Spatiotemporal single-cell RNA sequencing reveals the role of steroid hormone pathway during chicken primordial follicle formation.

The size of the initial primordial follicle pool in the ovary depends on primordial follicle formation, which determines the female reproductive lifespan. However, the molecular regulation of primordial follicle formation in chickens remains unclear. In this study, the left ovaries of chickens were collected at 2 d posthatch (dph), 5.5 dph, and 10.5 dph to examine the formation of primordial follicles. Single-cell mRNA sequencing (scRNA-seq) and spatial transcriptomic analysis were performed to explore the ovarian microenvironment and identify regulatory pathways involved in the formation of primordial follicles in chickens. Histomorphological analysis of chicken ovary tissues revealed the presence of germ cell cysts at 1 dph, which began to disintegrate at 2 dph. Primordial follicles appeared at 5.5 dph and continued to develop into larger-diameter follicles. scRNA-seq and spatial transcriptomic analysis revealed 24 cellular clusters involved in chicken primordial follicle formation. The metabolic pathway of steroid hormone synthesis was found in pregranulosa and pretheca cells. Histological analysis showed that chicken ovaries did not form primordial follicles after the inhibition of the steroid hormone synthesis pathway by simvastatin or tamoxifen. In addition, mRNA transcriptomic and bioinformatics analyses revealed that GREB1 was a downstream gene of the steroid hormone synthesis pathway during the formation of chicken primordial follicles. This study provides a valuable foundation for investigating primordial follicle formation in avian species and optimizing their reproductive performance.

Exploring right ovary degeneration in duck and goose embryos by histology and transcriptome dynamics analysis.

BACKGROUND: The development of asymmetric chick gonads involves separate developmental programs in the left and right gonads. In contrast to the left ovary developing into a fully functional reproductive organ, the right ovary undergoes gradual degeneration. However, the molecular mechanisms underlying the the degeneration of the right ovary remain incompletely understood. In the present study, we investigated the histomorphological and transcriptomic changes in the right ovary of ducks and geese during the the embryonic stage up to post-hatching day 1. RESULT: Hematoxylin-eosin stainings revealed that the right ovary developed until embryonic day 20 in ducks (DE20) or embryonic day 22 in geese (GE22), after which it started to regress. Further RNA-seq analyses revealed that both the differentially expressed genes (DEGs) in ducks and geese right ovary developmental stage were significantly enriched in cell adhesion-related pathway (ECM-receptor interaction, Focal adhesion pathway) and Cellular senescence pathway. Then during the degeneration stage, the DEGs were primarily enriched in pathways associated with inflammation, including Herpes simplex virus 1 infection, Influenza A, and Toll-like receptor signaling pathway. Moreover, duck-specific DEGs showed enrichment in Steroid hormone biosynthesis, Base excision repair, and the Wnt signaling pathway, while geese-specifically DEGs were found to be enriched in apoptosis and inflammation-related pathways, such as Ferroptosis, Necroptosis, RIG-I-like receptor signaling pathway, and NOD-like receptor signaling pathway. These findings suggest that the degeneration process of the right ovary in ducks occurs at a slower pace compared to that in geese. Additionally, the observation of the left ovary of the geese varying degeneration rates in the right ovary after hatching indicated that the development of the left ovary may be influenced by the degeneration of the right ovary. CONCLUSION: The data presented in this study provide valuable insights into the dynamic changes in histological structure and transcriptome during the degeneration of the right ovary in ducks and geese. In addition, through the analysis of shared characteristics in the degeneration process of the right ovary in both ducks and geese, we have uncovered the patterns of degradation and elucidated the molecular mechanisms involved in the regression of the right ovary in poultry. Furthermore, we have also made initial discoveries regarding the relationship between the degeneration of the right ovary and the development of the left ovary.

Estrogen Receptor Gene 1 (ESR1) Mediates Lipid Metabolism in Goose Hierarchical Granulosa Cells Rather than in Pre-Hierarchical Granulosa Cells.

(1) Background: The role of estrogen receptor gene 1 (ESR1) in female reproduction and lipid metabolism has been extensively investigated. However, its contribution to lipid metabolism during the development of poultry follicles remains unclear. (2) Methods: This study aimed to explore the function of ESR1 via overexpressing (ESR1ov) and interfering (ESR1si) with its expression in pre-hierarchical granulosa cells (phGCs) and hierarchical granulosa cells (poGCs). (3) Results: We successfully cloned and obtained an 1866 bp segment of the full-length CDS region of the Sichuan white goose ESR1 gene. In phGCs of the ESR1ov and ESR1si groups, there were no significant changes compared to the control group. However, in poGCs, the ESR1ov group exhibited decreased lipid deposition, triglycerides, and cholesterol compared to the control group, while the ESR1si group showed increased lipid deposition, triglycerides, and cholesterol. The expression of APOB and PPARα was significantly reduced in the ESR1ov group compared to the ESR1ov-NC group. Moreover, significant changes in the expression of ACCα, DGAT1, SCD, CPT1, and ATGL were observed between the ESR1si and ESR1si-NC group. (4) Conclusions: These findings shed light on the function and molecular mechanism of ESR1 in lipid metabolism in goose poGCs, providing a better understanding of the physiological process of goose follicular development.

MiR-202-5p Regulates Geese Follicular Selection by Targeting BTBD10 to Regulate Granulosa Cell Proliferation and Apoptosis.

The regulation of granulosa cells (GCs) proliferation and apoptosis is the key step in follicular selection which determines the egg production performance of poultry. miR-202-5p has been reported to be involved in regulating the proliferation and apoptosis of mammalian ovarian GCs. However, its role in regulating the proliferation and apoptosis of goose GCs is still unknown. In the present study, the GCs of pre-hierarchical follicles (phGCs, 8-10 mm) and those of hierarchical follicles (hGCs, F2-F4) were used to investigate the role of miR-202-5p in cell proliferation and apoptosis during follicle selection. In phGCs and hGCs cultured in vitro, miR-202-5p was found to negatively regulate cell proliferation and positively regulate cell apoptosis. The results of RNA-seq showed that BTB Domain Containing 10 (BTBD10) is predicted to be a key target gene for miR-202-5p to regulate the proliferation and apoptosis of GCs. Furthermore, it is confirmed that miR-202-5p can inhibit BTBD10 expression by targeting its 3'UTR region, and BTBD10 was revealed to promote the proliferation and inhibit the apoptosis of phGCs and hGCs. Additionally, co-transfection with BTBD10 effectively prevented miR-202-5p mimic-induced cell apoptosis and the inhibition of cell proliferation. Meanwhile, miR-202-5p also remarkably inhibited the expression of Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Beta (PIK3CB) and AKT Serine/Threonine Kinase 1 (AKT1), while it was significantly restored by BTBD10. Overall, miR-202-5p suppresses the proliferation and promotes the apoptosis of GCs through the downregulation of PIK3CB/AKT1 signaling by targeting BTBD10 during follicular selection. Our study provides a theoretical reference for understanding the molecular mechanism of goose follicular selection, as well as a candidate gene for molecular marker-assisted breeding to improve the geese' egg production performance.

miR-202-5p Inhibits Lipid Metabolism and Steroidogenesis of Goose Hierarchical Granulosa Cells by Targeting ACSL3.

miRNAs are critical for steroidogenesis in granulosa cells (GCs) during ovarian follicular development. We have previously shown that miR-202-5p displays a stage-dependent expression pattern in GCs from goose follicles of different sizes, suggesting that this miRNA could be involved in the regulation of the functions of goose GCs; therefore, in this study, the effects of miR-202-5p on lipid metabolism and steroidogenesis in goose hierarchical follicular GCs (hGCs), as well as its mechanisms of action, were evaluated. Oil Red O staining and analyses of intracellular cholesterol and triglyceride contents showed that the overexpression of miR-202-5p significantly inhibited lipid deposition in hGCs; additionally, miR-202-5p significantly inhibited progesterone secretion in hGCs. A bioinformatics analysis and luciferase reporter assay indicated that Acyl-CoA synthetase long-chain family member 3 (ACSL3), which activates long-chain fatty acids for the synthesis of cellular lipids, is a potential target of miR-202-5p. ACSL3 silencing inhibited lipid deposition and estrogen secretion in hGCs. These data suggest that miR-202-5p exerts inhibitory effects on lipid deposition and steroidogenesis in goose hGCs by targeting the ACSL3 gene.

Mechanism of SCD Participation in Lipid Droplet-Mediated Steroidogenesis in Goose Granulosa Cells.

Stearoyl-CoA desaturase (SCD) is a key enzyme catalyzing the rate-limiting step in monounsaturated fatty acids (MUFAs) production. There may be a mechanism by which SCD is involved in lipid metabolism, which is assumed to be essential for goose follicular development. For this reason, a cellular model of SCD function in goose granulosa cells (GCs) via SCD overexpression and knockdown was used to determine the role of SCD in GC proliferation using flow cytometry. We found that SCD overexpression induced and SCD knockdown inhibited GCs proliferation. Furthermore, ELISA analysis showed that SCD overexpression increased the total cholesterol (TC), progesterone, and estrogen levels in GCs, while SCD knockdown decreased TC, progesterone, and estrogen levels (p < 0.05). Combining these results with those of related multi-omics reports, we proposed a mechanism of SCD regulating the key lipids and differentially expressed gene (DEGs) in glycerophospholipid and glycerolipid metabolism, which participate in steroidogenesis mediated by the lipid droplet deposition in goose GCs. These results add further insights into understanding the lipid metabolism mechanism of goose GCs.

Integrated mRNA and miRNA transcriptome analysis provides novel insights into the molecular mechanisms underlying goose pituitary development during the embryo-to-hatchling transition.

It is well established that the endocrine system plays a pivotal role in preparing the avian embryos for the abrupt switch from chorioallantoic to pulmonary respiration during the critical embryo-to-hatchling transition. However, as the master gland of the endocrine system, there has been little research focusing on the molecular mechanisms controlling the development and function of the pituitary gland during the peri-hatch period in birds. In the present study, we aimed to determine the genome-wide mRNA and miRNA transcriptome profiles of the pituitary during the embryo-to-hatchling transition period from embryonic day 22 (E22) to post-hatching day 6 (P6) in the goose (Anser cygnoides). Of note, expression of Anser_cygnoides_newGene_32456 and LOC106031011 were significantly different among these 4 stages (i.e., E22, E26, P2, and P6). Meanwhile, the neuroactive ligand-receptor interaction pathway was significantly enriched by the DEGs commonly identified among three pairwise comparisons. At the miRNA transcriptome level, there were not commonly identified DE miRNAs among these 4 stages, while the 418 of their predicted target genes were mutually shared. Both the target genes of DE miRNAs in each comparison and these 418 shared target genes were significantly enriched in the ECM-receptor interaction and focal adhesion pathways. In the predicted miRNA-mRNA interaction networks of these 2 pathways, novel_miRNA_467, novel_miRNA_154, and novel_miRNA_340 were the hub miRNAs. In addition, multiple DE miRNAs also showed predicted target relationships with the DEGs associated with extracellular matrix (ECM) components. Among them, expression of novel_miR_120, tgu-miR-92-3p, and novel_miR_398 was significantly negatively correlated with that of LAMC3 (laminin subunit gamma3), suggesting that these miRNAs may regulate pituitary tissue remodeling and functional changes through targeting LAMC3 during development. These identified DE mRNAs and miRNAs as well as their predicted interaction networks involved in regulation of tissue remodeling and cellular functions were most likely to play critical roles in facilitating the embryo-to-hatchling transition. These results provide novel insights into the early developmental process of avian pituitary gland and will help better understand the underlying molecular mechanisms.

Lipidomics profiling of goose granulosa cell model of stearoyl-CoA desaturase function identifies a pattern of lipid droplets associated with follicle development.

BACKGROUND: Despite their important functions and nearly ubiquitous presence in cells, an understanding of the biology of intracellular lipid droplets (LDs) in goose follicle development remains limited. An integrated study of lipidomic and transcriptomic analyses was performed in a cellular model of stearoyl-CoA desaturase (SCD) function, to determine the effects of intracellular LDs on follicle development in geese. RESULTS: Numerous internalized LDs, which were generally spherical in shape, were dispersed throughout the cytoplasm of granulosa cells (GCs), as determined using confocal microscopy analysis, with altered SCD expression affecting LD content. GC lipidomic profiling showed that the majority of the differentially abundant lipid classes were glycerophospholipids, including PA, PC, PE, PG, PI, and PS, and glycerolipids, including DG and TG, which enriched glycerophospholipid, sphingolipid, and glycerolipid metabolisms. Furthermore, transcriptomics identified differentially expressed genes (DEGs), some of which were assigned to lipid-related Gene Ontology slim terms. More DEGs were assigned in the SCD-knockdown group than in the SCD-overexpression group. Integration of the significant differentially expressed genes and lipids based on pathway enrichment analysis identified potentially targetable pathways related to glycerolipid/glycerophospholipid metabolism. CONCLUSIONS: This study demonstrated the importance of lipids in understanding follicle development, thus providing a potential foundation to decipher the underlying mechanisms of lipid-mediated follicle development.

Comparative Analysis of Testicular Histology and lncRNA-mRNA Expression Patterns Between Landes Geese (Anser anser) and Sichuan White Geese (Anser cygnoides).

Landes geese and Sichuan White geese are two important genetic materials for commercial goose breeding. However, the differences in the male reproductive capacity between these two breeds and the potential molecular mechanisms and associated key genes have not been reported to date. The present study compared the testicular histology and mRNA-long non-coding RNA (lncRNA) expression patterns to reveal the differences in male reproductive performance between Sichuan White geese and Landes geese, as well as to explore the underlying molecular mechanisms. Histological results showed that the testicular organ index, semen volume, and long diameter of seminiferous tubules of Landes geese were significantly larger than those of Sichuan White geese. Analyses of mRNA-lncRNA expression profile showed that compared with Sichuan White geese, a total of 462 differentially expressed mRNAs (DEGs) (173 up-regulated and 289 down-regulated) and 329 differentially expressed lncRNAs (DE lncRNAs) (280 up-regulated, 49 down-regulated) were identified in Landes geese. Among these DEGs, there were 10 spermatogenesis-related and highly expressed (FPKM > 10) DEGs. Except for SEPP1, all of these DEGs were significantly up-regulated in the testes of Landes geese. Functional enrichment analysis indicated that the pathway related to metabolism progress and phosphoinositol signal is vitally responsible for differences in male reproductive performance between Landes geese and Sichuan White geese. These results show that compared with Sichuan White geese, the spermatogenesis in the testis of Landes geese was more active, which may be mainly related to the inositol phosphate signal. These data contribute to a better understanding of the mechanisms underlying different male reproductive performances between Landes geese and Sichuan White geese. This knowledge might eventually provide a theoretical basis for improving male reproductive performance in geese.

The immune and metabolic changes with age in giant panda blood by combined transcriptome and DNA methylation analysis.

Giant panda (Ailuropoda melanoleuca) is an endangered mammalian species. Exploring immune and metabolic changes that occur in giant pandas with age is important for their protection. In this study, we systematically investigated the physiological and biochemical indicators in blood, as well as the transcriptome, and methylation profiles of young, adult, and old giant pandas. The white blood cell (WBC), neutrophil (NEU) counts and hemoglobin (HGB) concentrations increased significantly with age (young to adult), and some indicators related to blood glucose and lipids also changed significantly with age. In the transcriptome analysis, differentially expressed genes (DEGs) were found in comparisons of the young and adult (257), adult and old (20), young and old (744) groups. Separation of the DEGs into eight profiles according to the expression trend using short time-series expression miner (STEM) software revealed that most DEGs were downregulated with age. Functional analysis showed that most DEGs were associated with disease and that these DEGs were also associated with the immune system and metabolism. Furthermore, gene methylation in giant pandas decreased globally with age, and the expression of CCNE1, CD79A, IL1R1, and TCF7 showed a highly negative correlation with their degree of methylation. These results indicate that the giant panda's immune function improves gradually with age (young to adult), and that changes in the methylation profile are involved in the effects of age on immune and metabolic functions. These results have important implications for the understanding and conservation of giant pandas.

Paternal weight of ducks may have an influence on offspring' small intestinal function and cecal microorganisms.

BACKGROUND: In animals, many factors affect the small intestinal function and cecal microorganisms, including body weight and genetic background. However, whether paternal weight impacts the small intestinal function and cecal microorganisms remains unknown to date. The current study used Nonghua sheldrake to estimate the effect of paternal weight on the intestine of the offspring by evaluating differences in small intestinal morphology, digestive enzyme activity, and cecal microorganisms between the offspring of male parents with high body weight (group H) and that of male parents with low body weight (group L). RESULTS: The results of the analysis of small intestinal morphology showed that the villus height of the jejunum of group H ducks was higher than that of group L ducks, and the difference was significant for ducks at 10 weeks of age. Moreover, the villus height/crypt depth of the duodenum in group H significantly exceeded that of group L at a duck age of 2 weeks. The amylase activity in the jejunum content of group H exceeded that of group L at 5 and 10 weeks of age. Furthermore, the proportion of the Firmicutes to Bacteroidetes was significantly higher in group H (duck age of 2 weeks). Among the genera with a relative abundance exceeding 1%, the relative abundances of genera Desulfovibrio, Megamonas, Alistipes, Faecalibacterium, and Streptococcus observed in group H were significantly different between group H and group L. CONCLUSIONS: For the first time, this study identifies the effect of paternal weight on offspring small intestinal function and cecal microorganisms. Consequently, this lays a foundation for further research on the relationship between male parents and offspring intestinal function.

miR-26a is Involved in Glycometabolism and Affects Boar Sperm Viability by Targeting PDHX.

miR-26a is associated with sperm metabolism and can affect sperm motility and apoptosis. However, how miR-26a affects sperm motility remains largely unknown. Our previous study indicated that the PDHX gene is predicted to be a potential target of miR-26a, which is responsible for pyruvate oxidative decarboxylation which is considered as a key step for connecting glycolysis with oxidative phosphorylation. In this study, we first reported a potential relationship between miR-26a and PDHX and their expressions in fresh, frozen-thawed, and epididymal boar sperm. Then, sperm viability and survival were determined after transfection of miR-26a. mRNA and protein expression level of PDHX in the liquid-preserved boar sperm after transfection were also determined by RT-qPCR and Western Blot (WB). Our results showed that expression level of PDHX was significantly increased during sperm transit from epididymal caput to corpus and cauda. Similarly, expression of PDHX was significantly higher (P < 0.05) in fresh sperm as compared to epididymal cauda and frozen-thawed sperm. However, the expression of miR-26a in epididymal corpus sperm was significantly higher (P < 0.05) than that of caput and cauda sperm. Furthermore, after transfection of boar sperm with miR-26a mimic and inhibitor under liquid storage, the lowest and highest sperm viability was observed in miR-26a mimic and inhibitor treatment (P < 0.05), respectively. The protein levels of PDHX, after 24 and 48 h of transfection of miR-26a mimics and inhibitor, were notably decreased and increased (P < 0.05), respectively, as compared to negative control (NC) group. In conclusion, the novel and enticing findings of our study provide a reasonable evidence that miR-26a via PDHX, a link between glycolysis and oxidative phosphorylation, could regulate the glycometabolic pathway which eventually affect boar sperm viability and survival.

Comparative Analysis of MicroRNA and mRNA Profiles of Sperm with Different Freeze Tolerance Capacities in Boar (Susscrofa) and Giant Panda (Ailuropodamelanoleuca).

Post-thawed sperm quality parameters vary across different species after cryopreservation. To date, the molecular mechanism of sperm cryoinjury, freeze-tolerance and other influential factors are largely unknown. In this study, significantly dysregulated microRNAs (miRNAs) and mRNAs in boar and giant panda sperm with different cryo-resistance capacity were evaluated. From the result of miRNA profile of fresh and frozen-thawed giant panda sperm, a total of 899 mature, novel miRNAs were identified, and 284 miRNAs were found to be significantly dysregulated (195 up-regulated and 89 down-regulated). Combined analysis of miRNA profiling of giant panda sperm and our previously published data on boar sperm, 46, 21 and 4 differentially expressed (DE) mRNAs in boar sperm were believed to be related to apoptosis, glycolysis and oxidative phosphorylation, respectively. Meanwhile, 87, 17 and 7 DE mRNAs in giant panda were associated with apoptosis, glycolysis and oxidative phosphorylation, respectively. Gene ontology (GO) analysis of the targets of DE miRNAs showed that they were mainly distributed on membrane related pathway in giant panda sperm, while cell components and cell processes were tied to the targets of DE miRNAs in boar sperm. Finally, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DE mRNAs indicated that most of these DE mRNAs were distributed in membrane signal transduction-related pathways in giant panda sperm, while those in boar sperm were mainly distributed in the cytokine-cytokine receptor interaction pathway and inflammatory related pathways. In conclusion, although the different freezing extenders and programs were used, the DE miRNAs and mRNAs involved in apoptosis, energy metabolism, olfactory transduction pathway, inflammatory response and cytokine-cytokine interactions, could be the possible molecular mechanism of sperm cryoinjury and freeze tolerance.

Exploration of miRNA and mRNA Profiles in Fresh and Frozen-Thawed Boar Sperm by Transcriptome and Small RNA Sequencing.

Due to lower farrowing rate and reduced litter size with frozen-thawed semen, over 90% of artificial insemination (AI) is conducted using liquid stored boar semen. Although substantial progress has been made towards optimizing the cryopreservation protocols for boar sperm, the influencing factors and underlying mechanisms related to cryoinjury and freeze tolerance of boar sperm remain largely unknown. In this study, we report the differential expression of mRNAs and miRNAs between fresh and frozen-thawed boar sperm using high-throughput RNA sequencing. Our results showed that 567 mRNAs and 135 miRNAs were differentially expressed (DE) in fresh and frozen-thawed boar sperm. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the majority of DE mRNAs were enriched in environmental information processing such as cytokine-cytokine receptor interactions, PI3K-Akt signaling, cell adhesion, MAPK, and calcium signaling pathways. Moreover, the targets of DE miRNAs were enriched in significant GO terms such as cell process, protein binding, and response to stimuli. In conclusion, we speculate that DE mRNAs and miRNAs are heavily involved in boar sperm response to environment stimuli, apoptosis, and metabolic activities. The differences in expression also reflect the various structural and functional changes in sperm during cryopreservation.

High throughput small RNA and transcriptome sequencing reveal capacitation-related microRNAs and mRNA in boar sperm.

BACKGROUND: Capacitation, a prerequisite for oocyte fertilization, is a complex process involving series of structural and functional changes in sperms such as membrane modifications, modulation of enzyme activities, and protein phosphorylation. In order to penetrate and fertilize an oocyte, mammalian sperms must undergo capacitation. Nevertheless, the process of sperm capacitation remains poorly understood and requires further elucidation. In the current study, via high throughput sequencing, we identified and explored the differentially expressed microRNAs (miRNAs) and mRNAs involved in boar sperm capacitation. RESULTS: We identified a total of 5342 mRNAs and 204 miRNAs that were differentially expressed in fresh and capacitated boar sperms. From these, 12 miRNAs (8 known and 4 newly identified miRNAs) and their differentially expressed target mRNAs were found to be involved in sperm capacitation-related PI3K-Akt, MAPK, cAMP-PKA and Ca2+signaling pathways. CONCLUSIONS: Our study is first to provide the complete miRNA and transcriptome profiles of boar sperm. Our findings provide important insights for the understanding of the RNA profile in boar sperm and future elucidation of the underlying molecular mechanism relevant to mammalian sperm capacitation.

Transcriptome Sequencing Reveals the Differentially Expressed lncRNAs and mRNAs Involved in Cryoinjuries in Frozen-Thawed Giant Panda (Ailuropoda melanoleuca) Sperm.

Sperm cryopreservation and artificial insemination are important methods for giant panda breeding and preservation of extant genetic diversity. Lower conception rates limit the use of artificial insemination with frozen-thawed giant panda sperm, due to the lack of understanding of the cryodamaging or cryoinjuring mechanisms in cryopreservation. Long non-coding RNAs (lncRNAs) are involved in regulating spermatogenesis. However, their roles during cryopreservation remain largely unexplored. Therefore, this study aimed to identify differentially expressed lncRNAs and mRNAs associated with cryodamage or freeze tolerance in frozen-thawed sperm through high throughput sequencing. A total of 61.05 Gb clean reads and 22,774 lncRNA transcripts were obtained. From the sequencing results, 1477 significantly up-regulated and 1,396 significantly down-regulated lncRNA transcripts from fresh and frozen-thawed sperm of giant panda were identified. GO and KEGG showed that the significantly dysregulated lncRNAs and mRNAs were mainly involved in regulating responses to cold stress and apoptosis, such as the integral component of membrane, calcium transport, and various signaling pathways including PI3K-Akt, p53 and cAMP. Our work is the first systematic profiling of lncRNA and mRNA in fresh and frozen-thawed giant panda sperm, and provides valuableinsights into the potential mechanism of cryodamage in sperm.

Polymorphisms in the Egl nine homolog 3 (EGLN3) and Peroxisome proliferator activated receptor-alpha (PPARα) genes and their correlation with hypoxia adaptation in Tibetan chickens.

Peroxisome proliferator activated receptor-alpha (PPARα) and Egl nine homolog 3 (EGLN3) play critical roles in facilitating the adaptation to a hypoxic environment. However, the relationship between EGLN3 and PPARα variants and hypoxic adaptation remains poorly understood in Tibetan chickens. To better understand the effects of genetic variation, we sequenced exons of PPARα and EGLN3 in 138 Lowland chickens (LC) from 7 breeds that were located in Emei, Miyi, Shimian, Wanyuan, Pengxian, and Muchuan in the Sichuan province, and Wenchang in the Hainan province (altitudes for these locations are below 1800 meters). Total 166 Tibetan chickens (TC) from 7 subpopulations that were located in Shigatse, Lhoka, Lhasa, Garze, Aba, Diqing and Yushu in the Tibetan area were also sequenced (altitudes greater than 2700 meters). One single-nucleotide polymorphism (rs316017491, C > T) was identified in EGLN3 and was shared by TC and LC with no significant difference for allele frequencies between them (P > 0.05). Six single-nucleotide polymorphisms (SNP1, A29410G; SNP2, rs13886097; SNP3, T29467C; SNP4, rs735915170; SNP5, rs736599044; and SNP6, rs740077421) including one non-synonymous mutation (SNP2, T > C) were identified in PPARα. This is the first report of SNP1 and SNP3. There was a difference between TC and LC for allele frequencies (P <0.01), except for SNP1, SNP4, and SNP5) The fix index statistic test indicated that there was population differentiation between TC and LC for SNP2, SNP3, and SNP6 in PPARα (P < 0.05). Phylogenetic analysis showed that the genetic distance among chickens, finch and great tit were close for both EGLN3 and PPARα. Bioinformatics analysis of PPARα showed that SNP2 leads to an amino acid substitution of Ile for Met, which results in the protein being more likely to be hydrolyzed. Thus, genetic variation in PPARα may play a role in the ability of TC to adapt to a high altitude environment; however we were unable to identify a relationship between polymorphisms in EGLN3 and environmental adaptability.