Exploration of intrinsic and extrinsic apoptotic pathways in zearalenone-treated rat sertoli cells.

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin produced mainly by Fusarium. ZEA causes reproductive disorders and is both cytotoxic and genotoxic in animals; however, little is known regarding the molecular mechanism(s) leading to ZEA toxicity. Sertoli cells are somatic cells that support the development of spermatogenic cells. The objective of this study was to explore the effects of ZEA on the proliferation, apoptosis, and necrosis of rat Sertoli cells to uncover signaling pathways underlying ZEA cytotoxicity. ZEA reduced the proliferation of rat Sertoli cells in a dose-dependent manner, as indicated by a CCK8 assay, while flow cytometry revealed that ZEA caused both apoptosis and necrosis. Immunoblotting revealed that ZEA treatment increased the ratio of Bax/Bcl-2, as well as the expression of FasL and caspases-3, -8, and -9, in a dose-dependent manner. Collectively, these data suggest that ZEA induced apoptosis and necrosis in rat Sertoli cells via extrinsic and intrinsic apoptotic pathways. This study provides new insights into the molecular mechanisms by which ZEA exhibits cytotoxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1731-1739, 2016.

Zearalenone exposure affects mouse oocyte meiotic maturation and granulosa cell proliferation.

Zearalenone (ZEN) is a metabolite of Fusarium and is a common contaminant of grains and foodstuffs. ZEN acts as a xenoestrogen and is considered to be cytotoxic, tissue toxic, and genotoxic, which causes abortions and stillbirths in humans and animals. Since estrogens affect oocyte maturation during meiosis, in this study we investigated the effects of ZEN on mouse oocyte meiotic maturation and granulosa cell proliferation. Our results showed that ZEN-treated oocyte maturation rates were decreased, which might be due to the disrupted cytoskeletons: (1) ZEN treatment resulted in significantly more oocytes with abnormal spindle morphologies; (2) actin filament expression and distribution were also disrupted after ZEN treatment, which was confirmed by the aberrant distribution of actin regulatory proteins. In addition, cortical granule-free domains (CGFDs) were disrupted after ZEN treatment, which indicated that ZEN may affect mouse oocyte fertilization capability. ZEN reduced mouse granulosa cell proliferation in a dose-dependent manner as determined by MTT assay and TUNEL apoptosis analysis, which may be another cause for the decreased oocyte maturation. Thus, our results demonstrated that exposure to zearalenone affected oocyte meiotic maturation and granulosa cell proliferation in mouse.

Oocyte quality in mice is affected by a mycotoxin-contaminated diet.

Mycotoxins, such as deoxynivalenol (DON), zearalenone (ZEN), and aflatoxin (AF), are commonly found in many food commodities and may impair the growth and reproductive efficiency of animals and humans. We investigated the effects of a mycotoxin-contaminated diet on mouse oocyte quality. Maize contaminated with DON (3.875 mg/kg), ZEN (1,897 µg/kg), and AF (806 µg/kg) was incorporated into a mouse diet at three different levels (0, 15, and 30% w/w). After 4 weeks, ovarian and germinal vesicle oocyte indices decreased in mycotoxin-fed mice. Oocytes from these mice exhibited low developmental competence with reduced germinal vesicle breakdown and polar body extrusion rates. Embryo developmental competence also showed a similar pattern, and the majority of embryos could not develop to the morula stage. Actin expression was also reduced in both the oocyte cortex and cytoplasm, which was accompanied by decreased expression of the actin nucleation factors profilin-1 and mDia1. Moreover, a large percentage of oocytes derived from mice that were fed a mycotoxin-contaminated diet exhibited aberrant spindle morphology, a loss of the cortical granule-free domain, and abnormal mitochondrial distributions, which further supported the decreased oocyte quality. Thus, our results demonstrate that mycotoxins are toxic to the mouse reproductive system by affecting oocyte quality.

[G protein-coupled receptor 3: a key factor in the regulation of the nervous system and follicle development].

G protein-coupled receptors (GPCRs), the largest cell surface receptor superfamily, are involved in many physiological and pathological processes. G protein-coupled receptor 3 (Gpr3) is a newly discovered sphingosine 1-phosphate receptor, which directly or indirectly takes part in regulating the processes of nervous system and follicle development in the vertebrates. As a potential therapeutic drug target for a variety of neurological diseases and premature ovarian failure, its physiological function and biological mechanisms deserve further studies. In this paper, we reviewed the functions of Gpr3 in the processes of nervous system development and ovarian follicular development in the vertebrates.

Mycotoxin-containing diet causes oxidative stress in the mouse.

Mycotoxins which mainly consist of Aflatoxin (AF), Zearalenone (ZEN) and Deoxynivalenol (DON) are commonly found in many food commodities. Although each component has been shown to cause liver toxicity and oxidative stress in several species, there is no evidence regarding the effect of naturally contained multiple mycotoxins on tissue toxicity and oxidative stress in vivo. In the present study, mycotoxins-contaminated maize (AF 597 µg/kg, ZEN 729 µg/kg, DON 3.1 mg/kg maize) was incorporated into the diet at three different doses (0, 5 and 20%) to feed the mice, and blood and tissue samples were collected to examine the oxidative stress related indexes. The results showed that the indexes of liver, kidney and spleen were all increased and the liver and kidney morphologies changed in the mycotoxin-treated mice. Also, the treatment resulted in the elevated glutathione peroxidase (GPx) activity and malondialdehyde (MDA) level in the serum and liver, indicating the presence of the oxidative stress. Moreover, the decrease of catalase (CAT) activity in the serum, liver and kidney as well as superoxide dismutase (SOD) activity in the liver and kidney tissue further confirmed the occurrence of oxidative stress. In conclusion, our data indicate that the naturally contained mycotoxins are toxic in vivo and able to induce the oxidant stress in the mouse.

Sphingosine 1-phosphate acts as an activator for the porcine Gpr3 of constitutively active G protein-coupled receptors.

We cloned the complete coding sequences of porcine Gpr3, Gpr6, and Gpr12 genes. Further, on the basis of their high levels of sequence similarity, these genes are identified as a subfamily of G protein-coupled receptors. These putative protein sequences also showed high sequence identity with other mammalian orthologs, including several highly conserved motifs. A wide expression of the Gpr3 gene in pigs was observed through tissue distribution analysis by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR, specially in the brain, pituitary, fat, liver and oocyte, where its strong expression was observed. The Gpr3 gene was found to be located on chromosome 6 and a single exon coded for the entire open-reading frame. Expression of porcine Gpr3 in HEK293 cells resulted in constitutive activation of adenylate cyclase (AC) similar in amplitude to that produced by fully stimulated G(s)-coupled receptors. Moreover, sphingosine 1-phosphate (S1P) could increase AC activation via the constitutively active Gpr3 receptor. When a Gpr3-green fluorescent protein (GFP) construct was expressed in HEK293 cells, GFP-labeled Gpr3 protein was shown to be localized in the plasmalemma and subcellular membranes. After S1P treatment, agonist-mediated internalization could be visualized by confocal microscopy. In short, our findings suggest the porcine Gpr3, Gpr6, and Gpr12 genes as a subfamily of G protein-coupled receptors, and porcine Gpr3 was a constitutively active G protein-coupled receptor. Constitutive activation of AC and agonist-mediated internalization of Gpr3 receptor could be modulated by the S1P, suggesting that S1P might act as an activator for porcine Gpr3 receptor.

Effect of interrupted endogenous BMP/Smad signaling on growth and steroidogenesis of porcine granulosa cells.

Bone morphogenetic proteins (BMPs) play a critical role in the growth and steroidogenesis of granulosa cells (GCs). BMP signals act through membrane-bound heteromeric serine/threonine kinase receptors. Upon ligand binding, BMPs activate intracellular Smad proteins and regulate growth and apoptosis in various cell types. The objective of this study was to demonstrate the effects of BMP/Smad signal on growth and steroidogenesis of porcine GCs. A strategy of RNA interference (RNAi)-mediated 'gene silencing' of Smad4, a core molecule mediating the intracellular BMP/Smad signal transduction pathways, was used to interrupt endogenous BMP/Smad signaling. Results indicate that Smad4-small interfering RNA (siRNA) caused specific inhibition of Smad4 mRNA and protein expression after transfection. Interrupted endogenous BMP/Smad signaling significantly inhibited growth, and induced apoptosis of porcine GCs, while decreasing estradiol production. In addition, interrupted BMP/Smad signaling significantly (P<0.05) changed the expression of Cyclin D2, CDK4, Bcl-2, and Cyp19a1. These findings provide new insights into how BMP/Smad signaling regulates the growth and steroidogenesis of porcine GCs.

[Signal transduction of BMP/Smad and its relationship with mammalian folliculogenesis].

BMPs belong to the transforming growth factor-b superfamily. BMPs have been proved to have extensive biological functions in mammals, including growth regulation, cell proliferation and differentiation. More and more evidence has shown that BMPs play a key role in fertility, especially in folliculogenesis in female mammals. Smad proteins are intra-cellular signaling transduction molecules of BMP family, which can transduce the BMP signal from the cell membrane to the nucleus. In this review, BMPs, BMP/Smad-related signal transduction and the regulation of BMP activity were summarized, and the regulatory roles of BMP/Smad signal transduction pathway in folliculogenesis were discussed.

The correlation of reproduction-related gene expression with germ cell number in DM and PLL gilts.

In this study, the ovarian germ cell number was counted in 3-week-old Duroc x Meishan (DM, n=30) and PIC x (Landrace x Large White) (PLL, n=53) gilts, and the mRNA expression levels of four reproduction-related genes were investigated by quantitative RT-PCR. Correlation of germ cell number with the expression level of these genes was analyzed. Results showed that the germ cell number of DM was significantly higher than that of PLL gilts (P<0.01), although there was no significant difference between the ovarian weight of DM and PLL gilts (P=0.269). No significant correlation existed between germ cell number and ovarian weight in the two gilt groups (R=0.335, P=0.07; R=0.119, P=0.398, respectively). A significant correlation was found between the germ cell number and expression level of ESR and IGF1R mRNA in DM gilts (R=0.648, P<0.05; R=0.757, P<0.01, respectively), but the correlation between the germ cell number and expression level of FSHR and INHBA mRNA did not reach statistical significance. Significant correlation was found between the germ cell number and the expression level of ESR, FSHR, and IGF1R mRNA in PLL gilts (R=0.435, P<0.01; R=0.438, P<0.01; R=0.292, P<0.05, respectively), but not with INHBA mRNA in PLL gilts.

[Linkage analysis of five genes in pigs using radiation hybrid clone panel].

RH (radiation hybrid) has proved to be an effective method in constructing human genome maps (including ESTs, STSs and microsatellites). In this study, based on the information of five human genes (FMR1, IDS, FATE, BGN, F8A) on the X chromosome, the linkage relationship of these five genes in pigs were analyzed by a panel of 96 radiation hybrid cell lines. The results showed that FMR1, IDS, FATE, BGN, F8A were in the same linkage group, when LOD was set at 4. When LOD was set at 5, FMR1 and IDS were in the one group, FATE and BGN in the other group, and F8A was in a group by itself.

[Effects of microsatellite DNA markers on meat quality traits in pig chromosome 13].

In this reseanch, 7 microsatellite DNA loci linked with PPAR gene were selected from the published genetic map of chromosome 13 in pig,and polymorphisms of these microsatellites in 100 samples from Sutai pigs (Duroc x Erhualian) populations were detected. Results revealed that the number of alleles were 6-9, heterozygosity 0.59 - 0.81, polymorphism information content 0.51 - 0.76. Effects of S0021, SW1937, SW482, S0222, S0293, S0281 and SWR2054 on meat quality traits were analyzed with PROC GLM of SAS. Results showed that the effects of S0021 on pH value and SW937 on water-holding capacity reached a significant level at P < 0.01 respectively. The effect of S0293 on tenderness and SW482 on BFT were also significant (P < 0.05). S0222, S0281 and SWR2054 had no significant effect on the 7 selected meat qualitytraits (P>0.05).

[Analysis of gene expression information in the fat and muscle tissues of pig].

An in silico study was developed to detect the gene expression profile of pig fat and muscle tissues by using the porcine EST resources and human gene sequences,and thus provided candidate information for basic genetic analysis in meat quality improvement of pig. In this study,a BLAST search was performed to identify homologies between the cDNA sequences of human genes and the ESTs sequences of pig,and the high homologous records were screened out. Four Java programs were developed to retrieve and collect sequences, and analyze the BLAST alignment results. By statistical analysis, it was found that there were at least 2002 genes expressed in the fat and muscle tissues of pig, and 1087 in the fat tissue, 1205 in the muscle respectively (290 genes co-expressed in the two tissues). The top-ranking records were screened out,and meantime, 114 basic active genes (BAGs) were found to express in the two tissues,80 in the fat tissue and 34 in the muscle tissue respectively. The top 10 records were described in the paper. This study was summarized in relation to current meat quality improvement of pig at molecular level.

[Mechanisms of epigenetic reprogramming in Mammalian resonstructed embryos produced by nuclear transfer.].

Circumstantial studies indicated that incomplete or inappropriate epigenetic reprogramming of donor nuclei was likely to be the primary reason for failures in nuclear transfer. In this view, we discussed the roles of several epigenetic mechanisms, including DNA methylation, chromatin remodeling, imprinting and X chromosome inactivation, telomere maintenance, and epigenetic inheritance in the observed abnormalities in clones from different species. Understanding the mechanisms underlying epigenetic reprogramming control will help us resolve the inherent problems in nuclear transfer technology and make its applications promising.

[Comparative study on lipogenic and lipolytic gene expression in intramuscular fat tissue between growing Erhualian and large white pigs].

To investigate the mechanism of difference in intramuscular fat deposition between Erhualian and Large White pigs,single tube relative-quantitative RT-PCR method was used to investigate the development patterns of lipogenic (ACX, LPL, ME) and lipolytic (HSL) gene expression with 18S internal standard control. Sixteen Large White boars and twenty Erhualian boars were selected and raised according to normal nutrition standard respectively. The animals were selected randomly and slaughtered at 15 kg, 40 kg, 60 kg and 90 kg for Large White pigs and at 18 kg, 40 kg, 60 kg, 80 kg and 90 kg for Erhualian pigs respectively; with four animals of each breed at each time. The supraspinatus and semimembranosus muscles were removed for total RNA extraction and longisimus dorsi muscle for intramuscular fat (IMF) analysis using ether extract method. The results showed: (1) The property of IMF between Erhualian and Large White boars was similar during early growing period (before 40 kg) (P > 0.05) ,thereafter, IMF level of Erhualian boars increased dramatically to 4% at 60 kg and over 5% at 90 kg while Large White boars kept steadily at about 2% (P < 0.05); (2) The pattern of lipogenic and lypolytic gene expression was similar between semimembranosus and supraspinatus muscle in each breed; (3) The tendency for LPL and ME mRNA expression coincided with that of IMF development among 20 approximately 60 kg in Erhualian pigs. The results suggest that the development of IMF between 20 kg and 60 kg in Erhualian pigs may play a meaningful role in deposition of IMF,and that the expression of ME and LPL mRNA may contribute to fast sediment of IMF in Erhualian pigs.