Contractions were observed in blastocysts developed from 2-cell embryos collected from mice of different ages, in order to evaluate the influence of aging of dams on the quality of blastocysts, i.e. their ability to hatch, based on contractions. The zona pellucida of cultured blastocysts collected from mice of different ages was ultrastructurally observed to examine the relationship between hatching and contractions or zonae pellucidae. Although the rate of development from 2-cell embryos to blastocysts did not differ by dams’ ages, the hatching rate was significantly lower in the blastocysts from mice aged 30 days (31.3%) and 360 days (25.0%) than in those from mice aged 60 to 90 days (53.8%). The number of weak contractions was significantly fewer in the blastocysts from mice aged 360 days (4.23 times), compared with those in the blastocysts from mice aged 30 days (5.53 times) and 60 to 90 days (5.50 times). The number of strong contractions was significantly larger in the blastocysts from mice aged 30 days (2.03 times) than in those from mice aged 60 to 90 days (1.37 times) and 360 days (1.50 times). The zona pellucida showed a dense and homogeneous fiber-network in the blastocysts from mice aged 30 and 60 to 90 days, but showed a rough and irregular fiber-network in the blastocysts from mice aged 360 days. The thickness of zonae pellucidae significantly differed among blastocysts collected from mice of different ages, and the zona pellucida was particularly thin in the blastocysts from mice aged 360 days (0.79 μm). From these results, it was confirmed that 2-cell embryos collected from young or aged mice can develop to blastocysts at a high rate, but their ability to hatch is poor. A low hatching ability is thought to be due to frequent strong contractions observed in the blastocysts from mice aged 30 days, and to a small number of weak contractions seen in the blastocysts from mice aged 360 days. In addition, it was inferred that blastocyst hatching is closely related to the number of weak contractions, rather than the structure or thickness of the zona pellucida.
Uterine contraction in sows during the estrous and puerperal periods, and in sows with multiple follicular cysts (MFC), were measured using a modified balloon method. On the basis of the graphs thus obtained, the frequency of uterine contractions, their average intensity and Planimeter (Pl) values were compared. In the puerperal period, contraction waves were completely different during suckling and not suckling period. Suckling stimuli affected contraction frequency and Pl values significantly (P<0.01). Immediately after parturition, the frequency of uterine contractions, their average intensity and Pl values tended to be high and decreased with the passage of time. For sows with weak contractions that required assistance in farrowing, intensity and Pl values were significantly low (P<0.05). For sows with small multiple follicular cysts (SMFC) and normal estrus, clear uterotonic activity was observed. Uterine contractile frequencies in sows with SMFC were slightly less frequent than in estrous sows, but greater contractions were observed, showing higher Pl values (P<0.05). These contractions also lasted a long duration. Moreover, intermittent uterine contractions were observed during the puerperal stage, whereas persistent uterotonic activity in SMFC and estrous sows.
The technology of mouse embryonic stem (ES) cells is clearly proving to be a very successful method for producing transgenic mice. Although there has been considerable efforts to establish ES cell lines from other animals, most of all lines obtained, so far, have been derived from mice. Here, we examined effects of allogenetic and xenogenetic feeders on the establishment of ES cells from mice and rats. We first used three types of feeder cells on the establishment of rat ES cells; mouse primary embryonic fibroblsts (M-PEF), rat primary embryonic fibroblasts (R-PEF) and a mouse embryonic fibroblast cell line, SL10. We could gain some rat ES cell lines only on R-PEF. RES-DA1 cell line from DA rat strain, has the same morphology as mouse ES cells, alkaline phosphatase activity and the 4C9 antigen. We, moreover, examined effects of M-PEF and R-PEF feeders on the establishment of mouse ES cells in C57BL/6 mice. The mouse ES cell line could be established only on M-PEF feeders. Both rat and mouse ES cell lines had pluripotenciality. These results suggested that the establishment of ES cell lines is more effective on allogenetic feeder cells than on xenogenetic cells.
The aims of this study were to determine profiles of plasma immunoreactive β-endorphin (ir-β-EP) during peripartum period in dairy cows, and to examine whether dystocia, retained placenta or body condition affects the plasma ir-β-EP level. Blood samples were collected from 21 Holstein-Friesian cows from day 180 of pregnancy until first ovulation. The cows were classified into two groups based on calving conditions and ir-β-EP patterns at parturition. Seven out of the 21 cows had normal parturition and 14 cows had abnormal parturition (dystocia and/or retained placenta). The proportion of cows which had ir-β-EP elevation at parturition tended to be higher in cows with abnormal parturition (11/14; 79%) than in those with normal parturition (3/7; 42%). Plasma cortisol level increased at parturition both in cows with ir-β-EP elevation and in cows without ir-β-EP elevation at parturition. Postpartum ir-β-EP concentrations in plasma were similar between cows with normal parturition and those with abnormal parturition, and between cows which had lost their body condition score (BCS) after calving and those which had maintained their BCS. In conclusion, abnormal parturition may affect plasma ir-β-EP level at parturition but not postpartum period.
Transrectal ovarian ultrasonography was performed to examine follicular and luteal dynamics during the estrous cycle in four adult female Shiba goats. Detectable follicles and the corpora lutea in both ovaries were monitored daily for 44 days using a B-mode scanner with a 7.5 MHz transrectal transducer. Blood samples were collected by jugular venipuncture at the time of ultrasonography and analyzed for estradiol-17β and progesterone by radioimmunoassay to monitor ovarian activity. Three to six follicles, whose diameters ranged from 1 to 7 mm, were observed throughout the estrous cycle; a few follicles grew to more than 5 mm in diameter and most of them atrophied during the luteal phase. Ovulatory follicles appeared from 5 days before ovulation coinciding with the start of the regression of corpora lutea, and then grew rapidly into 5.5 ± l.0 mm (mean ± SD) in diameter in parallel with the increase in the plasma concentration of estradiol-17β. The corpora lutea, 6.1 ± 1.3 mm in major axis, were detected from 3.0 ± 1.6 days after ovulation and stopped growing on 6 to 8 days after ovulation, with a major axis of 8.1 ± 2.2 mm. The mean length of the major axis of fully developed corpora lutea remained constant until 14 days after ovulation and then decreased rapidly to 5.2 ± 1.7 mm on the day of the next ovulation. In the early and functional luteal phase, there was a positive correlation between mean length of the total major axis of corpora lutea and mean plasma concentration of progesterone, whereas in the late luteal phase, the mean length of the total major axis of corpora lutea decreased slower than the progesterone levels. The present study demonstrated the ovarian follicular and luteal dynamics throughout the estrous cycle, indicating that ultrasonography is a reliable means for monitoring ovarian activity in Shiba goats.
To clarify the role of the extrahypothalamic input to the preoptic area (POA)-medial basal hypothalamus (MBH) on spontaneous ovulation, the dorsal cut of the POA-MBH was performed on various days and ova were counted in female rats. An anterior half-circle cut (anterior dorsal cut; ADC) was performed at the dorsal of the POA on the day of proestrus. As a result, ovulation was seen on the day of estrus when ADC was performed in the evening (18:00-19:30 h) but did not occur when performed in the morning (10:00-11:30 h). Furthermore, the suppressive effect of ADC was observed, even when cut was performed in the evening 2-5 days before estrus. These results suggest that the dorsal input to the POA-MBH plays an important role in ovulation-triggering mechanisms. In addition, about 7 days after the ADC, regular estrous cycle and normal ovulation were seen. Furthermore, destruction of the medial or bilateral lateral septum was done in the morning of proestrus, to investigate the role of gonadotropin-releasing hormone (GnRH) neurons in these areas. Lesions in the septal area had no effect, suggesting that the inhibitory effect on ovulation of the ADC is not due to interruption of the fibers of the septum including GnRH neurons.
The protein compositions of uterine secretions in young and aged hamsters during early pregnancy were studied by polyacrylamide slab-gel electrophoresis. Uterine luminal fluids were collected by flushing with a small amount of saline. A broad area of very rapidly migrating proteins, which was undetectable in plasma samples, appeared in the uterine fluids on Days 4 and 5 of pregnancy, which is when implantation occurs in this species. More protein bands were noted in this region in aged females in contrast to those of young females. Further, in the uterine fluids of aged hamsters, a post-albumin protein band disappeared at estrus and on Day 1 and the protein compositions in the post-transferrin region differed between the young and aged females. The qualitative changes in the protein content of the uterine fluids may account for the high rate of embryonic loss in aged hamsters.
This study was conducted to examine the possibility of performing nuclear transplantation using parthenogenetically derived embryos as donor nuclei (karyoplasts) to support embryonic development after nuclear transplantation. The recipient oocytes (cytoplasts) were enucleated and then activated after maturation, and the donor blastomeres were obtained from 8- to 16-cell stage embryos of parthenogenetically activated and fertilized embryos. The fusion, cleavage and blastocyst rates of reconstructed embryos that received fresh parthenogenetic blastomeres were 90%, 96% and 13%, respectively. The in vitro development of these reconstructed embryos was not significantly different (P>0.05) from the reconstructed embryos, which received IVF-derived blastomeres (94%, 95% and 21%, for fusion, cleavage and blastocyst rates, respectively). In contrast, these values decreased, when the reconstructed embryos received frozen-thawed parthenogenetic blastomeres (74%, 93% and 0%, for fusion, cleavage and blastocyst rates, respectively). Moreover, the development of reconstructed embryos that received parthenogenetic blastomeres showed some fragmentation at the compaction stage, unincorporation of blastomeres and multiple cavities (blastocoeles) in the blastocyst stage. This preliminary study has demonstrated the developmental potency of parthenogenetic bovine embryo nuclei, which could develop to the blastocyst stage when transplanted into enucleated oocytes.
Nuclear magnetic resonance (NMR) microscopy is a magnetic resonance imaging technique with an enhanced spatial resolution. In this study, fresh ovaries of mature rats immersed in phosphate buffered saline (PBS) were examined by NMR microscopy. Histological sections corresponding to NMR microimages were prepared, and components in ovaries were identified in microimages by comparison with light microscopic images. Signal intensities of antral follicles, corpora lutea (CL), stroma, adjacent fat tissue and PBS were measured in images with different echo times (TE) and analyzed. As TE increased, signal intensities decreased in CL and connective tissue including the stroma, while those in PBS and follicles changed less. Fat showed significantly high signals compared with other components at each TE. Stroma showed significantly low signals than other components except PBS and follicles at TE of 5 msec. There were no significant differences among CL, follicles and PBS at TE of 5 msec. At TE of 20 msec, however, there were significant differences in signal intensities between each components in the rank order fat > PBS > follicles > CL > stroma. Significant regression of signal intensity was seen on TE in follicles, CL and stroma. NMR microscopy is considered to be a useful tool to investigate the physiological function of rat ovaries.