MFN2 regulates progesterone biosynthesis and proliferation of granulosa cells during follicle selection in hens.

Follicle selection in hens refers to a biological process that only one small yellow follicle (SYF) is selected daily or near-daily for following hierarchical development (from F5/F6 to F1) until ovulation. MFN2 is a kind of GTPases located on the mitochondrial outer membrane, which plays a crucial role in mitochondrial fusion. This study aimed to elucidate the role of MFN2 in proliferation and progesterone biosynthesis of granulosa cells (GCs) during follicle selection in hens. The results showed that GCs began to produce progesterone (P4) after follicle selection, accompanied with changes from multi-layer with flat cells to single layer with cubic cells. MFN2 was detected in GCs of follicles from SYF to F1. After follicle selection, the expression level of MFN2 in GCs upregulated significantly, accompanied with increases in P4 biosynthesis, ATP production, mitochondrial DNA (mtDNA) copy numbers of granulosa cells. FSH (80 ng/mL) facilitated the effects of P4 biosynthesis and secretion, ATP production, mtDNA copy numbers, cell proliferation and the MFN2 transcription of granulosa cells from F5 (F5G) in vitro. However, FSH treatment did not promote P4 secretion in granulosa cells from SYF (SYFG) in vitro. Meanwhile, we observed that change fold of MFN2 transcription, ATP production, mtDNA copy numbers and cell proliferation rate in F5G after treatment with FSH were greater than those in SYFG. Furthermore, expression levels of MFN2 protein and messenger RNA in F5G were significantly higher than those in SYFG after treatment with FSH. P4 biosynthesis, ATP production, mtDNA copy numbers as well as cell proliferation reduced significantly in F5G with MFN2 knockdown. Oppositely, P4 biosynthesis, ATP production, mtDNA copy numbers and cell proliferation increased significantly in SYFG after the overexpression of MFN2. Our results suggest that the upregulation of MFN2 may be involved in the initiation of P4 biosynthesis, and promotion of GCs proliferation during follicle selection.

Improved the in vitro blastocyst development in oocytes recovered from cyclic buffaloes.

This study examined the impact of cyclicity (with or without cycle corpus luteum; CL) on oocyte quality and embryonic development in buffaloes. We collected oocytes from the ovaries of slaughtered buffaloes (N = 158 cyclic; n = 316 ovaries and N = 177 acyclic; n = 353 ovaries). Blood progesterone concentration and number of oocytes per ovary were higher in cyclic buffaloes. Cyclic buffalo ovaries produce higher oocytes with I + II and fewer III + IV grades. Oocytes from cyclic buffaloes had a higher maturation rate based on cumulus expansion, cleavage rate and embryo development to the 8-cell, morula and blastocyst stages than acyclic buffaloes. In conclusion, oocytes recovered from the ovaries of the cyclic buffaloes showed improved oocyte competence and subsequent in vitro blastocyst development.

Comparative proteomic analysis of seminal plasma exosomes in buffalo with high and low sperm motility.

BACKGROUND: Exosomes are nanosized membranous vesicles secreted by various types of cells, which facilitate intercellular communication by transporting bioactive compounds. Exosomes are abundant in biological fluids including semen, and their protein composition and the potential of seminal plasma exosomes (SPEs) as fertility biomarkers were elucidated in humans, however, little information is available regarding buffalo (Bubalus bubalis). Here, we examined protein correlation between spermatozoa, seminal plasma (SP), and SPEs, and we compared and analyzed protein differences between high-motility (H-motility) and low-motility (L-motility) SPEs in buffalo. RESULTS: SPEs were concentrated and purified by ultracentrifugation combined with sucrose density gradient centrifugation, followed by verification using western blotting, nanoparticle tracking analysis, and transmission electron microscopy. Protein composition in spermatozoa, SP and SPEs, and protein difference in H- and L-motility SPEs were identified by LC-MS/MS proteomic analysis and were functionally analyzed through comprehensive bioinformatics. Many SPEs proteins originated from spermatozoa and SP, and nearly one third were also present in spermatozoa and SP. A series of proteins associated with reproductive processes including sperm capacitation, spermatid differentiation, fertilization, sperm-egg recognition, membrane fusion, and acrosome reaction were integrated in a functional network. Comparative proteomic analyses showed 119 down-regulated and 41 up-regulated proteins in L-motility SPEs, compared with H-motility SPEs. Gene Ontology (GO) enrichment of differentially expressed proteins (DEPs) showed that most differential proteins were located in sperm and vesicles, with activities of hydrolase and metalloproteinase, and were involved in sperm-egg recognition, fertilization, single fertilization, and sperm-zona pellucida binding processes, etc. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that differential proteins were mainly involved in the PPRP signaling pathway, calcium signaling pathway, and cAMP signaling pathway, among others. Furthermore, 6 proteins associated with reproduction were validated by parallel reaction monitoring analysis. CONCLUSION: This study provides a comprehensive description of the seminal plasma exosome proteome and may be of use for further screening of biomarkers associated with male infertility.

Quantitative proteomics analysis of chicken embryos reveals key proteins that affect right gonadal degeneration in females.

In birds, embryonic gonads of females develop in a way different from mammals, with the left one develops into a functional ovary, while the right one degenerates during embryogenesis. Here, we examined the proteomics profiles of the female and male left and right gonads at embryonic day 6.5 (E6.5) with the label free tandem mass spectrometry proteomics technique. The relative protein abundance of the left and right gonads of female and male embryos was determined to identify their differential proteins. Overall, a total of 7726 proteins were identified, of which 79 and 54 proteins were significantly different in female and male right gonads compared with female left gonads and male left gonads respectively. Bioinformatics analysis showed that the proteins DMRT1, ZFPM2, TSHZ3 were potentially associated with the degeneration of the right gonads in female embryos. The proteomics in this study provide clues for further elucidation of the pathways of sex determination, sex differentiation, and right gonadal degeneration in birds.

Proteomic analysis identifies potential markers in small white and small yellow follicle development in chickens.

Extensive knowledge of follicular development is imperative for improving egg production in chickens. The functional role of follicles to produce oocytes (eggs) is well recognised; however, specific markers associated with follicle development have been poorly explored. Therefore, a tandem mass tag based proteomic technique was used to identify the status of the proteome of small white follicles (1-4mm) and small yellow follicles (6-8mm). Analysis of differentially expressed proteins (DEP, Fold Change>1.2, P -value<0.05) demonstrated a total of 92 proteins (n =92), of which 35 (n =35) were upregulated and 57 were downregulated. DEP were further used for gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathways. The GO analysis found that DEP were mainly associated with the RNA metabolic process, cellular component organisation, peptide biosynthetic process and protein folding, thereby suggesting a key role in the follicle development process. Kyoto Encyclopedia of Genes and Genomes enrichment pathway analysis of the DEP substantiated the findings of GO analysis and described that DEP are involved in regulation of the cytoskeleton, carbon metabolism and amino acid biosynthesis. The validation of proteomic data through real-time quantitative polymerase chain reaction suggested HSPA8, HSPA2, SOD1 and FKPB3 as potential markers of small white and small yellow follicle development. This study demonstrates an understanding of proteome dynamics and represents the most comprehensive information on the entire Guangxi Ma chicken follicular proteome.

Effect of addition of l-carnitine to cryopreservation extender on rabbit post-thaw semen parameters, antioxidant capacity, mitochondrial function, apoptosis and ultrastructure changes.

l-Carnitine (LC) is considered to be a natural antioxidant agent that could be used to improve the efficiency of reproduction. However, the precise machinery of the effect of LC supplementation on frozen-thawed rabbit sperm has not been evaluated. Thus, the aim of this study was to evaluate the effect of the addition of LC to a freezing medium on parameters and ultrastructure changes in frozen-thawed rabbit sperm. Rabbit bucks (7 months of age) were involved, and semen was collected using the artificial vagina method. Pooled rabbit semen was cryopreserved in a tris yolk fructose extender without any supplement (LC0, control group) or with LC at levels of 1, 2 or 4 mM (LC1, LC2 and LC4, respectively). The samples were then loaded into 0.25-ml straws and frozen over liquid nitrogen vapours before being plunged into the liquid nitrogen and stored at -196°C until evaluation. Data showed that the addition of LC significantly increased sperm motility, viability and membrane function, while sperm abnormalities decreased (p < .001). Sperm-like apoptosis (early, late and necrosis spermatozoa) was lower in the LC4 group compared with the other groups. l-Carnitine addition significantly enhanced the total antioxidant capacity and superoxide dismutase and glutathione peroxide activities and significantly reduced the protein carbonyl and malondialdehyde levels compared with the control group. Moreover, electron microscopy images demonstrated that LC addition (2 or 4 mM) preserved the acrosome and plasma membrane and protected the ultrastructure integrity of the cryopreserved spermatozoa in relation to the control group. Spermatozoa treated with LC exhibited higher mitochondria membrane potential (MMP) values compared with the control group. We conclude that the addition of LC (4 mM) to the freezing extender enhanced the quality, increased the antioxidant capabilities, preserved the ultrastructure integrity and reduced lipid and protein peroxidation as well as increased MMP activity of frozen-thawed rabbit sperm.

Integrated Analysis of Quantitative Proteome and Transcriptional Profiles Reveals the Dynamic Function of Maternally Expressed Proteins After Parthenogenetic Activation of Buffalo Oocyte.

Maternal-effect genes are especially critical for early embryonic development after fertilization and until massive activation of the embryonic genome occurs. By applying a tandem mass tag (TMT)-labeled quantitative proteomics combined with RNA sequencing approach, the proteome of the buffalo was quantitatively analyzed during parthenogenesis of mature oocytes and the two-cell stage embryo. Of 1908 quantified proteins, 123 differed significantly. The transcriptome was analyzed eight stages (GV, MII, 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst) of Buffalo using the RNA sequencing approach, and a total of 3567 unique genes were identified to be differently expressed between all consecutive stages of pre-implantation development. Validation of proteomics results (TUBB3, CTNNA1, CDH3, MAP2K1), which are involved in tight junction and gap junction, revealing that the maternal expression of the proteins possibly plays a role in the formation of cellular junctions firstly after parthenogenetic activation. Correlation and hierarchical analyses of transcriptional profiles and the expression of NPM2 and NLRP5 mRNA of buffalo in vitro developed oocytes and parthenogenetic embryos indicated that the "maternal-to-zygotic transition" (MZT) process might exist in the model of parthenogenesis, which is similar to a normally fertilized embryo, and may occur between the 8-cell to 16-cell stage. These data provide a rich resource for further studies on maternal proteins and genes and are conducive to improving nuclear transfer technology.

Association between variants in vitamin D-binding protein gene and vitamin D deficiency among pregnant women in china.

BACKGROUND: The prevalence of vitamin D deficiency and insufficiency is extremely high in pregnant women worldwide. However, the association between single nucleotide polymorphisms (SNPs) in vitamin D metabolic pathway genes and 25-hydroxyvitamin D (25(OH)D) concentration among Chinese pregnant women is seldom reported. The risk of adverse neonatal outcomes due to maternal vitamin D deficiency has not been well investigated. METHODS: A total of 815 pregnant women and 407 infants were enrolled in this study. Serum 25(OH)D concentration was detected. DNA was extracted from the maternal blood for genotyping genetic SNPs in vitamin D pathway. An XGBoost model was established based on SNPs combined with external variables. RESULTS: Mean serum 25(OH)D level was 15.67 ± 7.98 ng/mL among the pregnant women. Seventy-five percent of pregnant women had 25(OH)D deficiency in China. SNPs of GC (rs17467825, rs4588, rs2282679, rs2298850, and rs1155563) were significantly associated with maternal 25(OH)D concentration. The influence of variants of rs17467825, rs4588, rs2282679, and rs2298850 on maternal 25(OH)D might be modified by vitamin D supplementation and sunshine exposure. An XGBoost model was established for monitoring 25(OH)D status in pregnant women and provided clinical advice to reduce the risk of 25(OH)D deficiency. Mothers with 25(OH)D deficiency hinted a risk for macrosomia. CONCLUSION: A high prevalence of vitamin D deficiency in China has been confirmed. A clinical model was established to guide pregnant women to supplement vitamin D according to genotype. Furthermore, we suggest the effect of maternal vitamin D status on the risk of macrosomia.

Proteomic analysis demonstrates that parthenogenetically activated swamp buffalo embryos have dysregulated energy metabolism.

The comprehensive understanding of early embryo development is essential to optimize in vitro culture conditions. Protein expression landscape of parthenogenetically produced embryo remains unexplored. This study aimed to investigate the protein expression dynamics with a particular focus on energy metabolism throughout the early developmental stages of parthenogenetic buffalo embryos. For this purpose, we performed iTRAQ-based quantitative mass spectrometry and identified 280 proteins common in all stages. A total of 933 proteins were identified during the proteomics analysis. The data depicted that morula and blastocyst had distinct protein expression dynamics as compared to 2- to 16-cell-stage embryo. KEGG pathway analysis showed 23 proteins belonging to energy metabolism appeared in the data. Study of energy metabolism-related protein's expression pattern demonstrated that there was asynchrony in proteins related to glycolysis throughout the examined developmental stages. The expression pattern of pyruvate kinase mutase (PKM), an essential protein of glycolysis, indicated a slightly decreasing trend from 2-cell-stage embryo to blastocyst, and it was supported by expression of proteins involved in lactate production (LDHA and LDHB) suggesting the decreasing rate of aerobic glycolysis (Warburg Effect) at morula and blastocyst stage. The increased Warburg Effect is considered as the hallmark of proliferating cells or embryo at the blastocyst stage. Furthermore, the proteins involved in the citric acid cycle also showed down-regulation at the blastocyst stage, indicating a lesser role of oxidative phosphorylation at this stage. Therefore, it could be divulged from the study that there may be an irregular pattern of energy metabolism in early parthenogenetic embryos. Further studies are recommended to understand this phenomenon.

Seasonality and photoperiod influence in vitro production of buffalo embryos.

Buffalo is considered short-day breeder in tropical and subtropical part of the world and seasonality and photoperiodism impart major influence on its fertility. However, its impact on in vitro embryo production (IVEP) remains elusive. Therefore, this study investigated the effect of seasonal variations and photoperiodism on morphological and molecular parameters of IVEP in buffalo. For this purpose, we conducted two different experiments on the oocytes obtained by aspirating follicles from abattoir derived ovaries. In Exp. I, retrospective analysis was performed for oocyte recovery, blastocyst and hatching rate, during four consecutive seasonal periods (i.e. January-March, April-June, July-September and October-December). In Exp. II, oocytes from peak breeding and non-breeding seasons were subjected to 24 hr in vitro maturation and evaluated for polar body extrusion to assess maturation rate. Results showed that embryo development was markedly low during second quarter (April-June) and maximum during fourth quarter (October-December) of the year; referred as non-breeding and breeding seasons, respectively. Comparative data analysis demonstrated that poor oocyte quality is major reason for lesser efficiency of embryo production during non-breeding season than peak breeding season as suggested by poor oocyte recovery (2.31 ± 0.10 vs. 3.65 ± 0.27) and maturation rate (33.32 ± 2.1 vs. 63.15 ± 7.31). Subsequently, comparative gene expression analysis of blastocysts during peak breeding season significantly upregulated pluripotency gene (OCT-4) and downregulated heat shock protein 90, as compared to non-breeding season. Therefore, it could be divulged from the present study that seasonal variations and photoperiodism have profound effect on oocyte quality and subsequent embryo development. It is recommended to find suitable additives for in vitro maturation that could mitigate seasonal effects.

Maternal transcription profiles at different stages for the development of early embryo in buffalo.

Maternal mRNAs deposited in the egg during oogenesis are critical during the development of early embryo, before the activation of the embryonic genome. However, there is little known about the dynamic expression of maternally expressed genes in mammals. In this study, we made buffalo parthenogenesis as our research model to analyse maternal transcription profiles of pre-implantation embryo in buffalo using RNA sequencing. In total, 3,567 unique genes were detected to be differentially expressed among all constant stages during early embryo development (FPKM > 0). Interestingly, a total of 10,442 new genes were discovered in this study, and gene ontology analysis of the new differentially expressed genes indicated that the new genes have a wide cellular localization and are involved in many molecular functions and biological processes. Moreover, we identified eight clusters that were only highly expressed in a particular developmental stage and enriched a number of GO terms and KEGG pathways that were related to specific stage. Furthermore, we identified 1,530 hub genes (or key members) from the maternally expressed gene networks, and these hub genes were involved in 11 stage-specific modules. After visualization using Cytoscape 3.2.1 software, we obtained complex interaction network of hub genes, indicating the highly efficient cooperation between genes during the development in buffalo embryos. Further research of these genes will greatly deepen our understanding of embryo development in buffalo. Collectively, this research lays the foundation for future studies on the maternal genome function, buffalo nuclear transfer and parthenogenetic embryonic stem cells.

Proteomics Analysis Reveals that Warburg Effect along with Modification in Lipid Metabolism Improves In Vitro Embryo Development under Low Oxygen.

The molecular mechanism regulating embryo development under reduced oxygen tension remains elusive. This study aimed to identify the molecular mechanism impacting embryo development under low oxygen conditions. Buffalo embryos were cultured under 5% or 20% oxygen and were evaluated according to their morphological parameters related to embryo development. The protein profiles of these embryos were compared using iTRAQ-based quantitative proteomics. Physiological O2 (5%) significantly promoted blastocyst yield, hatching rate, embryo quality and cell count as compared to atmospheric O2 (20%). The embryos in the 5% O2 group had an improved hatching rate of cryopreserved blastocysts post-warming (p < 0.05). Comparative proteome profiles of hatched blastocysts cultured under 5% vs. 20% O2 levels identified 43 differentially expressed proteins (DEPs). Functional analysis indicated that DEPs were mainly associated with glycolysis, fatty acid degradation, inositol phosphate metabolism and terpenoid backbone synthesis. Our results suggest that embryos under physiological oxygen had greater developmental potential due to the pronounced Warburg Effect (aerobic glycolysis). Moreover, our proteomic data suggested that higher lipid degradation, an elevated cholesterol level and a higher unsaturated to saturated fatty acid ratio might be involved in the better cryo-survival ability reported in embryos cultured under low oxygen. These data provide new information on the early embryo protein repertoire and general molecular mechanisms of embryo development under varying oxygen levels.

Maturation of buffalo oocytes in vitro with acetyl-L-carnitine improves cryotolerance due to changes in mitochondrial function and the membrane lipid profile.

The effects of acetyl-l-carnitine (ALC) supplementation during IVM on subsequently vitrified buffalo oocytes were evaluated, followed by determination of the mitochondrial DNA copy number, measurement of mitochondrial membrane potential (MMP) and identification of the lipid profile of oocyte membranes as markers of oocyte quality after vitrification. Supplementation with ALC during IVM significantly improved the rates of oocyte cleavage and morula and blastocyst formation, and increased MMP after vitrification compared with unsupplemented vitrified oocytes (P<0.05). Using a bidirectional orthogonal projection to latent structures discriminant analysis based on positive ion matrix-assisted laser desorption ionisation time-of-flight mass spectrometry data, five phospholipid ions (m/z 728.7 (phosphatidylcholine (PC) 32:3), 746.9 (PC 32:5), 760.6 (PC 34:1), 768.8 (PC P-36:3) and 782.6 (PC 36:4); P<0.05) were identified as significantly more abundant in fresh oocytes than in unsupplemented vitrified oocytes. Meanwhile, three phospholipid ions (m/z 734.6 (PC 32:0), 760.6 (PC 34:1), and 782.6 (PC 36:4); P<0.05) were more abundant in ALC-supplemented vitrified oocytes than in unsupplemented vitrified oocytes. Therefore, supplementation with ALC during IVM may improve buffalo oocyte quality after vitrification by enhancing mitochondrial function and altering the phospholipid composition of vitrified oocyte membranes.

The effects of growth factors on proliferation of spermatogonial stem cells from Guangxi Bama mini-pig.

The objective of this study was to investigate the effects of different growth factors on the proliferation of Bama mini-pig spermatogonial stem cells (SSCs) in vitro. The growth factors glial cell line-derived neurotrophic factor (GDNF), leukaemia inhibitory factor (LIF), GDNF family receptor alpha-1 (GFRα1) and basic fibroblast growth factor (bFGF) were investigated. The SSCs were seeded on SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) feeder layers. Cultivation of the cells were subjected to a factorial design of the growth factors GDNF + bFGF, GDNF + bFGF + GFRα1, LIF + bFGF and LIF + bFGF + GFRα1. The SSCs could propagate for 25 passages in the medium adding GDNF + bFGF + GFRα1, 22 passages in the medium adding GDNF + bFGF, 6 passages in the medium adding LIF + bFGF, or LIF + bFGF + GFRα1. qRT-PCR analysis showed that the highest mRNA expression levels of NANOG, POU5F, DDX4, GFRα1 and UCHL1 were detected in the group adding GDNF + bFGF + GFRα1. The SSCs from the group adding GDNF + bFGF + GFRα1 also showed UCHL1-, DBA- and CDH1-positive staining. Moreover, Stra8 and Scp3 expression, and haploid peak were detected after induction of the SSCs from the group adding GDNF + bFGF + GFRα1. In conclusion, pig SSCs could be maintained for long term in the presence of GDNF, bFGF, and GFRα1.

Spermatogenesis-associated proteins at different developmental stages of buffalo testicular seminiferous tubules identified by comparative proteomic analysis.

The testicular seminiferous tubules contain Sertoli cells and different types of spermatogenic cells. They provide the microenvironment for spermatogenesis, but the precise molecular mechanism of spermatogenesis is still not well known. Here, we have employed tandem mass tag coupled to LC-MS/MS with the high-throughput quantitative proteomics technology to explore the protein expression from buffalo testicular seminiferous tubules at three different developmental stages (prepuberty, puberty, and postpuberty). The results show 304 differentially expressed proteins with a ≥2-fold change, and bioinformatics analysis indicates that 27 of these may be associated with spermatogenesis. Expression patterns of seven selected proteins were verified via Western blot and quantitative RT-PCR analysis, and further cellular localizations of these proteins by immunohistochemical or immunofluorescence analysis. Taken together, the results provide potential molecular markers of spermatogenesis and provide a rich resource for further studies on male reproduction regulation.

Derivation of chicken induced pluripotent stem cells tolerant to Newcastle disease virus-induced lysis through multiple rounds of infection.

BACKGROUND: Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a devastating disease of poultry and wild birds. ND is prevented by rigorous biocontainment and vaccination. One potential approach to prevent spread of the virus is production of birds that show innate resistance to NDV-caused disease. Induced pluripotent stem cell (iPSC) technology allows adult cells to be reprogrammed into an embryonic stem cell-like state capable of contributing to live offspring and passing on unique traits in a number of species. Recently, iPSC approaches have been successfully applied to avian cells. If chicken induced pluripotent stem cells (ciPSCs) are genetically or epigenetically modified to resist NDV infection, it may be possible to generate ND resistant poultry. There is limited information on the potential of ciPSCs to be infected by NDV, or the capacity of these cells to become resistant to infection. The aim of the present work was to assess the characteristics of the interaction between NDV and ciPSCs, and to develop a selection method that would increase tolerance of these cells to NDV-induced cellular damage. RESULTS: Results showed that ciPSCs were permissive to infection with NDV, and susceptible to virus-mediated cell death. Since ciPSCs that survived infection demonstrated the ability to recover quickly, we devised a system to select surviving cells through multiple infection rounds with NDV. ciPSCs that sustained 9 consecutive infections had a statistically significant increase in survival (up to 36 times) compared to never-infected ciPSCs upon NDV infection (tolerant cells). Increased survival was not caused by a loss of permissiveness to NDV replication. RNA sequencing followed by enrichment pathway analysis showed that numerous metabolic pathways where differentially regulated between tolerant and never-infected ciPSCs. CONCLUSIONS: Results demonstrate that ciPSCs are permissive to NDV infection and become increasingly tolerant to NDV under selective pressure, indicating that this system could be applied to study mechanisms of cellular tolerance to NDV.

Development, characterization and optimization of a new suspension chicken-induced pluripotent cell line for the production of Newcastle disease vaccine.

Traditionally, substrates for production of viral poultry vaccines have been embryonated eggs or adherent primary cell cultures. The difficulties and cost involved in scaling up these substrates in cases of increased demand have been a limitation for vaccine production. Here, we assess the ability of a newly developed chicken-induced pluripotent cell line, BA3, to support replication and growth of Newcastle disease virus (NDV) LaSota vaccine strain. The characteristics and growth profile of the cells were also investigated. BA3 cells could grow in suspension in different media to a high density of up to 7.0 × 10(6) cells/mL and showed rapid proliferation with doubling time of 21 h. Upon infection, a high virus titer of 1.02 × 10(8) EID50/mL was obtained at 24 h post infection using a multiplicity of infection (MOI) of 5. In addition, the cell line was shown to be free of endogenous and exogenous Avian Leukosis viruses, Reticuloendotheliosis virus, Fowl Adenovirus, Marek's disease virus, and several Mycoplasma species. In conclusion, BA3 cell line is potentially an excellent candidate for vaccine production due to its highly desirable industrially friendly characteristics of growing to high cell density and capability of growth in serum free medium.

Comparative Proteomic Analysis of Mature and Immature Oocytes of the Swamp Buffalo (Bubalus bubalis).

Maternal protein components change markedly during mammalian oogenesis. Many of these proteins have yet to be characterized and verified. In this study, a proteomics approach was used to evaluate changes in proteins during oogenesis in the Swamp Buffalo (Bubalus bubalis). Proteins from 500 immature oocytes and 500 in vitro matured oocytes were subjected to two-dimensional electrophoresis, and more than 400 spots were detected. Image analysis indicated that 17 proteins were differentially expressed between the two groups. Eight proteins were identified by mass spectrometry. In mature oocytes, three proteins were down-regulated: major vault protein (MVP), N-acetyllactosaminide ß-1,6-N-acetylglucosaminyl-transferase (GCNT-2), and gem-associated protein (GEMIN)8, whereas five other proteins, heat shock protein (HSP)60, Ras-responsive element-binding protein 1 (RREB-1), heat shock cognate 71 kDa protein (HSC71), hemoglobin subunit α (HBA), and BMP-2-inducible protein kinase (BMP-2K), were up-regulated. The expression profiles of HSP60 and GEMIN8 were further verified by Western blotting. The changes in HSP60 protein expression demonstrate the increasing need for mitochondrial protein importation to facilitate macromolecular assembly during oocyte maturation. The down-regulation of GEMIN8 production implies that RNA splicing is impaired in mature oocytes.

Proteome Profile and Quantitative Proteomic Analysis of Buffalo (Bubalusbubalis) Follicular Fluid during Follicle Development.

Follicular fluid (FF) accumulates in the antrum of the ovarian follicle and provides the microenvironment for oocyte development. FF plays an important role in follicle growth and oocyte maturation. The FF provides a unique window to investigate the processes occurring during buffalo follicular development. The observed low quality of buffalo oocytes may arise from the poor follicular microenvironment. Investigating proteins found in buffalo FF (BFF) should provide insight into follicular development processes and provide further understanding of intra-follicular maturation and oocytes quality. Here, a proteomic-based approach was used to analyze the proteome of BFF. SDS-PAGE separation combined with mass spectrometry was used to generate the proteomic dataset. In total, 363 proteins were identified and classified by Gene Ontology terms. The proteins were assigned to 153 pathways, including signaling pathways. To evaluate difference in proteins expressed between BFF with different follicle size (small, <4 mm; and large, >8 mm), a quantitative proteomic analysis based on multi-dimensional liquid chromatography pre-fractionation tandem Orbitrap mass spectrometry identification was performed. Eleven differentially expressed proteins (six downregulated and five upregulated in large BFF) were identified and assigned to a variety of functional processes, including serine protease inhibition, oxidation protection and the complement cascade system. Three differentially expressed proteins, Vimentin, Peroxiredoxin-1 and SERPIND1, were verified by Western blotting, consistent with the quantitative proteomics results. Our datasets offers new information about proteins present in BFF and should facilitate the development of new biomarkers. These differentially expressed proteins illuminate the size-dependent protein changes in follicle microenvironment.

ZPAC is required for normal spermatogenesis in mice.

The ubiquitin-proteasome pathway, involved in genetic recombination and sex-chromosome silencing during meiosis, plays critical roles in the specification of germ-line stem cells and the differentiation of gametes from gonocytes. Zygote-specific proteasome assembly chaperone (ZPAC) is expressed in the early mouse embryo, where it is important for progression of the mouse maternal-to-zygotic transition. The role of ZPAC during spermatogenesis in the adult gonads, however, remains unknown. In this study, rapid amplification of cDNA ends was used to determine the Zpac cDNA sequence, a 1584-bp transcript that includes a putative 1122-bp open reading frame coding for a 373 amino acid protein. Western blot and immunohistochemistry revealed that ZPAC was specifically expressed in gonads. To further dissect the function of ZPAC during spermatogenesis, we employed PiggyBac-based RNA interference vectors for transgenesis combined with cell transplantation to deplete Zpac during spermatogenesis. This RNAi-mediate depletion in Zpac expression disrupted normal spermatogenesis from spermatogonial stem cells. Two independent yeast two-hybrid screens further revealed an interaction between ZPAC and SYCE1. Together, these data suggest that ZPAC is required for normal spermatogenesis in mice.