Background: Stellera chamaejasmeLinn, an important poisonous plant of the China grassland, is toxic to humans and livestock. The rapid expansion ofS. chamaejasmehas greatly damaged the grassland ecology and, consequently, seriously endangered the development of animal husbandry. To draft efficient prevention and control measures, it has become more urgent to carry out research on its adaptive and expansion mechanisms in different unfavorable habitats at the genetic level. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used technique for studying gene expression at the transcript level; however, qRT-PCR requires reference genes (RGs) as endogenous controls for data normalization and only through appropriate RG selection and qRT-PCR can we guarantee the reliability and robustness of expression studies and RNA-seq data analysis. Unfortunately, little research on the selection of RGs for gene expression data normalization inS. chamaejasmehas been reported.Method: In this study, 10 candidate RGs namely,18S,60S,CYP,GAPCP1,GAPDH2,EF1B,MDH,SAND,TUA1, andTUA6, were singled out from the transcriptome database ofS. chamaejasme, and their expression stability under three abiotic stresses (drought, cold, and salt) and three hormone treatments (abscisic acid, ABA; gibberellin, GA; ethephon, ETH) were estimated with the programs geNorm, NormFinder, and BestKeeper.Result: Our results showed thatGAPCP1andEF1Bwere the best combination for the three abiotic stresses, whereasTUA6andSAND,TUA1andCYP,GAPDH2and60Swere the best choices for ABA, GA, and ETH treatment, respectively. Moreover,GAPCP1and60Swere assessed to be the best combination for all samples, and18Swas the least stable RG for use as an internal control in all of the experimental subsets. The expression patterns of two target genes (P5CS2andGI) further verified that the RGs that we selected were suitable for gene expression normalization.Discussion: This work is the first attempt to comprehensively estimate the stability of RGs inS. chamaejasme. Our results provide suitable RGs for high-precision normalization in qRT-PCR analysis, thereby making it more convenient to analyze gene expression under these experimental conditions.

This study investigated the effect of recombinant human lactoferrin (rhLF) on the premature ovarian failure (POF) of rats. After cyclophosphamide treatments, the POF rats were divided into the following groups: normal control group (NC), low-dose group (LD), medium-dose group (MD) and high-dose group (HD) of rhLF. After drug administrations, the ovarian indexes and hormonal levels were detected. After follicle number count, the proliferation and apoptosis were analyzed with the expressions of genes related with oogenesis, reactive oxygen species (ROS) production and apoptosis detected, followed by the calculation of oxidative stress and protein expressions. After 4-hydroperoxy cyclophosphamide (4-HC) treatments, the effect of rhLF on the proliferation, ROS production and gene expressions of primary rat granulosa cells (GCs) cultured in vitro were detected. After mating, the fertilities of POF rats were recorded. The result showed that the rhLF administrations up-regulated the ovarian index with the number of developing follicles increased and the decreases of hormonal levels conferred. The Ki-67 intensities of the MD and HD groups were up-regulated with the Tunnel intensities decreased. The rhLF treatments significantly promoted the expression of oogenesis, antioxidant and anti-apoptosis related genes. The expression of Bax and Caspase 3 were decreased with the expression of Bcl-2 up-regulated after rhLF administrations. The in vitro treatments of rhLF effectively conferred the toxicity of 4-HC on primary rat GCs. The fertility assessment showed the rhLF treatments up-regulated the offspring’s’ folliculogenesis, which confirmed the ameliorative role of rhLF on the POF damages via the inhibition of ROS production in GCs.

Regardless of the essential role of copper (Cu) in the physiological regulation process of mammalian reproduction, excessive exposure to Cu triggers the meiotic defects of porcine oocytes via compromising the mitochondrial functions. However, the connections between the excessive Cu exposure and meiotic defects of ovine oocytes have not been reported. In this study, the effect of copper sulfate (CuSO₄) exposure on the meiotic potentials of ovine oocytes was analyzed. Subsequently, the ameliorative effect of glutathione (GSH) supplementation on the meiotic defects of CuSO₄ exposed ovine oocytes was investigated. For these purposes, the in vitro maturation (IVM) of ovine cumulus oocyte complexes (COCs) was conducted in the presence of 5, 10, 20 and 40 μg/mL of CuSO₄ supplementation. Subsequently, different concentrations of GSH (2, 4 and 8 mM) were added to the IVM medium containing CuSO₄ solution. After IVM, the assay, including nuclear maturation, spindle organization, chromosome alignment, cytoskeleton assembly, cortical granule (CGs) dynamics, mitochondrial function, reactive oxygen species (ROS) generation, apoptosis, epigenetic modification and fertilization capacity of ovine oocytes were performed. The results showed that excessive Cu exposure triggered the meiotic defects of ovine oocytes via promoting the mitochondrial dysfunction related oxidative stress damage. Moreover, the GSH supplementation, not only ameliorated the decreased maturation potential and fertilization defect of CuSO₄ exposed oocytes, but inhibited the mitochondrial dysfunction related oxidative stress damage, ROS generation, apoptosis and altered H3K27me3 expression in the CuSO₄ exposed oocytes. Combined with the gene expression pattern, the finding in the present study provided fundamental bases for the ameliorative effect of GSH supplementation on the meiotic defects of CuSO₄ exposed oocytes via inhibiting the mitochondrial dysfunctions, further benefiting these potential applications of GSH supplementation in the mammalian IVM system and livestock breeding suffering from the excessive Cu exposure.

The aim of this study was to investigate the effect of bioactive peptides (BAPT) from animal sources on the development of mouse preantral follicles in vitro. Preantral follicles were isolated and randomly divided into the following groups: an untreated group (control) and three groups supplemented with 20, 40 and 60 μg/mL BAPT, respectively. After establishing the in vitro follicle culture, the gene expression levels and hormone levels were quantified. After in vitro maturation, the developmental rates, reactive oxygen species (ROS) production levels and mitochondrial distributions of MII oocytes were investigated, followed by the analyses of embryonic developmental rates after in vitro fertilization.The results showed that BAPT promoted the growth of mouse preantral follicles. Notably, after 14 d of in vitro culture, the levels of 17 β-estradiol and progesterone were up-regulated with BAPT treatments. Moreover, the expression levels of Oct4, Bmp15, GDF9, FOXO3, Zp3, FOXL2, Inhibin alpha, SOD2, Catalase, GPx and Bcl-2 in the developing follicles were significantly up-regulated after BAPT treatments (P < 0.05), while BAPT significantly inhibited the expression levels of BAX (P < 0.05). Following BAPT treatments, the ROS production levels of MII oocytes were decreased while the mitochondrial distributions were significantly enhanced. Furthermore, increased maturation rates, fertilization and embryonic developmental rates were found in these BAPT-treated groups (P < 0.05).These results demonstrated that BAPT significantly improved the development of preantral follicles in vitro by reducing ROS-dependent cellular damages and by enhancing mitochondrial distributions, thereby promoting the further applications of animal-derived BAPT in biomedical research.