Insights into restrictive cardiomyopathy from clinical and animal studies.

Cardiomyopathies are diseases that primarily affect the myocardium, leading to serious cardiac dysfunction and heart failure. Out of the three major categories of cardiomyopathies (hypertrophic, dilated and restrictive), restrictive cardiomyopathy (RCM) is less common and also the least studied. However, the prognosis for RCM is poor as some patients dying in their childhood. The molecular mechanisms behind the disease development and progression are not very clear and the treatment of RCM is very difficult and often ineffective. In this article, we reviewed the recent progress in RCM research from the clinical studies and the translational studies done on diseased transgenic animal models. This will help for a better understanding of the mechanisms underlying the etiology and development of RCM and for the design of better treatments for the disease.

Full term development of normal mice after transfer of IVF embryos derived from oocytes stored at room temperature for 1 day.

Early studies have shown that some mouse cumulus-oocyte complexes (COCs) stored at room temperature for 24 h still retained full developmental potential. In this study, we stored denuded mouse oocytes (DOs) at room temperature (25 degrees C) for 24 h and activated these oocytes with 10 mM SrCl2 or fertilized the oocytes by IVF. We found that nearly half of the DOs stored at room temperature for 1 day can be fertilized normally by IVF and that two foster mothers gave birth to seven pups. Embryos from stored oocytes were cultured in CZB medium with or without 1 microg/ml 17beta-estradiol (E2). The numbers of embryo that developed to morula/blastocyst stage after parthenogenetic activation and IVF were significantly increased when E2 was added to the culture (p<0.05). These results suggest that E2 might improve mouse embryo development in vitro. The birth of seven agouti pups and their healthy growth indicated that the storage of DOs at room temperature for 1 day may be a practical procedure for mammalian reproduction.

Development of normal mice after microinjection of round spermatids into oocytes stored at room temperature for one day.

Early studies have shown that some mouse cumulus-oocyte complexes (COCs) stored at room temperature for 24 hr still retained full developmental potential. In this study, we stored mouse COCs and denuded oocytes (DOs) at room temperature for 24 hr and activated these oocytes with 10 mM SrCl(2) or injected the oocytes with round spermatids. We found that DOs were better than COCs when stored at room temperature for 1 day and more normal oocytes were obtained when COCs were stored in more H-CZB medium at room temperature for 1 day. The rates of normal oocytes were significantly different after preservation with three schemes (90.01%, 55.81%, and 86.70%, P < 0.05). Our results also indicated that oocytes stored at room temperature for 1 day were fertilized normally (extrusion of the second polar body and formation of male and female pronuclei [PN]) after microinjection of round spermatid nuclei, and that the existence of cumulus cells (CCs) during oocyte storage did not significantly influence the early cleavage but had a detrimental effect on later embryo development and full-term development. After fertilization, most embryos developed to two-cell stage after being cultured for 24 hr, and the development rates of four- to eight-cell embryos between two experiments were similar. However, the rates of morula/blastocyst formation were significantly different (47.44% and 26.27%, respectively, P < 0.05). The birth of four healthy pups from stored DOs indicated that the storage of DOs at room temperature for 1 day might become a practical procedure in mammalian reproduction.

Time course of meiotic progression after transferring primary spermatocyte into ooplasm at different stages.

This study attempted to investigate the time course of meiotic progression after transferring primary spermatocyte (PS) into ooplasm at different maturing stages. In present experiments, PSs were introduced into maturing ooplasts or oocytes by electrofusion. Higher fusion rate was obtained by phytohemagglutinin (PHA) agglutination than by perivitelline space (PVS) insertion. When the ooplasms prepared at 0, 2, 5, and 8.5 hr of in vitro maturation (IVM) were used as recipients and PSs were used as donors, the reconstructed cells extruded the first polar body (PB1) approximately 8.5, 7, 5.5, and 3 hr after electrofusion, respectively. Especially, when ooplasm cultured for 8.5 hr in vitro after GV removal was fused with PS, the PB1 was emitted 7-11 hr after electrofusion. Additionally, the PB1 extrusions of GV and pro-MI oocytes fertilized with PSs were 2.5 hr earlier than control oocytes. The results suggest that (1) PSs undergo the first meiosis in different time courses when introduced into ooplasm at different maturing stages; (2) GV material plays an important role in determining the timing of PB1 extrusion; and (3) first meiotic division of GV and pro-MI oocytes can be accelerated by introducing PS.

Interspecies nuclear transfer of Tibetan antelope using caprine oocyte as recipient.

Interspecies nuclear transfer is an invalulable tool for studying nucleus-cytoplasm interactions; and at the same time, it provides a possible alternative to clone endangered animals whose oocytes are difficult to obtain. In the present study, we investigated the possibility of cloning Tibetan antelope embryos using abattoir-derived caprine oocytes as recipients. Effects of culture conditions, enucleation timing, and donor cell passages on the in vitro development of Tibetan antelope-goat cloned embryos were studied. Maternal to zygotic transition timing of interspecies Tibetan antelope embryos was also investigated using two types of cloned embryos, Tibetan antelope-rabbit and Tibetan antelope-goat embryos. Our results indicate that: (1) goat oocyte is able to reprogram somatic cells of different genus and supports development to blastocyst in vitro. (2) Coculture system supported the development of Tibetan antelope-goat embryos to blastocyst rate stage (4.0%), while CR1aa alone did not. (3) When MII phase enucleated caprine cytoplast and TII phase enucleated caprine cytoplast were used as recipients, the fusion rate and blastocyst rate of hybrid embryos were not statistically different (73.9% vs. 67.4%; 4.0% vs. 1.1%). (4) When donor cells at 3-8 passages were used, 2.9% hybrid embryos developed to blastocysts, while none developed to blastocysts when cells at 10-17 passages were used. (5) There may be a morula-to-blastocyst block for Tibetan antelope-goat, while there may be an 8- to 16-cell block for Tibetan antelope-rabbit embryos.

RNA Interference as a tool to study the function of MAD2 in mouse oocyte meiotic maturation.

Spindle checkpoint proteins control entry into anaphase and chromosome segregation. As a member of spindle checkpoint proteins, MAD2 takes a central role in the regulation of anaphase onset and genome integrity. Here, we used MAD2 siRNA transfection approach to study MAD2 functions during mouse oocyte meiotic maturation in vitro. Real-time PCR and laser scanning confocal microscopy showed that we successfully downregulated MAD2 transcript and protein expression. We further demonstrated that MAD2 downregulation resulted in a shortened duration of meiosis I and meiotic spindle abnormality, suggesting the function of MAD2 in mouse oocyte meiotic maturation. We also showed that MAD2 interference to some extent decreased GVBD rate, but increased apoptosis in mouse oocytes. In conclusion, our study shows that siRNA transfection is an effective tool to study MAD2 functions, and our results provide further evidence for the role of MAD2 as a spindle checkpoint protein in mouse oocytes.

Increased Th1/Th2 (IFN-gamma/IL-4) Cytokine mRNA ratio of rat embryos in the pregnant mouse uterus.

Somatic cell nuclei can be dedifferentiated in ooplasm from another species, and interspecies cloned embryos can be implanted into the uteri of surrogates. However, no full pregnancies have been achieved through interspecific mammalian cloning. Rat blastocysts transferred into mouse uteri provide a unique model for studying the causes of interspecific pregnancy failure. In this study, intraspecific pregnancy (mouse-mouse) and interspecific pregnancy (rat-mouse) models were established. On Day 9 of pregnancy, the fetoplacental units were separated from the uterine implantation sites and the expression of messenger (m)RNA was quantitated by real-time PCR. We compared the mRNA expression levels of type-1 T helper (Th1) and type-2 T helper (Th2) cytokines, interferon-gamma (IFN-gamma), and interleukin-4 (IL-4) in fetoplacental units between intraspecific and interspecific pregnancy groups. The mRNA expression of IFN-gamma in the fetoplacental units of the interspecific pregnancy group was significantly higher than that of the intraspecific pregnancy group (P<0.05). The mRNA expression of IL-4 in the interspecific pregnancy group was significantly lower than that in the intraspecific pregnancy group (P<0.05). We also analyzed the ratio of IFN-gamma/IL-4 mRNA, and an increased IFN-gamma/IL-4 mRNA ratio was observed in the interspecific pregnancy compared with that in the intraspecific pregnancy group. The IFN-gamma and IL-4 mRNA expressions indicate that there is a Th1/Th2 imbalance in the feto-maternal interface of interspecific pregnancies. Bias of Th1 cytokine dominance may be a barrier to reproductive success between species.

NuMA distribution and microtubule configuration in rabbit oocytes and cloned embryos.

The assembly of microtubules and the distribution of NuMA were analyzed in rabbit oocytes and early cloned embryos. Alpha-tubulin was localized around the periphery of the germinal vesicle (GV). After germinal vesicle breakdown (GVBD), multi-arrayed microtubules were found tightly associated with the condensed chromosomes and assembled into spindles. After the enucleated oocyte was fused with a fibroblast, microtubules were observed around the introduced nucleus in most reconstructed embryos and formed a transient spindle 2-4 h post-fusion (hpf). A mass of microtubules surrounded the swollen pseudo-pronucleus 5 hpf and a normal spindle was formed 13 hpf in cloned embryos. NuMAwas detected in the nucleus in germinal vesicle-stage oocytes, and it was concentrated at the spindle poles in both meiotic and mitotic metaphase. In both donor cell nucleus and enucleated oocyte cytoplasm, NuMA was not detected, while NuMA reappeared in pseudo-pronucleus as reconstructed embryo development proceeded. However, no evident NuMA staining was observed in the poles of transient spindle and first mitotic spindle in nuclear transfer eggs. These results indicate that NuMA localization and its spindle pole tethering function are different during rabbit oocyte meiosis and cloned embryo mitosis.

Rabbit oocyte cytoplasm supports development of nuclear transfer embryos derived from the somatic cells of the camel and Tibetan antelope.

This study was designed to examine the ability of rabbit metaphase II oocyte cytoplasm to support the development of interspecies nuclear transfer embryos reconstructed using donor nuclei from different species. Skin fibroblast cells from a camel and Tibetan antelope were used as donor nuclei. As a first step, we investigated the efficiency of different activation protocols by comparing the parthenogenetic development of rabbit oocytes. The protocol that yielded the highest blastocyst rate was used to activate the reconstructed embryos in nuclear transfer experiments. In addition, the effect of donor cell serum starvation on the development of the reconstructed embryo was also examined. More than half of the karyoplast-cytoplast couplets could be fused, and about one third of the reconstructed embryos were capable of completing first cleavage, regardless of the species of donor nuclei. Some of the cleaving reconstructed embryos were even capable of progressing further and developing to the blastocyst stage (1.4-8.7% for the Tibetan antelope and 0-7.5% for the camel, respectively). Our results suggest that the mechanisms regulating early embryo development may be conserved among mammalian species and some factors existing in rabbit oocyte cytoplasm for somatic nucleus reprogramming and dedifferentiation may not be species-specific. Rabbit oocyte cytoplasm can reprogram donor nuclei regardless of the origin of the nucleus and support in vitro development to an advanced stage.

In vitro culture and mtDNA fate of ibex-rabbit nuclear transfer embryos.

Rabbit oocyte can be used as the recipient in interspecies somatic cell nuclear transfer (iSCNT). This work was undertaken in order to study the developmental competence of Capra ibex somatic cells reprogrammed by rabbit oocytes and the fate of mitochondria in iSCNT embryos. Metaphase II (MII) oocytes from superovulated rabbit were used as nuclear recipients. The nuclear donors were Capra ibex somatic cells with different proliferative status: population doubling time (PDL) = 15 +/- 2 (group 1), 35 +/- 2 (group 2), 55 +/- 2 (group 3) and 70 +/- 2 (group 4). Oocytes reconstructed with electrical pulses (2.1kV/cm, 10 micros, 2 times) were activated (1.4kV, 20 micros, 2 times) and then cultured in Medium199 containing 10% fetal bovine serum at 38.5 degrees C, 5% CO2 in air. In groups 1, 2, 3 and 4, the fusion rates were 35.83%, 66.03%, 65.40% and 35.35%, respectively. Similar cleavage rates were observed among the four groups. However, the developmental potential to morula/blastocyst from early nuclear donor embryos (16.42%/10.45%) was significantly higher (p < 0.05) than in terminal donor embryos (9.52%/3.81%). Polymerase chain reaction analysis of the mitochondrial (mt) DNA cytb gene demonstrated that mtDNAs from ibex and rabbit could be detected at various developmental stages before implantation. In conclusion, our results provide some original information about rescuing Capra ibex using the iSCNT technique. These results indicate that: (1) enucleated rabbit oocytes make Capra ibex fibroblast nuclei reprogramme; (2) the proliferative status of donor cells affects the efficiency of iSCNT; and (3) rabbit ooplasm rescues the donor-derived mtDNAs, resulting in mtDNA heteroplasmy before implantation.