The Expression and Epigenetic Characteristics of the HSF2 Gene in Cattle-Yak and the Correlation with Its Male Sterility.

Aberrant expression of the heat shock proteins and factors was revealed to be closely associated with male reproduction. Heat shock factor 2 (HSF2) is a transcription factor that is involved in the regulation of diverse developmental pathways. However, the role and the corresponding molecular mechanism of HSF2 in male cattle-yak sterility are still poorly understood. Therefore, the aim of this study was to obtain the sequence and the biological information of the cattle-yak HSF2 gene and to investigate the spatiotemporal expression profiles of the locus during the development of cattle-yak testes. Additionally, the differential expression was analyzed between the cattle-yak and the yak, and the methylation of corresponding promoter regions was compared. Our results showed an additional 54 bp fragment and a missense mutation (lysine to glutamic acid) were presented in the cattle-yak HSF2 gene, which correlated with enriched expression in testicular tissue. In addition, the expression of the HSF2 gene showed dynamic changes during the growth of the testes, reaching a peak in adulthood. The IHC indicated that HSF2 protein was primarily located in spermatocytes (PS), spermatogonia (SP), and Sertoli cells (SC) in cattle-yak testes, compared with the corresponding cells of cattle and the yak. Furthermore, bisulfite-sequencing PCR (BSP) revealed that the methylated CpG sites in the promoter region of the cattle-yak HSF2 were more numerous than in the yak counterpart, which suggests hypermethylation of this region in the cattle-yak. Taken together, the low expression abundance and hypermethylation of HSF2 may underpin the obstruction of spermatogenesis, which leads to male cattle-yak infertility. Our study provided a basic guideline for the HSF2 gene in male reproduction and a new insight into the mechanisms of male cattle-yak sterility.

Molecular characterization and expression profile of the ALDH1A1 gene and its functions in yak luteal cells.

The ALDH1A1 gene encodes a cytoplasmic member of the aldehyde dehydrogenase 1 family, which plays an important role in regulating animal reproductive performance, including estrus cycle and embryonic development. The aim of this study was to characterize ALDH1A1 activity in ovaries of 3-5 year-old yaks and to determine its effects on cell proliferation, apoptosis, and progesterone secretion in luteal cells (LCs). The coding sequence (CDS) of the ALDH1A1 gene was cloned by reverse transcription-PCR and immunohistochemical analysis was used to confirm localization of the ALDH1A1 protein in the ovary. To assess the activity of ALDH1A1 in regulating progesterone secretion, si-ALDH1A1 was transfected into LCs in vitro and progesterone levels in LC supernatants were measured by ELISA. The interference efficiency was assessed by real-time quantitative PCR (RT-qPCR) and immunofluorescence staining, and cell proliferation and apoptosis were evaluated by EdU and TUNEL staining, respectively. The cloned ALDH1A1 sequence contained 1462 bp, encoding 487 amino acids. Immunohistochemical analysis showed that ALDH1A1 protein expression, which was significantly higher in LCs, was mainly found in antral follicles and the corpus luteum (CL). The expression of ALDH1A1 mRNA in LCs was effectively inhibited by si-ALDH1A1transfection, and progesterone secretion was markedly decreased along with the significant down-regulation of progesterone pathway-related genes, STAR, CYP11A1, CYP19A1, CYP17A1, 3ß-HSD, and HSD17B1. Knockdown of ALDH1A1 mRNA expression decreased cell proliferation and increased apoptosis in LCs. The mRNA expression of the proliferation-related genes, PCNA, CCND1, CCNB1 and CDC25A, was significantly down-regulated, while expression of the apoptosis-promoting CASP3 gene was significantly increased. In summary, we characterized the yak ALDH1A1 gene and revealed that ALDH1A1 knockdown promoted apoptosis, repressed cell proliferation, and decreased progesterone secretion by yak LCs, potentially by regulating the mRNA expression of genes related to proliferation, apoptosis, and progesterone synthesis and secretion.

Testis-specific knockout of Kdm2a reveals nonessential roles in male fertility but partially compromises spermatogenesis.

Lysine-specific histone demethylase 2 (Kdm2a) is a regulatory factor of histone modifications that participates in gametogenesis and embryonic development. The mis-regulation of Kdm2a can lead to aberrant gene expression, thereby contributing to abnormal cell proliferation, differentiation, apoptosis, and tumorigenesis. However, due to the potential confounding effects that are secondary to the loss of Kdm2a function from the soma in existing whole-animal mutants, the in vivo function of Kdm2a in spermatogenesis for male fertility remains unknown. Herein, we focus on exploring the spatiotemporal expression profile and biological functions of Kdm2a in the spermatogenesis and fertility of male mice. A testis-specific knockout Kdm2a model (Kdm2a cKO) was established by using the Stra8-Cre/loxP recombinase system to explore the roles of Kdm2a in male fertility. Our results showed that Kdm2a was ubiquitously expressed and dynamically distributed in multiple tissues and cell types in the testis of mice. Surprisingly, Kdm2a-deficient adult males were completely fertile and comparable with their control (Kdm2aflox/flox) counterparts. Despite the significantly reduced total number of sperm and density of seminiferous tubules in Kdm2a cKO testis accompanied by the degeneration of spermatogenesis, the fertilization ability and embryonic developmental competence of the Kdm2a cKO were comparable with those of their control littermates, suggesting that Kdm2a disruption did not markedly affect male fertility, at least during younger ages. Furthermore, Kdm2a homozygous mutants exhibited a lower total number and motility of sperm than the control group and showed notably affected serum 17ß-estradiol concentration. Interestingly, the transcriptome sequencing revealed that the loss of Kdm2a remarkably upregulated the expression level of Kdm2b. This effect, in turn, may induce compensative effects in the case of Kdm2a deficiency to maintain normal male reproduction. Together, our results reveal that Kdm2a shows spatiotemporal expression during testicular development and that its loss is insufficient to compromise the production of spermatozoa completely. The homologous Kdm2b gene might compensate for the loss of Kdm2a. Our work provides a novel Kdm2a cKO mouse allowing for the efficient deletion of Kdm2a in a testis-specific manner, and further investigated the biological function of Kdm2a and the compensatory effects of Kdm2b. Our study will advance our understanding of underlying mechanisms in spermatogenesis and male fertility.

Maternal Kdm2a-mediated PI3K/Akt signaling and E-cadherin stimulate the morula-to-blastocyst transition revealing crucial roles in early embryonic development.

Histone methylation plays an essential role in oocyte growth and preimplantation embryonic development. The modification relies on histone methyl-transferases and demethylases, and one of these, lysine-specific demethylase 2a (Kdm2a), is responsible for modulating histone methylation during oocyte and early embryonic development. The mechanism of how Kdm2a deficiency disrupts early embryonic development and fertility remains elusive. To determine if maternally deposited Kdm2a is required for preimplantation embryonic development, the expression profile of Kdm2a during early embryos was detected via immunofluorescence staining and RT-qPCR. The Kdm2a gene in oocytes was specifically deleted with the Zp3-Cre/LoxP system and the effects of maternal Kdm2a loss were studied through a comprehensive range of female reproductive parameters including fertilization, embryo development, and the number of births. RNA transcriptome sequencing was performed to determine differential mRNA expression, and the interaction between Kdm2a and the PI3K/Akt pathway was studied with a specific inhibitor and activator. Our results revealed that Kdm2a was continuously expressed in preimplantation embryos and loss of maternal Kdm2a suppressed the morula-to-blastocyst transition, which may have been responsible for female subfertility. After the deletion of Kdm2a, the global H3K36me2 methylation in mutant embryos was markedly increased, but the expression of E-cadherin decreased significantly in morula embryos compared to controls. Mechanistically, RNA-seq analysis revealed that deficiency of maternal Kdm2a altered the mRNA expression profile, especially in the PI3K/Akt signaling pathway. Interestingly, the addition of a PI3K/Akt inhibitor (LY294002) to the culture medium blocked embryo development at the stage of morula; however, the developmental block caused by maternal Kdm2a loss was partially rescued with a PI3K/Akt activator (SC79). In summary, our results indicate that loss of Kdm2a influences the transcriptome profile and disrupts the PI3K/Akt signaling pathway during the development of preimplantation embryo. This can result in embryo block at the morula stage and female subfertility, which suggests that maternal Kdm2a is a potential partial redundancy with other genes encoding enzymes in the dynamics of early embryonic development. Our results provide further insight into the role of histone modification, especially on Kdm2a, in preimplantation embryonic development in mice.

RFRP-3 Influences Apoptosis and Steroidogenesis of Yak Cumulus Cells and Compromises Oocyte Meiotic Maturation and Subsequent Developmental Competence.

RF amide-related peptide 3 (RFRP-3), a mammalian ortholog of gonadotropin-inhibitory hormone (GnIH), is identified to be a novel inhibitory endogenous neurohormonal peptide that regulates mammalian reproduction by binding with specific G protein-coupled receptors (GPRs) in various species. Herein, our objectives were to explore the biological functions of exogenous RFRP-3 on the apoptosis and steroidogenesis of yak cumulus cells (CCs) and the developmental potential of yak oocytes. The spatiotemporal expression pattern and localization of GnIH/RFRP-3 and its receptor GPR147 were determined in follicles and CCs. The effects of RFRP-3 on the proliferation and apoptosis of yak CCs were initially estimated by EdU assay and TUNEL staining. We confirmed that high-dose (10-6 mol/L) RFRP-3 suppressed viability and increased the apoptotic rates, implying that RFRP-3 could repress proliferation and induce apoptosis. Subsequently, the concentrations of E2 and P4 were significantly lower with 10-6 mol/L RFRP-3 treatment than that of the control counterparts, which indicated that the steroidogenesis of CCs was impaired after RFRP-3 treatment. Compared with the control group, 10-6 mol/L RFRP-3 treatment decreased the maturation of yak oocytes efficiently and subsequent developmental potential. We sought to explore the potential mechanism of RFRP-3-induced apoptosis and steroidogenesis, so we observed the levels of apoptotic regulatory factors and hormone synthesis-related factors in yak CCs after RFRP-3 treatment. Our results indicated that RFRP-3 dose-dependently elevated the expression of apoptosis markers (Caspase and Bax), whereas the expression levels of steroidogenesis-related factors (LHR, StAR, 3ß-HSD) were downregulated in a dose-dependent manner. However, all these effects were moderated by cotreatment with inhibitory RF9 of GPR147. These results demonstrated that RFRP-3 adjusted the expression of apoptotic and steroidogenic regulatory factors to induce apoptosis of CCs, probably through binding with its receptor GPR147, as well as compromised oocyte maturation and developmental potential. This research revealed the expression profiles of GnIH/RFRP-3 and GPR147 in yak CCs and supported a conserved inhibitory action on oocyte developmental competence.

The Biological Characteristics and Differential Expression Patterns of TSSK1B Gene in Yak and Its Infertile Hybrid Offspring.

This study aimed to investigate the spatially and temporally expressed patterns and biological characteristics of TSSK1B in male yaks and explore the potential correlation between TSSK1B and male sterility of the yak hybrid offspring (termed cattle-yak). First, the coding sequence (CDS) of TSSK1B was cloned by RT-PCR, and bioinformatics analysis was conducted with relevant software. Quantitative real-time PCR (RT-qPCR) was employed to detect the expression profile of TSSK1B in various tissues of male adult yaks, the spatiotemporal expression of TSSK1B in different stages of yak testes, and the differential expression of TSSK1B between yak and cattle-yak testes. The cellular localization of TSSK1B was determined by immunohistochemistry (IHC). Furthermore, the methylation status of the TSSK1B promoter region was analyzed by bisulfite-sequencing PCR (BSP). The results showed that TSSK1B was 1235 bp long, including 1104 bp of the CDS region, which encoded 367 amino acids. It was a conserved gene sharing the highest homology with Bos mutus (99.67%). In addition, the bioinformatics analysis revealed that TSSK1B was an unstable hydrophilic protein mainly containing the alpha helix of 34.06% and a random coil of 44.41%, with a transmembrane structure of 29 amino acids long. The RT-qPCR results demonstrated that TSSK1B was specifically expressed in yak testes compared with that in other tissues and especially highly expressed in adult yak testes. On the contrary, TSSK1B was hardly expressed in the testis of adult cattle-yak. IHC confirmed that TSSK1B protein was more strongly expressed in the testes of adult yaks than in their fetal and juvenile counterparts. Interestingly, nearly no expression was observed in the testes of cattle-yak compared with the corresponding testes of yak. Bisulfite-sequencing PCR (BSP) revealed that the methylated CpG sites in the TSSK1B promoter region of cattle-yak was significantly higher than that in the yak. Taken together, this study revealed that TSSK1B was specifically expressed in yak testes and highly expressed upon sexual maturity. Moreover, the rare expression in cattle-yak may be related to the hypermethylation of the promoter region, thereby providing a basis for further studies on the regulatory mechanism of TSSK1B in male cattle-yak sterility.

Oocyte-Specific Knockout of Histone Lysine Demethylase KDM2a Compromises Fertility by Blocking the Development of Follicles and Oocytes.

The methylation status of histones plays a crucial role in many cellular processes, including follicular and oocyte development. Lysine-specific demethylase 2a (KDM2a) has been reported to be closely associated with gametogenesis and reproductive performance, but the specific function and regulatory mechanism have been poorly characterized in vivo. We found KDM2a to be highly expressed in growing follicles and oocytes of mice in this study. To elucidate the physiological role of Kdm2a, the zona pellucida 3-Cre (Zp3-Cre)/LoxP system was used to generate an oocyte Kdm2a conditional knockout (Zp3-Cre; Kdm2aflox/flox, termed Kdm2a cKO) model. Our results showed that the number of pups was reduced by approximately 50% in adult Kdm2a cKO female mice mating with wildtype males than that of the control (Kdm2aflox/flox) group. To analyze the potential causes, the ovaries of Kdm2a cKO mice were subjected to histological examination, and results indicated an obvious difference in follicular development between Kdm2a cKO and control female mice and partial arrest at the primary antral follicle stage. The GVBD and matured rates of oocytes were also compromised after conditional knockout Kdm2a, and the morphological abnormal oocytes increased. Furthermore, the level of 17ß-estradiol of Kdm2a cKO mice was only 60% of that in the counterparts, and hormone sensitivity decreased as the total number of ovulated and matured oocytes decreased after superovulation. After deletion of Kdm2a, the patterns of H3K36me2/3 in GVBD-stage oocytes were remarkedly changed. Transcriptome sequencing showed that the mRNA expression profiles in Kdm2a cKO oocytes were significantly different, and numerous differentially expressed genes were involved in pathways regulating follicular and oocyte development. Taken together, these results indicated that the oocyte-specific knockout Kdm2a gene led to female subfertility, suggesting the crucial role of Kdm2a in epigenetic modification and follicular and oocyte development.

MicroRNA-34b-5p targets PPP1R11 to inhibit proliferation and promote apoptosis in cattleyak Sertoli cells by regulating specific signaling pathways.

Cattleyaks, a hybrid of (♂) and yak (♀), exhibit the marked productivity and adaptability of plateau, but suffer from male infertility. Small non-coding RNAs, especially miRNAs, play crucial roles in spermatogenesis and affect the growth of Sertoli cells (SCs). The objective of the present study was to explore the interaction between miR-34b-5p and protein phosphatase 1 regulatory inhibitor subunit 11 (PPP1R11) and its effect on cattleyak SCs. RT-qPCR was used to determine the expression pattern of miR-34b-5p and PPP1R11, while the cellular and subcellular localization of PPP1R11 was determined by immunohistochemistry and immunocytochemistry. The interaction between MiR-34b-5p and PPP1R11 was evaluated by immunofluorescence, proliferation, apoptosis, and western blotting assays. The potential binding sites between miR-34b-5p and PPP1R11 were uncovered through targeted search of an online database, and verified using a dual luciferase reporter system. Our data show that miR-34b-5p is differentially expressed in the testes and SCs of cattleyaks compared to yaks. Overexpression of miR-34b-5p in SCs suppressed proliferation and induced apoptosis, while the effects of miR-34b-5p knockdown were the reverse. The 3'UTR of PPP1R11 was identified as a potential target site of miR-34b-5p, and this was validated by online database searches and our data from the dual-luciferase reporter assay, and it displayed an inverse expression pattern to miR-34b-5p in SCs. The effects of silencing PPP1R11 by siRNA were similar to the results of miR-34b-5p upregulation, but significantly different from miR-34b-5p downregulation in cattleyak SCs. The effects with PPP1R11 overexpression were opposite, suggesting a novel biofunctional role of PPP1R11 inactivation in depressing cattleyak SCs growth. Lastly, we confirmed that miR-34b-5p inhibited PPP1R11 expression and induced apoptosis by regulating proliferation- and apoptosis-related genes in SCs. Thus, miR-34b-5p regulates the apoptosis and proliferation of cattleyak SCs via targeting PPP1R11, which can provide an innovative direction for exploring the mechanism of cattleyak male sterility.

MicroRNA-342-3p regulates yak oocyte meiotic maturation by targeting DNA methyltransferase 1.

MicroRNAs (miRNAs) play vital roles in the development of oocytes and ovarian follicles. We have previously shown differential expression of miR-342-3p during yak oocyte maturation. In this study, we investigated the role of miR-342-3p in meiotic maturation of yak oocytes and the underlying mechanism. The profile of ovarian DNA methyltransferase 1 (DNMT1) expression was investigated in yak by RT-qPCR and western blot analyses. The pattern of Dnmt1 expression in various meiotic stages (GV stage, MI stage and MII stage) of yak oocyte maturation was then measured by immunofluorescence staining. The interaction between Dnmt1 and miR-342-3p was verified by dual-luciferase reporter assay. Finally, miR-342-3p inhibitors were microinjected into yak cumulus-oocyte complex to evaluate the effects on oocyte maturation. MiR-342-3p expression was upregulated in oocytes during meiotic maturation, with significantly higher levels in the MII stage compared with the GV- and MI stages (p < .05), whereas the opposite pattern of Dnmt1 expression was detected. In the period to sexual maturity (3-year-old), DNMT1 showed an age-related pattern of ovarian expression at both the gene and protein levels. Immunohistochemistry analysis also indicated maturation-stage-related differences in DNMT1 expression in the ovarian follicles and corpus luteum, with expression predominantly detected in cumulus cells and oocytes. MiR-342-3p inhibitors effectively upregulated Dnmt1 expression and significantly inhibited oocyte meiotic maturation. Taken together, our results indicate that miR-342-3p plays a vital role in the meiotic maturation of yak oocytes by targeting the 3'-untranslated regions (UTR) of Dnmt1 and provide a new perspective on the mechanism of this process.

Biodegradable Black Phosphorus-based Nanomaterials in Biomedicine: Theranostic Applications.

Ascribe to the unique two-dimensional planar nanostructure with exceptional physical and chemical properties, black phosphorous (BP) as the emerging inorganic twodimensional nanomaterial with high biocompatibility and degradability has been becoming one of the most promising materials of great potentials in biomedicine. The exfoliated BP sheets possess ultra-high surface area available for valid bio-conjugation and molecular loading for chemotherapy. Utilizing the intrinsic near-infrared optical absorbance, BPbased photothermal therapy in vivo, photodynamic therapy and biomedical imaging has been realized, achieving unprecedented anti-tumor therapeutic efficacy in animal experiments. Additionally, the BP nanosheets can strongly react with oxygen and water, and finally degrade to non-toxic phosphate and phosphonate in the aqueous solution. This manuscript aimed to summarize the preliminary progresses on theranostic application of BP and its derivatives black phosphorus quantum dots (BPQDs), and discussed the prospects and the state-of-art unsolved critical issues of using BP-based material for theranostic applications.

Synthesis of Au NP@MoS2 Quantum Dots Core@Shell Nanocomposites for SERS Bio-Analysis and Label-Free Bio-Imaging.

In this work, we report a facile method using MoS2 quantum dots (QDs) as reducers to directly react with HAuCl4 for the synthesis of Au nanoparticle@MoS2 quantum dots (Au NP@MoS2 QDs) core@shell nanocomposites with an ultrathin shell of ca. 1 nm. The prepared Au NP@MoS2 QDs reveal high surface enhanced Raman scattering (SERS) performance regarding sensitivity as well as the satisfactory SERS reproducibility and stability. The limit of detection of the hybrids for crystal violet can reach 0.5 nM with a reasonable linear response range from 0.5 µM to 0.5 nM (R² ≈ 0.974). Furthermore, the near-infrared SERS detection based on Au NP@MoS2 QDs in living cells is achieved with distinct Raman signals which are clearly assigned to the various cellular components. Meanwhile, the distinguishable SERS images are acquired from the 4T1 cells with the incubation of Au NP@MoS2 QDs. Consequently, the straightforward strategy of using Au NP@MoS2 QDs exhibits great potential as a superior SERS substrate for chemical and biological detection as well as bio-imaging.

Multifunctional Nanoplatform Based on Black Phosphorus Quantum Dots for Bioimaging and Photodynamic/Photothermal Synergistic Cancer Therapy.

A multifunctional nanoplatform based on black phosphorus quantum dots (BPQDs) was developed for cancer bioimaging and combined photothermal therapy (PTT) and photodynamic therapy (PDT). BPQDs were functionalized with PEG chains to achieve improved biocompatibility and physiological stability. The as-prepared nanoparticles exhibite prominent near-infrared (NIR) photothermal and red-light-triggered photodynamic properties. The combined therapeutic application of PEGylated BPQDs were then performed in vitro and in vivo. The results demonstrate that the combined phototherapy significantly promote the therapeutic efficacy of cancer treatment in comparison with PTT or PDT alone. BPQDs could also serve as the loading platform for fluorescent molecules, allowing reliable imaging of cancer cells. In addition, the low cytotoxicity and negligible side effects to main organs were observed in toxicity experiments. The theranostic characteristics of PEGylated BPQDs provide an uplifting potential for the future clinical applications.

Facile synthesis of black phosphorus-Au nanocomposites for enhanced photothermal cancer therapy and surface-enhanced Raman scattering analysis.

Black phosphorus (BP), a new type of two-dimensional nanomaterial, has attracted crucial attention in recent years owing to its excellent properties and great potential in various chemical, physical, and biological fields. In this study, BP nanosheets loaded with Au nanoparticles (BP-Au NSs) are obtained by a one-step facile synthetic method. The Au nanostructures can not only enhance the photothermal efficiency of the nanocomposites, but also endow BP-Au NSs with the potential to act as effective surface-enhanced Raman scattering (SERS) substrates for Raman biodetection. Cancer photothermal therapy (PTT) has been carried out in vitro and in vivo using BP-Au NSs as nanoagents. Under irradiation by an 808 nm laser, BP-Au NSs are capable of producing sufficient hyperthermia to destroy cancer cells, and the transplanted tumors in most of the tumor-bearing mice disappeared; BP-Au NSs are more effective than bare BP nanosheets. The PTT effect can also be monitored by a Raman technique that benefits from the high SERS activity of the BP-Au NSs. The molecular fingerprint features of breast tumors before and after PTT treatment were clearly identified using SERS analysis. The theranostic applications of BP-Au NSs exhibit promising potential in biomedicine.

Biological pH sensing based on the environmentally friendly Raman technique through a polyaniline probe.

The biological pH plays an important role in various cellular processes. In this work, a novel strategy is reported for biological pH sensing by using Raman spectroscopy and polyaniline nanoparticles (PANI NPs) as the pH-sensitive Raman probe. It is found that the Raman spectrum of PANI NPs is strongly dependent on the pH value. The intensities of Raman spectral bands at 1225 and 1454 cm-1 increase obviously with pH value varying from 5.5 to 8.0, which covers the range of regular biological pH variation. The pH-dependent Raman performance of PANI NPs, as well as their robust Raman signals and sensitivities to pH, was well retained after the nanoparticles incorporated into living 4T1 breast adenocarcinoma cells. The data indicate that such PANI NPs can be used as an effective biological pH sensor. Most interestingly, the PANI spherical nanostructures can be acquired by a low-cost, metal-free, and one-pot oxidative polymerization, which gives them excellent biocompatibility for further biological applications.