from follicular phase oocytes (PA: 34.9+/-0.9%, 67.2+/-3.9; SCNT: 34.6+/-2.7%, 47.5+/-2.9). TUNEL assay and Hoechst 33342 staining revealed that percentage of blastocysts showing total apoptosis did not differ among the groups. However, luteal phase oocyte-derived blastocysts had the highest incidence of nuclear fragmentation. Among cloned blastocysts that showed the signs of apoptosis, the highest index of total apoptosis was observed in prepubertal oocyte-derived blastocysts (5.2+/-0.7). Blastocysts derived from luteal phase oocytes showed the lowest TUNEL index (2.0+/-0.5). The present study therefore, indicates that the sexual maturity and reproductive phase of cytoplast donor significantly influences the developmental ability, apoptosis and quality of blastocysts produced by SCNT or PA. Oocytes from sexually mature sows in luteal phase of their reproductive cycle may be better cytoplast recipients for SCNT.     

Optimal age of sexual maturity and limitations of juvenile survival in mammals

Although the life history evolution of small- and large-bodied mammals seems to be governed by different factors, the both shows relative neonate size and juvenile survival to be slightly dependent on body mass. I propose a hypothesis that natural selection simultaneously maximizes a time to maturity (minimizes somatic growth rate) and a number of newborn survived to reproduction. In this case optimal juvenile survival of large-bodied mammals must be close to e-beta and that of small-bodied approximately e-(1 + beta), where beta is the slope of the regression of log annual fecundity on log annual juvenile mortality. Analysis of vital characters for 71 mammal species revealed the slope to be close to unity. As a result frequency distribution of log juvenile survival shows bimodality which coincides well with predicted optimal survival for large- and small-bodies species. It is shown that the relative neonate size can be directly proportional to the juvenile survival and inversely proportional to the lifetime offspring production irrespective of mortality factors.     

不同供体细胞及其处理对猪核移植重构胚体外发育的影响

系统探讨了体细胞的组织来源及培养代数对猪核移植重构胚发育的影响。体外成熟培养40~44 h的猪卵母细胞去核后, 将经血清饥饿(0.5%FBS)培养2~9天、0.1 mg/L Aphidicolin(APD)培养+0.5% FBS培养2~9天或一般培养法(10% FBS)培养的卵丘细胞、颗粒细胞、输卵管上皮细胞和耳皮成纤维细胞, 直接注射到去核的卵母细胞质中, 或注射到卵周隙中, 再经电融合(100 V/mm, 30 [mu]s, 电脉冲1次)构建重构胚。重构胚以钙离子载体A23817 或电脉冲结合6-DMAP 激活处理, 体外培养6天。耳皮成纤维细胞和颗粒细胞经0.1 mg/L APD + 0.5% FBS培养处理后的重组胚卵裂率, 均高于血清饥饿和一般培养处理的同种供体细胞(P<0.01)。卵丘细胞、颗粒细胞经0.1 mg/L APD + 0.5% FBS处理后进行核移植的分裂率和发育率均高于输卵管上皮细胞和耳皮成纤维细胞(P<0.05)。以猪颗粒细胞为核供体时, 电融合法的重构胚分裂率显著高于胞质内注入法(P<0.05), 但囊胚发育率无显著差异(P>0.05)。培养3代和6代的猪颗粒细胞以及培养6代和10代的耳皮成纤维细胞, 其具有正常二倍染色体的细胞比例均无显著差异(P>0.05); 以这2种细胞不同培养代数做供体进行核移植时, 各代之间核移胚的体外分裂率、囊胚发育率无显著差异(P>0.05)。这些结果表明: (1) 猪耳皮成纤维细胞和颗粒细胞经培养传代所建立起来的细胞系相对比较稳定; (2) 0.1 mg/L APD预培养处理供体细胞能提高猪体细胞核移植的效果, 血清饥饿培养则无明显效果; (3) 猪颗粒细胞和耳皮成纤维细胞等均可做供核细胞, 核移植后都能得到体细胞克隆的囊胚, 但前者的效果略优于后者, 且其核移植效果不受供核细胞培养代数的影响; (4) 电融合核移植胚胎的发育率高于胞质内直接注入法, 但两者的总体效率相近。     

Effect of asynchronous transfer and oestrogen administration on survival and development of porcine embryos.

Results indicate that recovery of embryos on Days 11 and 13 of pregnancy was reduced for Day 5 embryos transferred to recipients on Day 6 of their oestrous cycle and was greatly reduced when embryos were transferred to recipients on Day 7 of the cycle (P less than 0.01). Administration of oestradiol-17 beta on Day 11 of the recipient's cycle did not appear to affect embryo development on Day 13. Day 6 embryos transferred to recipients on Day 8 of the oestrous cycle deteriorated rapidly within 24 h of transfer; there was no recovery of embryos from the uterus after 36 h. Treatment of pregnant gilts with 1 mg oestradiol-17 beta (i.v.) on Day 10.5 resulted in total embryonic loss by Day 23, but pregnancy rates of gilts treated with oestradiol-17 beta on Day 12 were similar to those of vehicle-treated gilts (60.6 vs. 71.4%).     

Ultrastructure of porcine embryos following development in vitro versus in vivo

The present study examined the ultrastructural appearance of porcine embryos from the four-cell stage to the blastocyst grown either in vivo or in vitro. Embryos were collected at slaughter from superovulated gilts and were fixed for transmission electron microscopy either immediately or after various periods of in vitro culture. In general, the morphology of in vivo and in vitro grown embryos was similar. In vivo grown four-cell stages contained dense fibrillar nucleoli. At the eight-cell stage the nucleoli possessed increasing amounts of chromatin and granules. In both stages the mitochondria were spherical or ovoid in shape and had only few cristae. In morulae and blastocysts the nucleoli were mainly of the fibrillogranular type, and the mitochondria were filamentous and possessed more cristae, of which many were tubular. Two major ultrastructural deviations were observed in about half of the in vitro cultured embryos. First, nucleolus-like structures were found outside the nuclei in the cytoplasm of blastomeres. These structures were spherical and composed of chromatin-like material containing characteristically a single large and several small vacuoles. The structures were frequently associated with profiles of smooth endoplasmic reticulum (SER). A second type of deviation was aggregates of SER appearing as spiral coils or multiangular complexes. Some embryos displayed both types of deviations. The physiological significance of these deviations remains speculative. They may be involved in the considerably reduced capability of porcine embryos to develop to piglets following in vitro culture.     

Effects of sexual differences of age at maturity and survival on population sex ratio

Summary Five demographical factors influencing the sex ratio of a population are classically considered. The influence of two of them is dependent on the longevity of individuals in the population. The effect of differential age at maturity between males and females is higher for animals with low annual survival, whereas the effect of differential annual survival between males and females is higher for animals with high annual survival. Such a conclusion applied to turtles, which are long life-span animals, allows us to retain differential survival between sexes as a major factor influencing the population sex ratio.     

Nuclear transfer of porcine embryos using cryopreserved delipated blastomeres as donor nuclei

Nuclear transfer protocol for the pig using cryopreserved delipated four- to eight-cell and morula stage embryos as nucleus donors was developed. Donor embryos, which had been delipated by micromanipulation following centrifugation for polarizing cytoplasmic lipid droplets, were cryopreserved with 1.5 M 1,2-propanediol and 0.1 M sucrose. Recipient cytoplasts were prepared from ovulated oocytes. Activation of oocytes could be induced more efficiently when electric stimulation was given 53 hr after the hCG injection or later (66–83%), compared with 52 hr or earlier (11–16%, P < 0.05), suggesting that aging after ovulation may be required for in vivo matured porcine oocytes to be activated by electric stimuli. Membrane fusion rates between donor blastomeres and enucleated oocytes were 88% (127/144) and 97% (56/58, P > 0.05) for the four- to eight-cell and morula stage embryos, respectively. In vitro developmental rates to the two-cell (53/100 vs. 35/65), four-cell (34/100 vs. 26/65), and morula stage (17/100 vs. 18/65) were the same between the nuclear transfer embryos with four- to eight-cell and morula nuclei. However, more embryos reconstituted with morula nuclei developed to blastocysts (15% vs. 6%, P < 0.05). These data demonstrated that blastomeres of cryopreserved, delipated porcine embryos can be used as donor nuclei for nuclear transfer. Frozen-thawed, delipated blastomeres can be efficiently isolated and fused, and therefore provide a useful source of donor nuclei. Mol. Reprod. Dev. 48:339–343, 1997. © 1997 Wiley-Liss, Inc.     

Factors affecting the survival of bisected sheep embryos in vivo

Two experiments were carried out to examine the effects of different factors on the survival of split sheep embryos. In Experiment 1, embryos collected on Day-6, Day-7 or Day-8 were bisected and transferred into recipient ewes in pairs. The proportions of Day-6, Day-7 and Day-8 demi-embryos developing to lambs were 26% (14 54 ), 30% (31 102 ) and 32% (24 74 ), respectively. Replacement of bisected late morula to expanded late blastocyst stage embryos into zonae did not affect their survival rate (P>0.5). The proportion of demi-embryos developing to lambs in recipients with two or more ovulations was higher (35%, 53 152 ) than in recipients with a single ovulation (21%, 16 78 ; P<0.05). In Experiment 2, Day-6 embryos were split with or without exposure to 0.25 M of sucrose and were transferred into recipients in pairs or singly. Exposure to 0.25 M of sucrose decreased the proportion of split embryos developing to lambs compared with that of the controls (31%, 22 70 vs 49%, 34 70 ; P<0.05). The effects of the number of demi-embryos transferred or the stage of development on the survival rate were not significant (P>0.05). The number of lambs born per original embryo was the highest when the embryos were split without exposure to sucrose and transferred into recipients singly (106%, 17 16 ).     

Donor and recipient genotype and heterosis effects on survival and prenatal growth of transferred mouse embryos

Reciprocal embryo transfers amongst two inbred strains (C3HeB/FeJ and SWR/J) and their F1 cross (C3SWF1) were used to examine donor and recipient genotype and heterosis effects on survival and prenatal growth of mouse embryos. Among inbred strains, significant recipient genotype effects were detected for both embryo survival (P less than 0.01) and prenatal growth (P less than 0.05), while no donor genotype effects were observed. The recipient effect on overall embryo survival was due to a higher proportion of C3H recipients maintaining pregnancy to term than SWR recipients (P less than 0.01), rather than survival within litters. Irrespective of their own genotype, embryos developing in C3H uteri achieved larger body weights (P less than 0.01) and longer tail lengths (P less than 0.05) at birth than did embryos developing in SWR uteri. Recipient heterosis was not significant, while donor heterosis was significant for prenatal growth traits (P less than 0.001).     

Distribution analysis of transferred donor cells in avian blastodermal chimeras.

Blastodermal chimeras were constructed by transferring quail cells to chick blastoderm. Contribution of donor cells to host were histologically analyzed utilizing an in situ cell marker. Of the embryos produced by injection of stage XI-XIII quail cells into stage XI-2 chick blastoderm, more than 50 percent were definite chimeras. The restriction on the spatial arrangement of donor cells was induced by varying the stage of host. Ectodermal chimerism was limited to the head region and no mesodermal chimerism was shown when the quail cells were injected into stage XI-XIII blastoderm. Mesodermal and ectodermal chimerisms were limited to the trunk, not to the head region, when the quail cells were injected into the stage XIV-2 blastoderm. In these chimeras, however, some of the injected quail cells formed ectopic epidermal cysts. Consequently, the stage XIV-2 blastoderm may become intolerant of the injected cells. Our results suggest that it is possible to obtain chimeras that have chimerism limited to a particular germ layer and region by varying the stage of donor cell injection. Injected quail cells contributed to endodermal tissues and primordial germ cells regardless of the injection site. The quail-chick blastodermal chimeras could be useful in the production of a transgenic chicken and in the investigation of immunological tolerance.     

Effects of alterations in the immunocompetent status of Mus musculus females on the survival of transferred Mus caroli embryos

The role of the immune system in promoting the midterm death of Mus caroli embryos transferred to the Mus musculus uterus was studied in vivo by transferring M. caroli blastocysts to recipients with altered immune status. Transfers of embryos to chimaeric mothers (Mus musculus in equilibrium Mus caroli), which were expected to be tolerant of species antigens, resulted in survival of M. musculus embryos but death of M. caroli embryos. The preferential survival of M. musculus embryos was explained by showing that M. musculus embryos can survive in the M. caroli uterus. Transfers to T cell-deficient mice of genotype nu/nu and to NK cell-deficient mice of genotype bg/bg as well as treatment of normal transfer recipients with Cyclosporin A or anti-Ia antiserum failed to prolong survival. However, immunization of recipients with M. caroli lymphocytes promoted more rapid and uniform failure of the interspecies pregnancy. Cytotoxic cells were detected in the resorbing embryos on Day 10.5 in immune pregnancies and on Day 12.5 in non-immune pregnancies and these cells were promiscuous in their pattern of lysis, showing equal reactivity against M. caroli, transfer recipient and 3rd party target cells. These experiments show that failure of M. caroli embryos in the M. musculus uterus is complex, but probably does not involve responses by classical cytotoxic T lymphocyte or natural killer cell pathways. Participation of the immune system in the resorption process, however, is confirmed and is associated with generation of promiscuous cytolytic cells.     

Effect of long-term selection for early postnatal growth rate on survival and prenatal development of transferred mouse embryos

Reciprocal embryo transfer procedures were performed among mouse selection lines to examine prenatal maternal effects on survival and development of transferred embryos. Mice were from generations 28 and 29 of an experiment to select for (i) increased body weight again from 0 to 10 days (E+); (ii) decreased body weight gain from 0 to 10 days (E-); or (iii) a randomly bred control line (C). A total of 118 embryo transfer procedures performed 12 h after conception resulted in 983 progeny born to 89 litters. There was a 39% overall embryo survival rate and 75% overall pregnancy success rate. Response to superovulation and oestrous synchronization was significantly lower (P < 0.01) in the E+ line. E+ individuals that did superovulate produced an average of 37 oocytes per flush, which was significantly higher than in the control line mice (29 oocytes per flush; P < 0.01). The ability to complete pregnancy successfully was not influenced by uterine environment or embryo-uterine interaction. In contrast, embryo survival in successful pregnancies was significantly affected by uterine environment. There were large maternal effects for body weight and tail length at birth; E+ recipients produced pups that were significantly larger than E- recipient pups (P < 0.01), which in turn were significantly larger than pups gestated by control recipients (P < 0.01).     

3H-uridine incorporation in early porcine embryos.

The present study investigated the ontogeny of 3H-uridine incorporation into RNA as a measure for RNA synthesis in preimplantation porcine embryos from the two-cell stage up to the stage of the newly hatched blastocyst. A total of 568 embryos were cultured in vitro for 3 hr in medium (KRB plus lamb serum) containing 9 microM 3H-uridine. After disruption of cell membranes, RNA was isolated on DEAE cellulose filters, and the radioactivity was taken as a measure for the rate of RNA synthesis. No RNA synthesis was detected at the two-cell stage. From the four-cell to the morula stage, 3H-uridine incorporation per embryo increased about ninefold (P less than 0.001); in blastocyst stages, the increase between developmental stages was not statistically significant. Hatched blastocysts had the highest genomic activity. On a per cell basis, 3H-uridine incorporation was not different from the four-cell stage up to the zona pellucida-intact blastocyst and amounted to 0.29-0.37 fmol 3H-uridine incorporation/cell/3 hr. In hatched blastocysts, 3H-uridine incorporation per blastomere was increased (P less than 0.01 compared with younger stages) and amounted to 0.86 fmol 3H-uridine incorporation/cell/3 hr. It is concluded that 1) the rate of uridine incorporation depends on the cell stage in zona pellucida-intact porcine embryos and 2) uridine incorporation per blastomere is significantly increased in hatched blastocysts compared with earlier stages.     

Influence of cell cycle stage of the donor nucleus on development of nuclear transplant rabbit embryos.

We evaluated the influence of the stage of the cell cycle of the donor nucleus on development in vitro of nuclear transplant rabbit embryos. The developmental potential of nuclei in early, mid-, and late stages of the cell cycle was determined. Duration of the G1 phase in early embryos was determined, and a procedure for reversibly synchronizing donor embryos in the G1 phase was developed. In addition, the extent of development in vitro of nuclear transplant embryos with donor nuclei synchronized in the G1 phase was evaluated. Development to blastocysts was greatly affected by the stage of the cycle of the donor nucleus. Use of early-stage nuclei led to 59% nuclear transplant blastocysts, whereas 32% and 3% were obtained with mid- and late-stage nuclear donors, respectively (p less than 0.001). The short duration of the G1 phase in 16- and 32-cell-stage embryos (approximately 30 min) necessitated a procedure for synchronizing blastomeres in the G1 phase. This entailed, first, a 10-h incubation in 0.5 micrograms/ml colcemid to arrest embryos in metaphase. After release from colcemid, embryos were allowed to cleave in 0.1 microgram/ml of the DNA synthesis inhibitor, aphidicolin, and remained blocked at the G1/S transition. This treatment was reversible, as assessed by the resumption of DNA synthesis, cleavage rate, and development to blastocysts of treated embryos. The beneficial effect of using early-stage donor blastomeres was confirmed by the enhanced rate of development of manipulated embryos to blastocysts with donor nuclei in the G1 phase (71%), as opposed to the late S phase (15%, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)