Single-cell RNA-sequencing analysis and characterisation of testicular cells in giant panda (Ailuropoda melanoleuca).

CONTEXT: The giant panda (Ailuropoda melanoleuca ) is a rare and endangered species to be preserved in China. The giant panda has a low reproductive capacity, and due to the scarcity of samples, studies on testes from giant panda are very limited, with little knowledge about the process of spermatogenesis in this species. AIMS: To establish the gene expression profiles in cells from the testis of a giant panda. METHODS: The 10×Genomics single-cell RNA-sequencing platform was applied to cells from the testis of an adult giant panda. KEY RESULTS: We identified eight testicular cell types including six somatic and two germ cell types from our single-cell RNA-sequencing datasets. We also identified the differentially expressed genes (DEGs) in each cell type, and performed functional enrichment analysis for the identified testicular cell types. Furthermore, by immunohistochemistry we explored the protein localisation patterns of several marker genes in testes from giant panda. CONCLUSIONS: Our study has for the first time established the gene expression profiles in cells from the testis of a giant panda. IMPLICATIONS: Our data provide a reference catalogue for spermatogenesis and testicular cells in the giant panda, laying the foundation for future breeding and preservation of this endangered species.

Profiling of miRNAs in porcine germ cells during spermatogenesis.

Spermatogenesis includes mitosis of spermatogonia, meiosis of pachytene spermatocytes and spermiogenesis of round spermatids. MiRNAs as a ~22 nt small noncoding RNA are involved in regulating spermatogenesis at post-transcriptional level. However, the dynamic miRNAs expression in the developmental porcine male germ cells remains largely undefined. In this study, we purified porcine spermatogonia, pachytene spermatocytes and round spermatids using a STA-PUT apparatus. A small RNA deep sequencing and analysis were conducted to establish a miRNAs profiling in these male germ cells. We found that 19 miRNAs were differentially expressed between spermatogonia and pachytene spermatocytes, and 74 miRNAs differentially expressed between pachytene spermatocytes and round spermatids. Furthermore, 91 miRNAs were upregulated, while 108 miRNAs were downregulated in spermatozoa. We demonstrated that ssc-miR-10a-5p, ssc-miR-125b, ssc-let-7f and ssc-miR-186 were highly expressed in spermatogonia, pachytene spermatocytes, round spermatids and spermatozoa respectively. The findings could provide novel insights into roles of miRNAs in regulation of porcine spermatogenesis.

Generation and characterization of giant panda induced pluripotent stem cells.

The giant panda (Ailuropoda melanoleuca) stands as a flagship and umbrella species, symbolizing global biodiversity. While traditional assisted reproductive technology faces constraints in safeguarding the genetic diversity of giant pandas, induced pluripotent stem cells (iPSCs) known for their capacity to differentiate into diverse cells types, including germ cells, present a transformative potential for conservation of endangered animals. In this study, primary fibroblast cells were isolated from the giant panda, and giant panda iPSCs (GPiPSCs) were generated using a non-integrating episomal vector reprogramming method. Characterization of these GPiPSCs revealed their state of primed pluripotency and demonstrated their potential for differentiation. Furthermore, we innovatively formulated a species-specific chemically defined FACL medium and unraveled the intricate signaling pathway networks responsible for maintaining the pluripotency and fostering cell proliferation of GPiPSCs. This study provides key insights into rare species iPSCs, offering materials for panda characteristics research and laying the groundwork for in vitro giant panda gamete generation, potentially aiding endangered species conservation.

Establishment of cell lines with porcine spermatogonial stem cell properties.

BACKGROUND: Spermatogonial stem cells (SSCs) are capable of both self-renewal and differentiation to mature functional spermatozoa, being the only adult stem cells in the males that can transmit genetic information to the next generation. Porcine SSCs hold great value in transgenic pig production and in establishment of porcine models for regenerative medicine. However, studies and applications of porcine SSCs have been greatly hampered by the low number of SSCs in the testis as well as the lack of an ideal stable long-term culture system to propagate porcine SSCs perpetually. RESULTS: In the present study, by lentiviral transduction of plasmids expressing the simian virus 40 (SV40) large T antigen into porcine primary SSCs, we developed two immortalized cell lines with porcine SSC attributes. The established cell lines, with the expression of porcine SSC and germ cell markers UCHL1, PLZF, THY1, VASA and DAZL, could respond to retinoic acid (RA), and could colonize the recipient mouse testis without tumor formation after transplantation. The cell lines displayed infinite proliferation potential, and have now been cultured for more than 7 months and passaged for over 35 times without morphological abnormalities. CONCLUSIONS: We have for the first time established porcine SSC lines that could provide abundant cell sources for mechanistic studies on porcine SSC self-renewal and differentiation, thereby facilitating development of an optimal long-term culture system for porcine primary SSCs and their application to animal husbandry and medicine.

YTHDF2 promotes spermagonial adhesion through modulating MMPs decay via m6A/mRNA pathway.

As the foundation of male fertility, spermatogenesis is a complicated and highly controlled process. YTHDF2 plays regulatory roles in biological processes through accelerating the degradation of target mRNAs. However, the function of YTHDF2 in spermatogenesis remains elusive. Here, we knocked out Ythdf2 in mouse spermatogonia via CRISPR/Cas9, and found that depletion of Ythdf2 mainly downregulated the expression of matrix metallopeptidase (MMPs), thus affecting cell adhesion and proliferation. m6A-IP-PCR and RIP-PCR analysis showed that Mmp3, Mmp13, Adamts1 and Adamts9 were modified with m6A and simultaneously interacted with YTHDF2. Moreover, inhibition of Mmp13 partially rescued the phenotypes in Ythdf2-KO cells. Taken together, YTHDF2 regulates cell-matrix adhesion and proliferation through modulating the expression of Mmps by the m6A/mRNA degradation pathway.

Profiling of miRNAs in porcine Sertoli cells.

BACKGROUND: Sertoli cells (SCs) create a specialized environment to support and dictate spermatogenesis. MicroRNAs (miRNAs), a kind of ~ 22 nt small noncoding RNAs, have been reported to be highly abundant in mouse SCs and play critical roles in spermatogenesis. However, the miRNAs of porcine SCs remain largely unknown. METHODS: We isolated porcine SCs and conducted small RNA sequencing. By comparing miRNAs in germ cells, we systematically analyzed the miRNA expression pattern of porcine SCs. We screened the highly enriched SC miRNAs and predicted their functions by Gene Ontology analysis. The dual luciferase assay was used to elucidate the regulation of tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) by ssc-miR-149. RESULTS: The analysis showed that 18 miRNAs were highly expressed in SCs and 15 miRNAs were highly expressed in germ cells. These miRNAs were predicted to mediate SC and germ cell functions. In addition, ssc-miR-149 played critical roles in SCs by targeting TRAF3. CONCLUSION: Our findings provide novel insights into the miRNA expression pattern and their regulatory roles of porcine SCs.

Meclofenamic acid represses spermatogonial proliferation through modulating m6A RNA modification.

BACKGROUND: N6-Methyladenosine (m6A), the most prevalent modification in mammalian mRNA, plays important roles in numerous biological processes. Several m6A associated proteins such as methyltransferase like 3 (METTL3), methyltransferase like 14 (METTL14), α-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5) and YTH domain containing 2 (YTHDC2) are involved in the regulation of spermatogenesis and oogenesis. However, the role of the first detected m6A demethylase, fat mass and obesity associate protein (FTO), in germ cells remains elusive. Elucidation of FTO roles in the regulation of germ cell fate will provide novel insights into the mammalian reproduction. METHODS: Mouse GC-1 spg cells were treated with the ester form of meclofenamic acid (MA2) to inhibit the demethylase activity of FTO. The cellular m6A and m6Am level were analyzed through high performance liquid chromatography combined with tandem mass spectrometry (HPLC/MS-MS). The cell apoptosis was detected via TUNEL and flow cytometry. The cell proliferation was detected through EdU and western blot. The mRNA level of core cyclin dependent kinases (CDKs) was quantified via q-PCR. RNA decay assay were performed to detect RNA stability. Dual fluorescence assay was conducted to study whether MA2 affects the expression of CDK2 dependent on the m6A modification at 3'UTR. RESULTS: MA2 significantly increased the cellular m6A level and down-regulated the expression of CDK1, CDK2, CDK6 and CdC25a, resulting in arrest of G1/S transition and decrease of cell proliferation. MA2 downregulated CDK2 mRNA stability. Additionally, mutation of the predicted m6A sites in the Cdk2-3'UTR could mitigated the degradation of CDK2 mRNA after MA2 treatment. CONCLUSION: MA2 affected CDKs expression through the m6A-dependent mRNA degradation pathway, and thus repressed spermatogonial proliferation.

Preparation of Yolk-Shell-Structured Cox Fe1-x P with Enhanced OER Performance.

The design and development of low-cost, highly efficient, and stable electrocatalysts to take the place of noble-metal catalysts for the oxygen evolution reaction (OER) remain a significant challenge. Herein, the synthesis of yolk-shell-structured binary transition metal phosphide Cox Fe1-x P with different Co/Fe ratios by phosphidation of a cobalt ferrite precursor is reported. The as-synthesized Cox Fe1-x P catalysts were used for the OER. All yolk-shell Cox Fe1-x P catalysts with different Co/Fe ratios showed much better performance than the corresponding solid catalyst. The formation of Co oxides on the catalyst surface during OER and the optimal Co/Fe ratio were found to be critical to their activity. Among the as-prepared Cox Fe1-x P catalysts, that with a Co/Fe ratio of 0.47/0.53 (Co0.47 Fe0.53 P) exhibited the best performance. Co0.47 Fe0.53 P has an overpotential of 277 mV at a current density of 10 mA cm-2 , a Tafel slope of 37 mV dec-1 , and superior stability in alkaline medium. The outstanding performance is partly ascribed to the transfer of valence electrons from Co to P and Fe. The Co0.47 Fe0.53 P matrix with excellent conductivity and Fe phosphate that is stable on the surface of the catalyst are also helpful for the OER performance. In addition, the yolk-shell structure of Co0.47 Fe0.53 P increases the contact area between electrolyte and catalyst. These characteristics of Co0.47 Fe0.53 P greatly improve its OER performance. This optimized binary transition metal phosphide provides a new approach for the design of nonprecious-metal electrocatalysts.

Rational Construction of Hierarchically Porous Fe-Co/N-Doped Carbon/rGO Composites for Broadband Microwave Absorption.

Developing lightweight and broadband microwave absorbers for dealing with serious electromagnetic radiation pollution is a great challenge. Here, a novel Fe-Co/N-doped carbon/reduced graphene oxide (Fe-Co/NC/rGO) composite with hierarchically porous structure was designed and synthetized by in situ growth of Fe-doped Co-based metal organic frameworks (Co-MOF) on the sheets of porous cocoon-like rGO followed by calcination. The Fe-Co/NC composites are homogeneously distributed on the sheets of porous rGO. The Fe-Co/NC/rGO composite with multiple components (Fe/Co/NC/rGO) causes magnetic loss, dielectric loss, resistance loss, interfacial polarization, and good impedance matching. The hierarchically porous structure of the Fe-Co/NC/rGO enhances the multiple reflections and scattering of microwaves. Compared with the Co/NC and Fe-Co/NC, the hierarchically porous Fe-Co/NC/rGO composite exhibits much better microwave absorption performances due to the rational composition and porous structural design. Its minimum reflection loss (RLmin) reaches - 43.26 dB at 11.28 GHz with a thickness of 2.5 mm, and the effective absorption frequency (RL ≤ - 10 dB) is up to 9.12 GHz (8.88-18 GHz) with the same thickness of 2.5 mm. Moreover, the widest effective bandwidth of 9.29 GHz occurs at a thickness of 2.63 mm. This work provides a lightweight and broadband microwave absorbing material while offering a new idea to design excellent microwave absorbers with multicomponent and hierarchically porous structures.

FTO Knockout Causes Chromosome Instability and G2/M Arrest in Mouse GC-1 Cells.

N 6-methyladenosine (m6A) is the most abundant modification on eukaryotic mRNA. m6A plays important roles in the regulation of post-transcriptional RNA splicing, translation, and degradation. Increasing studies have uncovered the significance of m6A in various biological processes such as stem cell fate determination, carcinogenesis, adipogenesis, stress response, etc, which put forwards a novel conception called epitranscriptome. However, functions of the fat mass and obesity-associated protein (FTO), the first characterized m6A demethylase, in spermatogenesis remains obscure. Here we reported that depletion of FTO by CRISPR/Cas9 induces chromosome instability and G2/M arrest in mouse spermatogonia, which was partially rescued by expression of wild type FTO but not demethylase inactivated FTO. FTO depletion significantly decreased the expression of mitotic checkpoint complex and G2/M regulators. We further demonstrated that the m6A modification on Mad1, Mad2, Bub1b, Cdk1, and Ccnb2 were directly targeted by FTO. Therefore, FTO regulates cell cycle and mitosis checkpoint in spermatogonia because of its m6A demethylase activity. The findings give novel insights into the role of RNA methylation in spermatogenesis.

αMSH prevents ROS-induced apoptosis by inhibiting Foxo1/mTORC2 in mice adipose tissue.

Alpha-melanocyte stimulating hormone (αMSH) is an important adenohypophysis polypeptide hormone that regulates body metabolic status. To date, it is well known that the disorder of hypothalamic αMSH secretion is related to many metabolic diseases, such as obesity and type II diabetes. However, the underlying mechanisms are poorly understood. In our study, we focused on the reactive oxygen species (ROS)-induced adipocyte apoptosis and tried to unveil the role of αMSH in this process and the signal pathway which αMSH acts through. Kunming white mice were used and induced to oxidative stress status by hydrogen peroxide (H2O2) injection and a significant reduction of αMSH were found in mice serum, while elevated ROS level and mRNA level of pro-apoptotic genes were observed in mice adipose tissue. What is more, when detect the function of αMSH in ROS-induced apoptosis, similar inhibitory trend was found with the oxidative stress inhibitor N-acetyl-L-cysteine (NAC) in ROS-induced adipocyte apoptosis and this trend is αMSH receptor melanocortin 5 receptor (MC5R) depended, while an opposite trend was found between αMSH and Foxo1, which is a known positive regulator of adipocyte apoptosis. Further, we found that the repress effect of αMSH in adipocytes apoptosis is acting through Foxo1/mTORC2 pathway. These findings indicate that, αMSH has a strong inhibitory effect on ROS-induced adipocyte apoptosis and underlying mechanism is interacting with key factors in mTOR signal pathway. Our study demonstrated a great role of αMSH in adipocyte apoptosis and brings a new therapeutic mean to the treatment of obesity and diabetes.

Expression of a grape (Vitis vinifera) bZIP transcription factor, VlbZIP36, in Arabidopsis thaliana confers tolerance of drought stress during seed germination and seedling establishment.

Drought is one of the most serious factors that limit agricultural productivity and there is considerable interest in understanding the molecular bases of drought responses and their regulation. While numbers of basic leucine zipper (bZIP) transcription factors (TFs) are known to play key roles in response of plants to various abiotic stresses, only a few group K bZIP TFs have been functionally characterized in the context of stress signaling. In this study, we characterized the expression of the grape (Vitis vinifera) group K bZIP gene, VlbZIP36, and found evidence for its involvement in response to drought and the stress-associated phytohormone abscisic acid (ABA). Transgenic Arabidopsis thaliana lines over-expressing VlbZIP36 under the control of a constitutive promoter showed enhanced dehydration tolerance during the seed germination stage, as well as in the seedling and mature plant stages. The results indicated that VlbZIP36 plays a role in drought tolerance by improving the water status, through limiting water loss, and mitigating cellular damage. The latter was evidenced by reduced cell death, lower electrolyte leakage in the transgenic plants, as well as by increased activities of antioxidant enzymes. We concluded that VlbZIP36 enhances drought tolerance through the transcriptional regulation of ABA-/stress-related genes.