Epigallocatechin gallate improves the quality of diabetic oocytes.

BACKGROUND: Maternal diabetes compromises the quality and developmental potential of oocytes. Therefore, it is important to study how to ameliorate the adverse effects of diabetes on oocyte quality. Epigallocatechin gallate (EGCG) has a variety of physiological activities, including anti-inflammatory, antioxidant, and anti-diabetes. In the present study, we evaluated the effect of EGCG on the maturation of diabetic oocytes in vitro. OBJECTIVE: Investigating the role of EGCG in restoring the adverse effects of diabetes on oocyte quality. METHODS: Diabetes mouse model was established by a single injection of streptozotocin (STZ). Oocytes were collected and matured in vitro with/without EGCG in M16 medium. RESULTS: Compared with control, diabetic oocytes have a higher frequency of spindle defects and chromosome misalignment, but EGCG effectively reduces the incidence of oocytes with abnormal spindle assembly and chromosome mismatches. Moreover, the abnormal mitochondrial membrane potential (MMP) of diabetic oocytes is significantly alleviated by EGCG, and the reduced expression of genes regulating mitochondrial fusion (Mfn1 and Mfn2) and fission (Drp1) in diabetic oocytes is significantly increased while EGCG is added. EGCG also decreases the higher level of reactive oxygen species (ROS) in diabetic oocytes that may be regulated by the increased expression of superoxide dismutase 1 (Sod1) and superoxide dismutase 2 (Sod2). EGCG can also reduce the DNA damage of diabetic oocytes. CONCLUSIONS: Our results suggest that EGCG, at least partially, improve the quality of diabetic oocytes.

Protocatechuic Acid Delays Postovulatory Oocyte Ageing in Mouse.

SCOPE: Tea is a popular beverage worldwide and has many health functions. Protocatechuic acid (PCA) is an important bioactive component of tea and has benefit to health. In some cases, oocytes after ovulation may miss the optimal fertilization time and enter a postovulatory ageing process. Therefore, to investigate the role of PCA in delaying oocyte ageing is aimed. METHODS AND RESULTS: Metaphase II (MII) oocytes aged in vitro are randomly divided into three groups: control, aged, and aged + PCA. PCA treatment (30 µM) reduces the fragmentation rate and the incidence of abnormal spindle morphology and chromosome misalignment of oocytes aged 24 h in vitro. The mitochondrial dysfunction of aged oocytes, such as decreased mitochondrial membrane potential and excessive accumulation of reactive oxygen (ROS), is also alleviated by PCA. PCA also delays apoptosis of aged oocytes, and improves the sperm binding capacity. Otherwise, aged oocytes treated with PCA have a higher fertilization rate and blastocyst rate compared with untreated aged oocytes in vitro. CONCLUSION: PCA is an important bioactive ingredient of tea that improves aged oocyte quality, suggesting that PCA is available to improve the quality of aged oocytes in vitro.

Epigallocatechin gallate improves meiosis maturation against Diazinon exposure in porcine oocytes.

Diazinon (DZN) is a refractory organophosphorus pesticide (OP) in the surrounding environment due to its overuse in agriculture. The antioxidant activity of Epigallocatechin gallate (EGCG) from green tea is at least 100 times greater than that of vitamin C. This study aimed to study the effects of DZN on the meiotic maturation of porcine oocytes, as well as the protective roles of EGCG. Firstly, the effects of DZN and EGCG on meiotic nuclear maturation of porcine oocytes were detected, and then embryonic development was investigated by chemical parthenogenetic activation. Next, the spindle assembly, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), DNA damage, and finally the early apoptosis of oocytes were examined by immunofluorescence staining. The results revealed that DZN exposure significantly reduced the quality of porcine oocytes, such as failure of nuclear and cytoplasmic maturation, evidenced by abnormal spindle assembly, disordered chromosome alignment, low MMP, observably increased ROS, severe DNA damage, and early apoptosis. Appropriate EGCG could significantly reduce all these defects caused by DZN. In conclusion, EGCG can help prevent the harm that DZN exposure can do. These findings offer convincing support for enhancing the oocyte quality from EGCG through daily ordinary beverages.

LncRNA8276 primes cell-cell adhesion for regulation of spermatogenesis.

BACKGROUND: Human sperm concentration and motility have dropped dramatically (50%) in the past few decades, and environmental factors are involved in this decline. Long non-coding RNAs (lncRNA) have been discovered to be involved in many cellular processes including spermatogenesis. OBJECTIVE: This investigation aimed to explore the role of lncRNA8276 in murine spermatogenesis. MATERIALS AND METHODS: The expression of lncRNA8276 was modified by knockdown or overexpression in mouse testes and spermatogonial stem cells (C18-4 cell line). Sperm quality was determined in the F0 and F1 generations of mice. Furthermore, the underlying mechanisms were studied through gene expression and/or protein expression of spermatogenesis-related genes and cell junction-related genes by different methods. RESULTS: In the current investigation, we discovered that sperm lncRNA8276 was decreased by NH3 /H2 S in three generations (F0, F1, and F2) of mouse sperm. In vivo testicular knockdown of lncRNA8276 led to a decline in sperm concentration and motility in both F0 (muF0) and F1 (muF1) generations Moreover, knockdown lncRNA8276 decreased the gene and protein levels of important genes related to cell-cell junctions and spermatogenesis. The data were further confirmed in mouse spermatogonia stem cell line C18-4 cells through knockdown of lncRNA8276. DISCUSSION AND CONCLUSION: Our study suggests that lncRNA8276 may be involved in cell-cell junction formation in the mouse testis to regulate spermatogenesis. It may be a target for the modification of spermatogenesis and male fertility, or male contraception. This investigation offers a potential therapeutic strategy for male infertility.

ß-carotene Rescues Busulfan Disrupted Spermatogenesis Through Elevation in Testicular Antioxidant Capability.

ß-carotene, precursor of vitamin A, is an excellent antioxidant with many beneficial properties. It is a lipid-soluble antioxidant and a very effective quencher of reactive oxygen species (ROS) to reduce the oxidative stress. In contrast to vitamin A, ß-carotene is not toxic even consumed in higher amount when it is delivered from natural plant products. Recently, we found that ß-carotene acts as a potential antioxidant in the oocyte to improve its quality. Even though many studies have been reported that ß-carotene has the beneficial contribution to the ovarian development and steroidogenesis, it is unknown the effects of ß-carotene on the spermatogenesis. This investigation aimed to explore the hypothesis that ß-carotene could improve spermatogenesis and the underlying mechanism. And we found that ß-carotene rescued busulfan disrupted spermatogenesis in mouse with the increase in the sperm concentration and motility. ß-carotene improved the expression of genes/proteins important for spermatogenesis, such as VASA, DAZL, SYCP3, PGK2. Moreover, ß-carotene elevated the testicular antioxidant capability by the elevation of the antioxidant glutathione and antioxidant enzymes SOD, GPX1, catalase levels. In conclusion, ß-carotene may be applied for the infertile couples by the improvement of spermatogenesis, since, worldly many couples are infertile due to the idiopathic failed gametogenesis (spermatogenesis).

Tet1 Deficiency Leads to Premature Reproductive Aging by Reducing Spermatogonia Stem Cells and Germ Cell Differentiation.

Ten-eleven translocation (Tet) enzymes are involved in DNA demethylation, important in regulating embryo development, stem cell pluripotency and tumorigenesis. Alterations of DNA methylation with age have been shown in various somatic cell types. We investigated whether Tet1 and Tet2 regulate aging. We showed that Tet1-deficient mice undergo a progressive reduction of spermatogonia stem cells and spermatogenesis and thus accelerated infertility with age. Tet1 deficiency decreases 5hmC levels in spermatogonia and downregulates a subset of genes important for cell cycle, germ cell differentiation, meiosis and reproduction, such as Ccna1 and Spo11, resulting in premature reproductive aging. Moreover, Tet1 and 5hmC both regulate signaling pathways key for stem cell development, including Wnt and PI3K-Akt, autophagy and stress response genes. In contrast, effect of Tet2 deficiency on male reproductive aging is minor. Hence, Tet1 maintains spermatogonia stem cells with age, revealing an important role of Tet1 in regulating stem cell aging.

Glucagon-like peptide (GCGL) is a novel potential TSH-releasing factor (TRF) in Chickens: I) Evidence for its potent and specific action on stimulating TSH mRNA expression and secretion in the pituitary.

Our recent study proposed that the novel glucagon-like peptide (GCGL), encoded by a glucagon-like gene identified in chickens and other lower vertebrates, is likely a hypophysiotropic factor in nonmammalian vertebrates. To test this hypothesis, in this study, we investigated the GCGL action on chicken pituitaries. The results showed that: 1) GCGL, but not TRH, potently and specifically stimulates TSH secretion in intact pituitaries incubated in vitro or in cultured pituitary cells monitored by Western blotting or a cell-based luciferase reporter assay; 2) GCGL (0.1nM-10nM) dose dependently induces the mRNA expression of TSHß but not 5 other hormone genes in cultured pituitary cells examined by quantitative real-time RT-PCR, an action likely mediated by intracellular adenylate cyclase/cAMP/protein kinase A and phospholipase C/inositol 1,4,5-trisphosphate/Ca(2+) signaling pathways coupled to GCGL receptor (GCGLR); 3) GCGLR mRNA is mainly localized in pituitary cephalic lobe demonstrated by in situ hybridization, where TSH-cells reside, further supporting a direct action of GCGL on thyrotrophs. The potent and specific action of GCGL on pituitary TSH expression and secretion, together with the partial accordance shown among the temporal expression profiles of GCGL in the hypothalamus and GCGLR and TSHß in the pituitary, provides the first collective evidence that hypothalamic GCGL is most likely to be a novel TSH-releasing factor functioning in chickens. The discovery of this novel potential TSH-releasing factor (GCGL) in a nonmammalian vertebrate species, ie, chickens, would facilitate our comprehensive understanding of the hypothalamic control of pituitary-thyroid axis across vertebrates.

Discovery of a novel functional leptin protein (LEP) in zebra finches: evidence for the existence of an authentic avian leptin gene predominantly expressed in the brain and pituitary.

Leptin (LEP) is reported to play important roles in controlling energy balance in vertebrates, including birds. However, it remains an open question whether an authentic "LEP gene" exists and functions in birds. Here, we identified and characterized a LEP gene (zebra finch LEP [zbLEP]) encoding a 172-amino acid precursor in zebra finches. Despite zbLEP showing limited amino acid sequence identity (26%-29%) to human and mouse LEPs, synteny analysis proved that zbLEP is orthologous to mammalian LEP. Using a pAH32 luciferase reporter system and Western blot analysis, we demonstrated that the recombinant zbLEP protein could potently activate finch and chicken LEP receptors (zbLEPR; cLEPR) expressed in human embryonic kidney 293 cells and enhance signal transducer and activator of transcription 3 phosphorylation, further indicating that zbLEP is a functional ligand for avian LEPRs. Interestingly, quantitative real-time RT-PCR revealed that zbLEP mRNA is expressed nearly exclusively in the pituitary and various brain regions but undetectable in adipose tissue and liver, whereas zbLEPR mRNA is widely expressed in adult finch tissues examined with abundant expression noted in pituitary, implying that unlike mammalian LEP, finch LEP may not act as an adipocyte-derived signal to control energy balance. As in finches, a LEP highly homologous to zbLEP was also identified in budgerigar genome. Strikingly, finch and budgerigar LEPs show little homology with chicken LEP (cLEP) previously reported, suggesting that the so-called cLEP is incorrect. Collectively, our data provide convincing evidence for the existence of an authentic functional LEP in avian species and suggest an important role of brain- and pituitary-derived LEP played in vertebrates.

Identification of the receptors for somatostatin (SST) and cortistatin (CST) in chickens and investigation of the roles of cSST28, cSST14, and cCST14 in inhibiting cGHRH1-27NH2-induced growth hormone secretion in cultured chicken pituitary cells.

Somatostatin receptors (SSTRs) are proposed to mediate the actions of somatostatin (SST) and its related peptide, cortistatin (CST), in vertebrates. However, the identity, functionality, and tissue expression of these receptors remain largely unknown in most non-mammalian vertebrates including birds. In this study, five SSTRs (named cSSTR1, cSSTR2, cSSTR3, cSSTR4, cSSTR5) were cloned from chicken brain by RT-PCR. Using a pGL3-CRE-luciferase reporter system, we demonstrated that activation of each cSSTR expressed in CHO cells by cSST28, cSST14 and cCST14 treatment could inhibit forskolin-induced luciferase activity of CHO cells, indicating the functional coupling of all cSSTRs to Gi protein(s). Interestingly, cSSTR1-4 expressed in CHO cells could be activated by cSST28, cSST14 and cCST14 with high potencies, suggesting that they may function as the receptors common for these peptides. In contrast, cSSTR5 could be potently activated by cSST28 only, indicating that it is a cSST28-specific receptor. Using RT-PCR, wide expression of cSSTRs was detected in chicken tissues including pituitary. In accordance with their expression in pituitary, cSST28, cSST14, and cCST14 were demonstrated to inhibit basal and novel cGHRH1-27NH2-induced GH secretion in cultured chicken pituitary cells dose-dependently (0-10nM) by Western blot analysis, suggesting the involvement of cSSTR(s) common for these peptides in mediating their inhibitory actions. Collectively, our study establishes a molecular basis to elucidate the roles of SST/CST in birds and provide insights into the roles of SST/CST in vertebrates, such as their conserved actions on pituitary.

Characterization of the novel duplicated PRLR gene at the late-feathering K locus in Lohmann chickens.

A partial duplication of the prolactin (PRL) receptor gene (designated as dPRLR) has been identified at the late-feathering (LF) K locus on chromosome Z of some chicken strains recently, implying that dPRLR is probably a candidate gene associated with LF development in chickens. However, little is known about the structure, functionality, and spatiotemporal expression of the dPRLR gene in chickens. In this study, using 3'-RACE and RT-PCR, the full-length cDNA of the dPRLR obtained from the kidneys of male Lohmann layer chickens carrying a K allele was cloned. The cloned dPRLR is predicted to encode a membrane-spanning receptor of 683 amino acids, which is nearly identical to the original PRLR, except for its lack of a 149-amino acid C-terminal tail. Using a 5× STAT5-Luciferase reporter system and western blot analysis, we demonstrated that dPRLR expressed in HepG2 cells could be potently activated by chicken PRL and functionally coupled to the intracellular STAT5 signaling pathway, suggesting that dPRLR may function as a novel receptor for PRL. RT-PCR assays revealed that similar to the original PRLR gene, dPRLR mRNA is widely expressed in all embryonic and adult tissues examined including the skin of male Lohmann chickens with a K allele. These findings, together with the expression of PRL mRNA detected in the skin of embryos at embryonic day 20 and 1-week-old chicks, suggest that skin-expressed dPRLR and PRLR, together with plasma and skin-derived PRL, may be involved in the control of the LF development of chicks at hatching. Moreover, the wide tissue expression of dPRLR implies that dPRLR may regulate other physiological processes of chickens carrying the K allele.

Molecular characterization of prolactin receptor (cPRLR) gene in chickens: gene structure, tissue expression, promoter analysis, and its interaction with chicken prolactin (cPRL) and prolactin-like protein (cPRL-L).

In this study, gene structure, tissue expression, and promoter usage of prolactin receptor (PRLR) and its interaction with prolactin (PRL) and the newly identified prolactin-like protein (PRL-L) were investigated in chickens. The results showed that (1) PRLR gene was found to consist of at least 25 exons by 5'-RACE and RT-PCR assays; (2) multiple PRLR 5'-UTR sequences different in exon composition were isolated from chicken liver or intestine by 5'-RACE and could be subdivided into type I and type II transcripts according to the first exon used (exon 1G or exon 1A); (3) PRLR Type I transcripts with exon 1G were detected to be predominantly expressed in adult kidney and small intestine by RT-PCR, implying their expression is likely controlled by a tissue-specific promoter (P1). By contrast, PRLR type II transcripts containing exon 1A are widely expressed in adult and embryonic tissues examined and their expression is controlled by a generic promoter (P2) near exon 1A, which was demonstrated to display promoter activities in cultured DF-1, HEK293 and LoVo cells by the dual-luciferase reporter assay; (4) Using a 5×STAT5-luciferase reporter system, cPRLR expressed in HepG2 cells was shown to be activated by recombinant cPRL and cPRL-L via interaction with PRLR membrane-proximal ligand-binding domain, suggesting that like cPRL, cPRL-L is also a functional ligand of cPRLR. Collectively, characterization of cPRLR gene helps to elucidate the roles of PRLR and its ligands in birds and provides insights into the regulatory mechanisms of PRLR expression conserved in birds and mammals.

Discovery of a novel glucagon-like peptide (GCGL) and its receptor (GCGLR) in chickens: evidence for the existence of GCGL and GCGLR genes in nonmammalian vertebrates.

Glucagon (GCG), glucagon-related peptides, and their receptors have been reported to play important roles including the regulation of glucose homeostasis, gastrointestinal activity, and food intake in vertebrates. In this study, we identified genes encoding a novel glucagon-like peptide (named GCGL) and its receptor (GCGLR) from adult chicken brain using RACE and/or RT-PCR. GCGL was predicted to encode a peptide of 29 amino acids (cGCGL(1-29)), which shares high amino acid sequence identity with mammalian and chicken GCG (62-66%). GCGLR is a receptor of 430 amino acids and shares relatively high amino acid sequence identity (53-55%) with the vertebrate GCG receptor (GCGR). Using a pGL3-CRE-luciferase reporter system, we demonstrated that synthetic cGCGL(1-29), but not its structurally related peptides, i.e. exendin-4 and GCG, could potently activate GCGLR (EC(50): 0.10 nm) expressed in Chinese hamster ovary cells, indicating that GCGLR can function as a GCGL-specific receptor. RT-PCR assay revealed that GCGL expression is mainly restricted to several tissues including various brain regions, spinal cord, and testes, whereas GCGLR mRNA is widely expressed in adult chicken tissues with abundant expression noted in the pituitary, spinal cord, and various brain regions. Using synteny analysis, GCGL and GCGLR genes were also identified in the genomes of fugu, tetraodon, tilapia, medaka, coelacanth, and Xenopus tropicalis. As a whole, the discovery of GCGL and GCGLR genes in chickens and other nonmammalian vertebrates clearly indicates a previously unidentified role of GCGL-GCGLR in nonmammalian vertebrates and provides important clues to the evolutionary history of GCG and GCGL genes in vertebrates.

Identification of the receptors for prolactin-releasing peptide (PrRP) and Carassius RFamide peptide (C-RFa) in chickens.

Prolactin-releasing peptide (PrRP) and its structurally related peptide, Carassius Arg-Phe-amide peptide (C-RFa), have been reported to play similar roles in regulating food intake and pituitary functions in vertebrates. However, the identity, functionality, and expression of the receptor(s) for PrRP and C-RFa remain largely unknown in nonmammalian vertebrates, including birds. In this study, three receptors homologous to mammalian PrRP receptor (PrRPR), named cPrRPR1, cPrRPR2, and cC-RFaR, respectively, were cloned from chicken brain by RT-PCR. Using a pGL3-NFAT-RE-luciferase reporter system, we demonstrated that cPrRPR1 and cPrRPR2 expressed in Chinese hamster ovarian cells could be activated by cPrRP20 and cC-RFa20 potently, whereas cC-RFaR could only be activated effectively by cC-RFa20 (EC50, 0.11 nM), indicating that cPrRPR1 and cPrRPR2 can function as common receptors for PrRP and C-RFa, whereas cC-RFaR is a receptor specific to C-RFa. Using a pGL3-CRE-luciferase reporter system, cPrRPR1, cPrRPR2, and cC-RFaR expressed in Chinese hamster ovarian cells were also shown to activate intracellular protein kinase A signaling pathway upon cC-RFa20 treatment (100 nM). Moreover, RT-PCR assay revealed that cPrRPR1, cPrRPR2, and cC-RFaR were widely expressed in most adult chicken tissues examined, including various regions of brain. These findings, together with evidence of PrRP and C-RFa encoded by separate genes in chicken, Xenopus, and zebrafish, and the differential expression of PrRP and C-RFa genes in chicken tissues, strongly suggest that PrRP and C-RFa may play similar yet distinctive roles in nonmammalian vertebrates, including chicken, and their actions are mediated by common receptor(s) or a specific C-RFa receptor.

Characterization of chicken secretin (SCT) and secretin receptor (SCTR) genes: a novel secretin-like peptide (SCT-LP) and secretin encoded in a single gene.

Secretin and the secretin receptor have been reported to play an important role in regulating pancreatic water and bicarbonate secretion in mammals; however, little is known about their expression, structure, and biological functions in non-mammalian vertebrates including birds. In this study, the full-length cDNAs encoding secretin and secretin receptor have first been cloned from duodenum of adult chickens. The putative chicken secretin receptor (cSCTR) is 449 amino acids in length and shares high sequence identity (58-63%) with its mammalian counterparts. Interestingly, chicken secretin cDNA encodes not only the secretin peptide (cSCT), but also a novel secretin-like peptide (cSCT-LP), which shares high amino acid identity with chicken (56%) and mammalian (48-52%) secretin. Using a pGL3-CRE-luciferase reporter system, we further demonstrated that both cSCT (EC(50): 0.31nM) and cSCT-LP (EC(50): 1.10nM), but not other structurally-related peptides, could potently activate cSCTR expressed in CHO cells, suggesting that both peptides may function as potential ligands for cSCTR. Using RT-PCR, the expression of secretin and secretin receptor in adult chicken tissues was also examined. Secretin was detected to be predominantly expressed in small intestine, while the mRNA expression of cSCTR was restricted to several tissues including gastrointestinal tract, liver, testis, pancreas and several brain regions. Collectively, results from present study not only established a molecular basis to elucidate the physiological roles of SCT, SCT-LP and SCTR in chickens, but also provide critical insights into structural and functional changes of secretin and its receptor during vertebrate evolution.