Regulatory Effects of the Kiss1 Gene in the Testis on Puberty and Reproduction in Hezuo and Landrance Boars.

Kisspeptin, a neuropeptide encoded by the Kiss1 gene, combines with its receptor Kiss1R to regulate the onset of puberty and male fertility by the hypothalamic-pituitary-gonadal axis. However, little is known regarding the expression signatures and molecular functions of Kiss1 in the testis. H&E staining revealed that well-arranged spermatogonia, spermatocytes, round and elongated spermatids, and spermatozoa, were observed in 4-, 6-, and 8-month-old testes compared to 1- and 3-month-old testes of Hezuo pigs; however, these were not observed in Landrance until 6 months. The diameter, perimeter, and cross-sectional area of seminiferous tubules and the perimeter and area of the tubular lumen increased gradually with age in both pigs. Still, Hezuo pigs grew faster than Landrance. The cloning results suggested that the Hezuo pigs' Kiss1 CDS region is 417 bp in length, encodes 138 amino acids, and is highly conserved in the kisspeptin-10 region. qRT-PCR and Western blot indicated that the expression trends of Kiss1 mRNA and protein were essentially identical, with higher expression levels at post-pubertal stages. Immunohistochemistry demonstrated that the Kiss1 protein was mainly located in Leydig cells and post-pubertal spermatogenic cells, ranging from round spermatids to spermatozoa. These studies suggest that Kiss1 is an essential regulator in the onset of puberty and spermatogenesis of boars.

Molecular characterization, expression patterns and cellular localization of BCAS2 gene in male Hezuo pig.

Background: Breast carcinoma amplified sequence 2 (BCAS2) participates in pre-mRNA splicing and DNA damage response, which is implicated in spermatogenesis and meiosis initiation in mouse. Nevertheless, the physiological roles of BCAS2 in the testes of large mammals especially boars remain largely unknown. Methods: In this study, testes were collected from Hezuo pig at three development stages including 30 days old (30 d), 120 days old (120 d), and 240 days old (240 d). BCAS2 CDS region was firstly cloned using RT-PCR method, and its molecular characteristics were identified using relevant bioinformatics software. Additionally, the expression patterns and cellular localization of BCAS2 were analyzed by quantitative real-time PCR (qRT-PCR), Western blot, immunohistochemistry and immunofluorescence. Results: The cloning and sequence analysis indicated that the Hezuo pig BCAS2 CDS fragment encompassed 678 bp open reading frame (ORF) capable of encoding 225 amino acid residues, and possessed high identities with some other mammals. The results of qRT-PCR and Western blot displayed that BCAS2 levels both mRNA and protein were age-dependent increased (p < 0.01). Additionally, immunohistochemistry and immunofluorescence results revealed that BCAS2 protein was mainly observed in nucleus of gonocytes at 30 d testes as well as nucleus of spermatogonia and Sertoli cells at 120 and 240 d testes. Accordingly, we conclude that BCAS2 is critical for testicular development and spermatogenesis of Hezuo pig, perhaps by regulating proliferation or differentiation of gonocytes, pre-mRNA splicing of spermatogonia and functional maintenance of Sertoli cells, but specific mechanism still requires be further investigated.

Cloning, expression analysis and localization of DAZL gene implicated in germ cell development of male Hezuo pig.

Deleted in azoospermia-like (DAZL) is essential for mammalian testicular function and spermatogenesis. To explore the molecular characterization, expression patterns, and cellular localization of the DAZL in Hezuo pig testes, testicular tissue was isolated from Hezuo pig at five development stages including 30 days old (30 d), 90 days old (90 d), 120 days old (120 d), 180 days old (180 d), and 240 days old (240 d). DAZL cDNA was first cloned using the RT-PCR method, and its molecular characterization was analyzed using relevant bioinformatics software. Subsequently, the expression patterns and cellular localization of DAZL were evaluated using quantitative real-time PCR (qRT-PCR), Western blot, and immunohistochemistry. The cloning and sequence analysis showed that the Hezuo pig DAZL cDNA fragment contained 888 bp open reading frame (ORF) capable of encoding 295 amino acid residues and exhibited high identities with some other mammals. The qRT-PCR and Western blot results indicated that DAZL was specifically expressed in Hezuo pig testes, and DAZL levels of both mRNA and protein were expressed at all five reproductive stages of Hezuo pig testes, with extremely significant higher expression levels in 90 d, 120 d, 180 d, and 240 d than those in 30 d (p < 0.01). Additionally, immunohistochemistry results revealed that DAZL protein was mainly localized in gonocytes at 30 d testes, primary spermatocytes, and spermatozoon at other developmental stages, and Leydig cells throughout five development stages. Together, these results suggested that DAZL may play an important role by regulating the proliferation or differentiation of gonocytes, development of primary spermatocytes and spermatozoon, and functional maintenance of Leydig cells in testicular development and spermatogenesis of Hezuo pig. Nevertheless, the specific regulatory mechanisms underlying these phenomena still requires further investigated and verified.

Nitrogen-doped carbonized polymer dots activated by alkalinity of arginine for multicolor multi-platform sensor.

Among carbon-based materials, carbon dots (CDs) are popular because of their stable optical properties and good biocompatibility. Their fluorescence properties often are tailored by size, surface modification, or heteroatom doping with multiple precursors. In this paper, the alkalinity of Arginine (Arg) acts as an activator to promote carbonation and increase luminescence efficiency. Meanwhile, Arg acts as a tetrahedron and plays another three roles in preparing amino-modified nitrogen-doped carbonized polymer dots (SA-NCPDs), in which Arg serves as carbon source, nitrogen-doped nitrogen source, and surface modifier. The NCPDs based only on arginine display narrow full width at half maximum (FWHM) of 54 nm. The SA-NCPDs present dual emission distribution in the UV and visible blue regions, respectively. The SA-NCPDs present multicolor emission, especially crimson emission in water under 77 K in different solvents. Besides, SA-NCPDs in different solvents and the prepared polyvinyl alcohol (PVA) composite film have different phosphorescence and long afterglow at 77 K. The excellent biocompatibility and stability of the SA-NCPDs imply it is a potential material in biomedicine. In addition, a multiplatform multicolor ratiometric sensor and visualized colorimetric sensor with high selectivity were constructed successfully for detecting Cu2+, H2O2, and OPD based on self-absorption.

Identification and Characterization of lncRNA and mRNA in Testes of Landrace and Hezuo Boars.

Chinese HZ boars are typical plateau miniature boars characterized by precocious puberty, which is closely related to testicular development and spermatogenesis. Accumulating evidence indicates that lncRNA is involved in the testicular development and regulation of spermatogenesis. However, little is known about the lncRNA precocious regulation in testicular development and spermatogenesis on early sexual maturity of HZ boars. Thus, we investigated the expression and characterization of lncRNA and mRNA in 30-day-old and 120-day-old HZ boar testes using transcriptome to explore precocious puberty. Landrace (LC) boar was treated as the control. Histological analyses indicated that HZ boar underwent puberty development at an earlier stage than LC boar and had achieved sexual maturity at 120 days old. RNA-Seq yielded a total of 187 lncRNAs and 984 mRNAs; these molecules were identified as possible candidates for precocious puberty. GO terms and KEGG pathways enrichment analyses revealed that the differentially expressed lncRNA and their targeted genes were involved in metabolic pathways regulating testis development and spermatogenesis, such as the PI3K-Akt, TGF-beta and Wnt pathways. Further screening, some lncRNA (such as LOC102166140, LOC110259451, and MSTRG.15011.2), and mRNA (such as PDCL2, HSD17B4, SHCBP1L, CYP21A2, and SPATA3) were found to be possibly associated with precocious puberty, which would add to our understanding of the molecular regulatory mechanisms of precocious puberty. This study provided valuable information for further study of the role of lncRNA and mRNA in the process of precocious puberty.

Significantly Red-Shifted Emissions of Nonconventional AIE Polymers Containing Zwitterionic Components.

Nonconventional luminescence polymers without any aromatic structures have attracted great interest from researchers due to their special structure and excellent biocompatibility. However, these materials mostly emit in the blue or green region, in which preparation of materials with long-wavelength (especially near-infrared) emission is still a great challenge. In this work, it is found that 2-(dimethyl amino) ethyl methacrylate (DMA) and itaconic anhydride (ITA) undergo a ring-opening reaction at room temperature, and subsequently generate zwitterionic compound (IDMA). Based on the clustering-triggered emission (CTE) mechanism, ionic bond can effectively promote the isolated electron-rich chromophores to form new emissive clusters with extended electron delocalization. Herein, two oligomers (P1 and P2) with different fluorescence emissions by controlling the concentration of zwitterionic monomers before polymerization are synthesized. It is worth noting that the maximum emission wavelength of P2 at high concentration is up to 712 nm, which is very rare in previous reports. In addition, the resulting oligomer (P2) shows typical aggregation-enhanced emission (AEE), excitation-dependent fluorescence, temperature-sensitive emission, and solvatochromism. The cytotoxicity assay demonstrates that P2 was low toxic to Huh7 and LM3 cells, and suitable for cell imaging. This research provides the possibility for rational molecular design and the feasibility of luminescence regulation.

Aggregation induced emission of amino-thiol capped gold nanoparticles (GNPs) through metal-amino-coordination.

Au11(SG)7 gold nanoparticles (GNPs) were synthesized from HAuCl4 using thiol compounds containing an amino group to serve as both the reducing agent and the ligand. A three-dimensional network structure (…Au-SNH2→Mn+⟵H2NS-Au…) was formed after the Mn+ (Pb2+, Cd2+, Zn2+ and Ag+) coordinated the gold nanoparticles through the amino group in the thiol ligand, which promoted aurophilicity (…Au…Au…) and induced GNP aggregation and emission. The differences in coordination between the amino group and metal ions resulted in different emission wavelengths (Pb2+, Cd2+, Zn2+ and Ag+: λex = 365 nm˜370 nm, λem = 580, 645, 630 and 565 nm). Aggregation induced emission of amino thiol capped GNPs via coordination of Pb2+ or Cd2+ can be used as a fluorescent sensor of the both metal ions (λex = 365 nm, λem = 580/645 nm) and were used for living bioimaging in vivo and in vitro.