Kehuan Lu

Development of bovine embryos after in vitro fertilization of oocytes with flow cytometrically sorted, stained and unsorted sperm from different bulls

The objective of this study was to determine the effect of staining bovine sperm, with or without flow cytometry, on in vitro fertilization of bovine oocytes and blastocyst development. Bovine oocytes (n=4273) were fertilized with frozen-thawed sperm from three bulls that was: stained with Hoechst 33342 and sorted (into X- or Y-chromosome-bearing sperm) with flow cytometry; stained but not sorted; and not stained or sorted (Control). Oocytes, aspirated from slaughterhouse ovaries, were matured in TCM199 (supplemented with 10% fetal calf serum and 15 ng FSH, 1.0 microg LH, 1.0 microg E2/ml) for 22-24h at 39 degrees C in 5% CO(2) in air with maximum humidity. Presumptive zygotes were removed from culture and placed in chemically defined medium (CDM-1) 6-7h after insemination and cultured for 65-66h. Embryos that had cleaved by 72h post-insemination were cultured an additional 96h in CDM-2 containing 0.12 IU insulin/ml. Cleavage and blastocyst rates per oocyte inseminated were recorded on Day 3 and Days 7-8 after insemination, respectively. There was no significant difference in blastocyst rate among the three types of sperm; however, cleavage rates with stained and sorted sperm (53.1%) and unsorted, stained sperm (59.9%) were lower (P<0.05) than Control sperm (69.7%). Furthermore, there were significant differences due to semen from different bulls in cleavage and blastocyst rates.

Membrane progestin receptor beta (mPR-β): A protein related to cumulus expansion that is involved in in vitro maturation of pig cumulus–oocyte complexes

A new group of putative membrane receptors have now been isolated from fish and other vertebrates, including human. These proteins are classified into three groups known as membrane progestin receptor alpha, beta and gamma (mPR-alpha, -beta and -gamma). In the present study we have investigated the role of mPR-beta in regulating in vitro maturation (IVM) of pig cumulus-oocyte complexes (COCs). RT-PCR and Western blot analysis indicated that COCs contain transcripts and proteins for mPR-beta. The levels of both transcripts and proteins increased between 0 and 20h IVM, but then decreased between 20 and 44h. The luteinizing hormone (LH) and follicle-stimulating hormone (FSH) did not affect mPR-beta expression during IVM. Immunofluorescence analysis indicated that the mPR-beta was localized in the plasma membrane of cumulus cell. However, in mouse embryonic fibroblasts (MEFs), mPR-beta was detected at the endoplasmic reticulum (ER) rather than the plasma membrane. Cumulus expansion was impaired significantly (P<0.05) when COCs were incubated in maturation medium containing 10% (v/v) anti-mPR-beta serum during IVM. Bioinformatics analysis predicted that mPR-beta had an ER retention motif and an endocytosis internalization motif. These results suggest that the mPR-beta is a molecule related to cumulus expansion and it might function by regulation of exocytosis. In conclusion, this is the first description of the expression patterns and subcellular localization of mPR-beta in COCs and might shed light on the function of the protein.

In vitro embryo production in buffalo (Bubalus bubalis) using sexed sperm and oocytes from ovum pick up

The objective was to explore the use of sexed sperm and OPU-derived oocytes in an IVP system to produce sex-preselected bubaline embryos. Oocytes were recovered from 20 fertile Murrah and Nili-Ravi buffalo cows by repeated (twice weekly) ultrasound-guided transvaginal ovum pick up (OPU), or by aspiration of abbatoir-derived bubaline ovaries, and subjected to IVF, using frozen-thawed sexed or unsexed bubaline semen. On average, 4.6 oocytes were retrieved per buffalo per session (70.9% were Grades A or B). Following IVF with sexed sperm, oocytes derived from OPU had similar developmental competence as those from abattoir-derived ovaries, in terms of cleavage rate (57.6 vs. 50.4%, P=0.357) and blastocyst development rate (16.0 vs. 23.9%, P=0.237). Furthermore, using frozen-thawed sexed versus unsexed semen did not affect rates of cleavage (50.5 vs. 50.9%, P=0.978) or blastocyst development (15.3 vs. 19.1%, P=0.291) after IVF using OPU-derived oocytes. Of the embryos produced in an OPU-IVP system, 9 of 34 sexed fresh embryos (26.5%) and 5 of 43 sexed frozen embryos (11.6%) transferred to recipients established pregnancies, whereas 7 of 26 unsexed fresh embryos (26.9%) and 6 out of 39 unsexed frozen embryos (15.4%) transferred to recipients established pregnancies. Eleven sex-preselected buffalo calves (10 females and one male) and 10 sexed buffalo calves (six females and four males) were born following embryo transfer. In the present study, OPU, sperm sexing technology, IVP, and embryo transfer, were used to produce sex-preselected buffalo calves. This study provided proof of concept for further research and wider field application of these technologies in buffalo.

Cloned Guangxi Bama minipig (Sus scrofa) and its offspring have normal reproductive performance.

Xenotransplantation is a rapidly expanding field of research, and cloned miniature pigs are considered to be good model animals for its development. Although many animal species have been cloned, the success rate is very low, especially in the pig. To optimize the protocols for somatic cell nuclear transfer in the Guangxi Bama minipig, the relationship between cell cycle synchronization and nuclear histone acetylation levels were investigated. The results showed that the cells were efficiently synchronized by either serum starvation or contact inhibition. The level of nuclear histone acetylation in G0/G1 donor cells had similar variation trends in serum starvation and contact inhibition groups. When the synchronized donor cells were introduced into the enucleated oocytes, 8.8% (serum starvation group) or 9.7% (contact inhibition group) of the reconstructed embryos developed to blastocysts. After embryo transfer, one healthy male Guangxi Bama minipig was obtained. To evaluate the fertility of the cloned pig and its offspring, a series of mating experiments were done. Ninety-eight F1 generation crossbred piglets were born, of which 93 piglets survived. Also, the F1 pigs gave birth to 22 F2 generation piglets, of which 14 piglets survived. In conclusion, a Guangxi Bama minipig was successfully cloned from cultured newborn male gonad fibroblast cells, and the cloned minipig and its offspring had normal fertility.

Sex-preselected buffalo (Bubalus bubalis) calves derived from artificial insemination with sexed sperm

Flow cytometry sorting of X- and Y-chromosome bearing sperm has been emerging as a promising technology to alter the sex ratio in progenies of mammals in the recent years. The objective of this study was to evaluate the efficiency of AI by using the sexed sperm to produce sex-preselected calves in buffalo species. A total of 43 buffalo cows were inseminated with X-sorted sperm, 30 of which were confirmed pregnant 3 mo following AI. In terms of conception rate, significant difference was observed between AI with sexed sperm derived from different bulls (P<0.05), but not between sexed and non-sexed sperm (P>0.05), nor between heifers and parous buffalo cows (P>0.05). A total of 29 sex-preselected calves, 24 females and 5 males, developed to term and were viable on delivery. Results of this study indicate the feasibility of the application of the sexing technology to accelerate the genetic improvement in swamp buffalo.

Fibroblasts from the new-born male testicle of Guangxi Bama mini-pig (Sus scrofa) can support nuclear transferred embryo development in vitro.

Miniature pigs are valuable for research in xenotransplantation and as models for investigating human diseases. Although many mammalian species have been cloned, the success rates have been very low, especially in the pig. In the present study, an attempt was made to optimize somatic cell nuclear transfer (SCNT) protocols for use in the production of the Guangxi Bama mini-pig. Firstly, mini-pig fibroblast cells from a new-born Guangxi Bama piglet were isolated and cultured. Cell type was identified by fluorescence immunocytochemistry (ICC); the cells expressed cimentin, but not cytoceratin and follicular stimulation hormone receptor (FSHR). Secondly, the optimal cell cycle synchronization protocol for treating fibroblast cells from the newborn piglets testicle was investigated by contact inhibition and serum starvation. When fibroblast cells were treated by contact inhibition, a higher fusion (66.0% vs. 58.3%, p > 0.05) and blastocyst production (20.8% vs. 15.1, p > 0.05) rates were obtained than with serum starvation. Thirdly, to examine the ability of old cells to be morphologically remodelled after activation, testicular fibroblasts (passage 10-14) were introduced into enucleated oocytes; enlarged nuclei were formed in most of the reconstructed embryos at 6 h and enlarged nuclei or distinct pseudopronuclei were formed in nearly all the reconstructed embryos at 12 h. The old donor cell could be morphologically remodelled correctly and was competent to support embryo development to the blastocyst in vitro. Fourthly, the in vitro development potential of the cloned embryos was investigated using two types of donor cell: ear fibroblasts and low or high passage testicular fibroblasts. The rate of fusion was highest using low passage testicle fibroblasts (84.5% vs. 69.8% and 80.0%, p < 0.05), as was development to the blastocyst stage (14.6% vs. 7.7% and 6.3%, p < 0.05). Finally, the effect of phytohaemagglutinin (PHA) on parthenogenetic and cloned embryo development was examined. The PHA had no significant effect on the parthenogenetic embryos, but cloned embryo development to the blastocyst stage was significantly increased by PHA (10 microg/ml), (13.4% vs. 5.6% and 5.6%, p < 0.05).

In vitro development of porcine transgenic nuclear-transferred embryos derived from newborn Guangxi Bama mini-pig kidney fibroblasts

Porcine transgenic cloning has potential applications for improving production traits and for biomedical research purposes. To produce a transgenic clone, kidney fibroblasts from a newborn Guangxi Bama mini-pig were isolated, cultured, and then transfected with red and green fluorescent protein genes using lipofectamine for nuclear transfer. The results of the present study show that the kidney fibroblasts exhibited excellent proliferative capacity and clone-like morphology, and were adequate for generation of somatic cell nuclear transfer (SCNT)-derived embryos, which was confirmed by their cleavage activity and blastocyst formation rate of 70.3% and 7.9%, respectively. Cells transfected with red fluorescent protein genes could be passed more than 35 times. Transgenic embryos cloned with fluorescent or blind enucleation methods were not significantly different with respect to cleavage rates (92.5% vs. 86.8%, p > 0.05) and blastocyst-morula rates (26.9% vs. 34.0%, p > 0.05), but were significantly different with respect to blastocyst rates (3.0% vs. 13.2%, p < 0.05). Cleavage (75.3%, 78.5% vs. 78.0%, p > 0.05), blastocyst (14.1%, 16.1% vs. 23.1%, p > 0.05) and morula/blastocyst rates (43.5%, 47.0% vs. 57.6%, p > 0.05) were not significantly different between the groups of transgenic cloned embryos, cloned embryos, and parthenogenetic embryos. This indicates that long-time screening by G418 caused no significant damage to kidney fibroblasts. Thus, kidney fibroblasts represent a promising new source for transgenic SCNT, and this work lays the foundation for the production of genetically transformed cloned Guangxi Bama mini-pigs.

RNA-Seq Profiling of Intact and Enucleated Oocyte SCNT Embryos Reveals the Role of Pig Oocyte Nucleus in Somatic Reprogramming

The specific molecular mechanisms involved in somatic reprogramming remain unidentified. Removal of the oocyte genome is one of the primary causes of developmental failure in cloned embryos, whereas intact oocyte shows stronger reprogramming capability than enucleated oocyte. To identify the reason for the low efficiency of cloning and elucidate the mechanisms involved in somatic reprogramming by the oocyte nucleus, we injected pig cumulus cells into 539 intact MII oocytes and 461 enucleated MII oocytes. Following activation, 260 polyploidy embryos developed to the blastocyst stage whereas only 93 traditionally cloned embryos (48.2% vs. 20.2%, P < 0.01) reached blastocyst stage. Blastocysts generated from intact oocytes also had more cells than those generated from enucleated oocytes (60.70 vs. 46.65, P < 0.01). To identify the genes that contribute to this phenomenon, two early embryos in 2-cell and 4-cell stages were collected for single-cell RNA sequencing. The two kinds of embryos were found to have dramatically different transcriptome profiles. Intact oocyte nuclear transfer embryos showed 1,738 transcripts that were up-regulated relative to enucleated cloned embryos at the 2-cell stage and 728 transcripts that were down-regulated (|log2Ratio| ≥ 5). They showed 2,941 transcripts that were up-regulated during the 4-cell stage and 1,682 that were down-regulated (|log2Ratio| ≥ 5). The most significantly enriched gene ontology categories were those involved in the regulation of binding, catalytic activity, and molecular transducer activity. Other genes that were notably up-regulated and expressed in intact oocyte nuclear transfer embryos were metabolic process. This study provides a comprehensive profile of the differences in gene expression between intact oocyte nuclear transfer embryos and traditional nuclear transfer embryos. This work thus paves the way for further research on the mechanisms underlying somatic reprogramming by oocytes.

Cholesterol induces proliferation of chicken primordial germ cells

Primordial germ cells (PGCs) are the precursors of sperm and eggs and may serve as suitable cells for use in research in developmental biology and transgenic animals. However, the long-term propagation of PGCs in vitro has so far been plagued by the loss of their germ cell characteristics. This is largely because of the scarcity of knowledge concerning cell division and proliferation in these cells and the poor optimization of the culture medium. The sonic hedgehog (SHH) signaling pathway is involved in proliferation of many types of cells, but little is known about its role in chicken PGCs. The results of the current study indicate that the proliferation of chicken PGCs increases significantly when cholesterol, a molecule that facilitates the trafficking of HH ligands, is supplemented in the culture medium. This effect was attenuated when an SHH antagonist, cyclopamine was added, suggesting the involvement of SHH signaling in this process. The characterization of PGCs treated with cholesterol has shown that these cells express germ-cell-related markers and retain their capability to colonize the embryonic gonad after re-introduction to vasculature of stage-15 HH embryos, indicating that proliferation of PGCs induced by cholesterol does not alter the germ cell characteristics of these cells.

Flow cytometric and near-infrared Raman spectroscopic investigation of quality in stained, sorted, and frozen-thawed buffalo sperm

Flow cytometry and Laser Tweezers Raman spectroscopy have been used to investigate Nili-Ravi buffalo (Bubalus bubalis) sperm from different samples (fresh, stained, sorted and frozen-thawed) of the flow-sorting process to optimize sperm sex sorting procedures. During the sorting and freezing-thawing processes, the two detection methods both indicated there were differences in mitochondrial activity and membrane integrity. Moreover, a dispersive-type NIR (Near Infrared Reflection) use of the Raman system resulted in the ability to detect a variety of sperm components, including relative DNA, lipid, carbohydrates and protein contents. The use of the Raman system allowed for PCA (principal components analysis) and DFA (discriminant function analysis) of fresh, stained, sorted and frozen-thawed sperm. The methodology, therefore, allows for distinguishing sperm from different samples (fresh, stained, sorted and frozen-thawed), and demonstrated the great discriminative power of ANN (artificial neural network) classification models for the differentiating sperm from different phases of the flow-sorting process. In conclusion, the damage induced by sperm sorting and freezing-thawing procedures can be quantified, and in the present research it is demonstrated that Raman spectroscopy is a valuable technology for assessing sperm quality.