RESUMEN
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.
Asunto(s)
Familia de Aldehído Deshidrogenasa 1 , Células Lúteas , Retinal-Deshidrogenasa , Animales , Bovinos/genética , Femenino , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Familia de Aldehído Deshidrogenasa 1/genética , Familia de Aldehído Deshidrogenasa 1/metabolismo , Apoptosis , Proliferación Celular , Regulación de la Expresión Génica/fisiología , Células Lúteas/metabolismo , Progesterona/metabolismo , Retinal-Deshidrogenasa/genética , Retinal-Deshidrogenasa/metabolismoRESUMEN
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.
RESUMEN
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.
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Fertilidad , Espermatogénesis , Testículo , Animales , Masculino , Ratones , Fertilidad/genética , Ratones Noqueados , Semen , Espermatogénesis/genética , Espermatozoides/metabolismo , Testículo/metabolismoRESUMEN
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.
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Infertilidad Femenina , Animales , Femenino , Ratones , Embarazo , Blastocisto , Cadherinas/metabolismo , Cadherinas/farmacología , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Infertilidad Femenina/veterinaria , Mórula , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
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.
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Células del Cúmulo , Oocitos , Animales , Femenino , Bovinos , Células del Cúmulo/metabolismo , Oocitos/metabolismo , Gonadotropinas/metabolismo , Mamíferos/metabolismo , ApoptosisRESUMEN
At present, a real objects-based full-color holographic system usually uses a digital single-lens reflex (DSLR) camera array or depth camera to collect data. It then relies on a spatial light modulator to modulate the input light source for the reconstruction of the 3-D scene of the real objects. However, the main challenges the high-quality holographic 3-D display faced were the limitation of generation speed and the low accuracy of the computer-generated holograms. This research generates more effective and accurate point cloud data by developing an RGB-D salient object detection model in the acquisition unit. In addition, a divided point cloud gridding method is proposed to enhance the computing speed of hologram generation. In the RGB channels, we categorized each object point into depth grids with identical depth values. The depth girds are divided into M × N parts, and only the effective parts will be calculated. Compared with traditional methods, the calculation time is dramatically reduced. The feasibility of our proposed approach is established through experiments.
RESUMEN
As a widely used detergent, anionic surfactant linear alkylbenzene sulfonates (LAS) is a common toxic pollutant in wastewater. In this study, Pseudomonas sp. strain H6 was isolated from activated sludge and municipal wastewater, which had good degradation effect on LAS. The results showed that strain H6 could grow with LAS as the sole carbon source. When the concentration of LAS was less than 100 mg/L, strain H6 could degrade more than 80% of the LAS within 24 h. Meanwhile, the growth of strain H6 increased with the increase of LAS concentration, reaching the maximum growth at the presence of 100 mg/L LAS. When the concentration of LAS was over 100 mg/L, strain H6's cell growth and degradation of LAS showed a downward trend due to the strong toxicity of LAS, and the degradation rate of LAS almost tended to zero with 500 mg/L LAS. Further mutagenesis analysis of strain H6 showed that positive mutation occurred under ultraviolet and nitrite mutagenesis with using ampicillin to increase the screening pressure, and the degradation rate of LAS was 44.91% higher than that of original strain.