Study on the expression patterns and function of JUNO and CD9 in bovine oocytes during in vitro maturation.

Fertilization proteins JUNO and CD9 play vital roles in sperm-egg fusion, but little is known about their expression patterns during in vitro maturation (IVM) and their function during in vitro fertilization (IVF) of bovine oocytes. In this study, qRT-PCR and immunofluorescence staining were used to detect the mRNA and protein expression levels of JUNO and CD9 genes in bovine oocytes and cumulus cells. Then, fertilization rate of MII oocytes treated with (i) JUNO antibody (1, 5 and 25 µg/ml) or (ii) CD9 antibody (1, 5 and 25 µg/ml) or (iii) CD9 antibody (5 µg/ml) + JUNO antibody (5 µg/ml) were recorded. Our results showed that the mRNA and protein expression levels of JUNO and CD9 genes significantly increased from bovine GV oocytes to MII oocytes, and similar mRNA expression patterns of JUNO and CD9 were also detected in cumulus cells. All groups of oocytes treated with CD9 antibody or JUNO antibody showed significantly decreased fertilization rates (p < .05). Particularly, the fertilization ability of oocytes treated with CD9 antibody (5 µg/ml) + JUNO antibody (5 µg/ml) sharply decreased to 3.48 ± 0.11%. In conclusion, our study revealed the expression levels of JUNO and CD9 genes in oocytes and cumulus cells increased during IVM of bovine oocytes, with JUNO protein playing a major role in the fertilization of bovine oocytes.

Knockdown of CLAUDIN-6 Inhibited Apoptosis and Induced Proliferation of Bovine Cumulus Cells.

This study aims to investigate the effects of CLAUDIN-6 (CLDN6) on cell apoptosis and proliferation of bovine cumulus cells (CCs). Immunofluorescence staining was used to localize CLDN6 protein in CCs. Three pairs of siRNA targeting CLDN6 and one pair of siRNA universal negative sequence as control were transfected into bovine CCs. Then, the effective siRNA was screened by real-time quantitative PCR (RT-qPCR) and Western blotting. The mRNA expression levels of apoptosis related genes (CASPASE-3, BAX and BCL-2) and proliferation related genes (PCNA, CDC42 and CCND2) were evaluated by RT-qPCR in CCs with CLDN6 knockdown. Cell proliferation, apoptosis and cell cycle were detected by flow cytometry with CCK-8 staining, Annexin V-FITC staining and propidium iodide staining, respectively. Results showed that the CLDN6 gene was expressed in bovine CCs and the protein was localized in cell membranes and cytoplasms. After CLDN6 was knocked down in CCs, the cell apoptosis rate significantly decreased and the pro-apoptotic genes BAX and CASPASE-3 were down-regulated significantly, whereas the anti-apoptotic gene BCL-2 was markedly up-regulated (p < 0.05). Additionally, CLDN6 knockdown significantly enhanced cell proliferation of CCs at 72 h after siRNA transfection. The mRNA levels of proliferation-related genes PCNA, CCND2 and CDC42 increased obviously in CCs with CLDN6 knockdown (p < 0.05). After CLDN6 was down-regulated, the percentage of CCs at S phase was significantly increased (p < 0.05). However, there was no remarkable difference in the percentages of cells at the G0/G1 phase and G2/M phase between CCs with or without CLDN6 knockdown (p > 0.05). Therefore, the expression of CLDN6 and its effects on cell proliferation, apoptosis and cell cycle of bovine CCs were first studied. CLDN6 low expression inhibited cell apoptosis, induced cell proliferation and cell cycle arrest of bovine CCs.

Thioredoxin-interacting protein regulates glucose metabolism and improves the intracellular redox state in bovine oocytes during in vitro maturation.

The extent of glucose metabolism during oocyte maturation is closely related to oocyte developmental potential. Thioredoxin-interacting protein (TXNIP) is an α-arrestin family protein that negatively regulates glucose uptake into cells. However, little information is available regarding the function of TXNIP in bovine oocytes. Accordingly, the present study was performed to investigate the influence of TXNIP on glucose metabolism in bovine oocytes during in vitro maturation. Pharmacological inhibition of TXNIP by azaserine enhanced glucose uptake and imparted a specific metabolic effect on glycolysis and pentose phosphate pathway (PPP). RNA interference (RNAi) was adopted to further determine the biological significance of TXNIP in regulating glucose metabolism. The maturation rate and the developmental competence of TXNIP siRNA-treated oocytes were significantly improved. Knockdown of TXNIP in bovine oocytes significantly increased glycolysis by increasing the activities of phosphofructokinase (PFK), pyruvate kinase, and lactate dehydrogenase; pyruvate and lactate production; and intracellular ATP level, as well as mitochondrial activity. Furthermore, glucose metabolism through PPP was also enhanced by TXNIP depletion, as TXNIP siRNA treatment promoted glucose-6-phosphate dehydrogenase (G6PDH) activity and NADPH content, and helped maintain a high level of glutathione and a low level of reactive oxygen species within the oocytes. Further studies revealed that inhibition of TXNIP resulted increases in glucose transporter 1 (GLUT1) expression, as well as PFK1 platelet isoform (PFKP) and G6PDH mRNA levels. These results reveal that TXNIP depletion promotes oocyte maturation by enhancing both glycolysis and the PPP. During in vitro maturation of bovine oocytes, TXNIP serves as a key regulator of glucose uptake by controlling GLUT1 expression.

Regulation of AMH, AMHR-II, and BMPs (2,6) Genes of Bovine Granulosa Cells Treated with Exogenous FSH and Their Association with Protein Hormones.

Anti-Mullerian hormone (AMH) is an important reproductive marker of ovarian reserve produced by granulosa cells (GCs) of pre-antral and early-antral ovarian follicles in several species, including cattle. This hormone plays a vital role during the recruitment of primordial follicles and follicle stimulating hormone (FSH)-dependent follicular growth. However, the regulatory mechanism of AMH expression in follicles is still unclear. In this study, we compared the expression of AMH, AMHR-II, BMP2, BMP6, FSHR, and LHCGR genes during follicular development. In-vitro expression study was performed with and without FSH for AMH, AMHR-II, BMP2, and BMP6 genes in bovine GCs which were isolated from 3-8 mm follicles. Association among the mRNA expression and hormone level was estimated. GCs were collected from small (3-8 mm), medium (9-12 mm) and large size (13 to 24 mm) follicles before, during onset, and after deviation, respectively. Further, mRNA expression, hormones (AMH, FSH, and LH), apoptosis of GCs, and cell viability were detected by qRT-PCR, ELISA, flow cytometry, and spectrophotometry. AMH, AMHR-II, BMP2, and FSHR genes were highly expressed in small and medium follicles as compared to large ones. In addition, the highest level of AMH protein (84.14 ± 5.41 ng/mL) was found in medium-size follicles. Lower doses of FSH increased the viability of bovine GCs while higher doses repressed them. In-vitro cultured GCs treated with FSH significantly increased the AMH, AMHR-II, and BMP2 expression levels at lower doses, while expression levels decreased at higher doses. We found an optimum level of FSH (25 ng/mL) which can significantly enhance AMH and BMP2 abundance (p < 0.05). In summary, AMH, AMHR-II, and BMP2 genes showed a higher expression in follicles developed in the presence of FSH. However, lower doses of FSH demonstrated a stimulatory effect on AMH and BMP2 expression, while expression started to decline at the maximum dose. In this study, we have provided a better understanding of the mechanisms regulating AMH, AMHR II, and BMP2 signaling in GCs during folliculogenesis, which would improve the outcomes of conventional assisted reproductive technologies (ARTs), such as superovulation and oestrus synchronization in bovines.

Melatonin Improves the Fertilization Capacity of Sex-Sorted Bull Sperm by Inhibiting Apoptosis and Increasing Fertilization Capacitation via MT1.

Little information is available regarding the effect of melatonin on the quality and fertilization capability of sex-sorted bull sperm, and even less about the associated mechanism. Sex-sorted sperm from three individual bulls were washed twice in wash medium and incubated in a fertilization medium for 1.5 h, and each was supplemented with melatonin (0, 10-3 M, 10-5 M, 10-7 M, and 10-9 M). The reactive oxygen species (ROS) and endogenous antioxidant activity (glutathione peroxidase (GPx); superoxide dismutase (SOD); catalase (CAT)), apoptosis (phosphatidylserine [PS] externalization; mitochondrial membrane potential (Δψm)), acrosomal integrity events (malondialdehyde (MDA) level; acrosomal integrity), capacitation (calcium ion [Ca2+]i level; cyclic adenosine monophosphate (cAMP); capacitation level), and fertilization ability of the sperm were assessed. Melatonin receptor 1 (MT1) and 2 (MT2) expression were examined to investigate the involvement of melatonin receptors on sex-sorted bull sperm capacitation. Our results show that treatment with 10-5 M melatonin significantly decreased the ROS level and increased the GPx, SOD, and CAT activities of sex-sorted bull sperm, which inhibited PS externalization and MDA levels, and improved Δψm, acrosomal integrity, and fertilization ability. Further experiments showed that melatonin regulates sperm capacitation via MT1. These findings contribute to improving the fertilization capacity of sex-sorted bull sperm and exploring the associated mechanism.

Exogenous glutathione improves intracellular glutathione synthesis via the γ-glutamyl cycle in bovine zygotes and cleavage embryos.

Excess reactive oxygen species (ROS) generated in embryos during in vitro culture damage cellular macromolecules and embryo development. Glutathione (GSH) scavenges ROS and optimizes the culture system. However, how exogenous GSH influences intracellular GSH and improves the embryo developmental rate is poorly understood. In this study, GSH or GSX (a stable GSH isotope) was added to the culture media of bovine in vitro fertilization embryos for 7 days. The cleavage rate, blastocyst rate, and total cell number of blastocysts were calculated. Similarly to GSH, GSX increased the in vitro development rate and embryo quality. We measured intracellular ROS, GSX, and GSH for 0-32-hr postinsemination (hpi) in embryos (including zygotes at G1, S, and G2 phases and cleaved embryos) cultured in medium containing GSX. Intracellular ROS significantly decreased with increasing intracellular GSH in S-stage zygotes (18 hpi) and cleaved embryos (32 hpi). γ-Glutamyltranspeptidase ( GGT) and glutathione synthetase ( GSS) messenger RNA expression increased in zygotes (18 hpi) and cleaved embryos treated with GSH, consistent with the tendency of overall GSH content. GGT activity increased significantly in 18 hpi zygotes. GGT and GCL enzyme inhibition with acivicin and buthionine sulfoximine, respectively, decreased cleavage rate, blastocyst rate, total cell number, and GSH and GSX content. All results indicated that exogenous GSH affects intracellular GSH levels through the γ-glutamyl cycle and improves early embryo development, enhancing our understanding of the redox regulation effects and transport of GSH during embryo culture in vitro.

Melatonin protects against paraquat-induced damage during in vitro maturation of bovine oocytes.

Paraquat (PQ), a broad-spectrum agricultural pesticide, causes cellular toxicity by increasing oxidative stress levels in various biological systems, including the reproductive system. PQ exposure causes embryotoxicity and reduces the developmental abilities of embryos. However, there is little information regarding the toxic effects of PQ on oocyte maturation. In this study, we studied the toxic effects of PQ exposure and the effects of melatonin on PQ-induced damage in bovine oocytes. PQ exposure disrupted nuclear and cytoplasmic maturation, which was manifested as decreased cumulus cell expansion, reduced first polar body extrusion, and abnormal distribution patterns of cortical granules and mitochondria. In addition, PQ treatment severely disrupted the ability of the resulted in vitro-produced embryos to develop to the blastocyst stage. Moreover, PQ exposure significantly increased the intracellular reactive oxygen species (ROS) level and early apoptotic rate, and decreased the glutathione (GSH) level, antioxidative CAT and GPx4 mRNA, and apoptotic-related Bcl-2/Bax mRNA ratio. These results indicated that PQ causes reproductive toxicity in bovine oocytes. Melatonin application resulted in significant protection against the toxic effects of PQ in PQ-exposed oocytes. The mechanisms underlying the role of melatonin included the inhibition of PQ-induced p38 mitogen-activated protein kinase (MAPK) activation, and restoration of abnormal trimethyl-histone H3 lysine 4 (H3K4me3) and trimethyl-histone H3 lysine 9 (H3K9me3) levels. These results reveal that melatonin serves as a powerful agent against experimental PQ-induced toxicity during bovine oocyte maturation and could form a basis for further studies to develop therapeutic strategies against PQ poisoning.

Detrimental effects of lipopolysaccharides on maturation of bovine oocytes.

OBJECTIVE: Gram-negative bacteria lipopolysaccharide (LPS) has been reported to be associated with uterine impairment, embryonic resorption, ovarian dysfunction, and follicle retardation. Here, we aimed to investigate the toxic effects of LPS on the maturation ability and parthenogenetic developmental competence of bovine oocytes. METHODS: First, we developed an in vitro model to study the response of bovine cumulus-oocyte complexes (COCs) to LPS stress. After incubating germinal vesicle COCs in 10 µg/mL of LPS, we analyzed the following three aspects: the expression levels of the LPS receptor toll-like receptor 4 (TLR4) in COCs, activities of intracellular signaling protein p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-kappa B (NF-κB); and the concentrations of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-6. Furthermore, we determined the effects of LPS on the maturation ability and parthenogenetic developmental competence of bovine oocytes. RESULTS: The results revealed that LPS treatment significantly elevated TLR4 mRNA and protein expression levels in COCs. Exposure of COCs to LPS also resulted in a marked increase in activity of the intracellular signaling protein p-p38 MAPK and NF-κB. Furthermore, oocytes cultured in maturation medium containing LPS had significantly higher concentrations of the proinflammatory cytokines IL-1ß, TNF-α, and IL-6. LPS exposure significantly decreased the first polar body extrusion rate. The cytoplasmic maturation, characterized by polar body extrusion and distribution of peripheral cortical granules, was significantly impaired in LPS-treated oocytes. Moreover, LPS exposure significantly increased intracellular reactive oxygen species levels and the relative mRNA abundance of the antioxidants thioredoxin (Trx), Trx2, and peroxiredoxin 1 in oocytes. Moreover, the early apoptotic rate and the release of cytochrome C were significantly increased in response to LPS. The cleavage, morula, and blastocyst formation rates were significantly lower in parthenogenetically activated oocytes exposed to LPS, while the incidence of apoptotic nuclei in blastocysts was significantly increased. CONCLUSION: Together, these results provide an underlying mechanism by which LPS impairs maturation potential in bovine oocytes.

Effects of epigallocatechin-3-gallate on bovine oocytes matured in vitro.

OBJECTIVE: Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols and is considered one of the most promising bioactive compounds in green tea because of its strong antioxidant properties. However, the protective role of EGCG in bovine oocyte in vitro maturation (IVM) has not been investigated. Therefore, we aimed to study the effects of EGCG on IVM of bovine oocytes. METHODS: Bovine oocytes were treated with different concentrations of EGCG (0, 25, 50, 100, and 200 µM), and the nuclear and cytoplasmic maturation, cumulus cell expansion, intracellular reactive oxygen species (ROS) levels, total antioxidant capacity, the early apoptosis and the developmental competence of in vitro fertilized embryos were measured. The mRNA abundances of antioxidant genes (nuclear factor erythriod-2 related factor 2 [NRF2], superoxide dismutase 1 [SOD1], catalase [CAT], and glutathione peroxidase 4 [GPX4]) in matured bovine oocytes were also quantified. RESULTS: Nuclear maturation which is characterized by first polar body extrusion, and cytoplasmic maturation characterized by peripheral and cortical distribution of cortical granules and homogeneous mitochondrial distribution were significantly improved in the 50 µM EGCG-treated group compared with the control group. Adding 50 µM EGCG to the maturation medium significantly increased the cumulus cell expansion index and upregulated the mRNA levels of cumulus cell expansion-related genes (hyaluronan synthase 2, tumor necrosis factor alpha induced protein 6, pentraxin 3, and prostaglandin 2). Both the intracellular ROS level and the early apoptotic rate of matured oocytes were significantly decreased in the 50 µM EGCG group, and the total antioxidant ability was markedly enhanced. Additionally, both the cleavage and blastocyst rates were significantly higher in the 50 µM EGCG-treated oocytes after in vitro fertilization than in the control oocytes. The mRNA abundance of NRF2, SOD1, CAT, and GPX4 were significantly increased in the 50 µM EGCG-treated oocytes. CONCLUSION: In conclusion, 50 µM EGCG can improve the bovine oocyte maturation, and the protective role of EGCG may be correlated with its antioxidative property.

Melatonin improves the fertilization capacity and developmental ability of bovine oocytes by regulating cytoplasmic maturation events.

Melatonin is a well-characterized antioxidant that has been successfully used to protect oocytes from reactive oxygen species during in vitro maturation (IVM), resulting in improved fertilization capacity and development ability. However, the mechanism via which melatonin improves oocyte fertilization capacity and development ability remains to be determined. Here, we studied the effects of melatonin on cytoplasmic maturation of bovine oocytes. In the present study, bovine oocytes were cultured in IVM medium supplemented with 0, 10-7 , 10-9 , and 10-11  mol/L melatonin, and the cytoplasmic maturation parameters of MII oocytes after IVM were investigated, including redistribution of organelles (mitochondria, cortical granules [CGs], and endoplasmic reticulum [ER]), intracellular glutathione (GSH) and ATP levels, expression of endogenous antioxidant genes (Cat, Sod1, and GPx), and fertilization-related events (IP3R1 distribution and expression of CD9 and Juno). Our results showed that melatonin significantly improved the cytoplasmic maturation of bovine oocytes by improving the normal distribution of organelles, increasing intracellular GSH and ATP levels, enhancing antioxidant gene expression levels, and modulating fertilization-related events, all of which resulted in increased fertilization capacity and developmental ability. Meanwhile, melatonin also increased the mRNA and protein expression levels of the Tet1 gene and decreased the Dnmt1 gene mRNA and protein levels in bovine oocytes, indicating that melatonin regulates the expression of the detected genes via demethylation. These findings shed insights into the potential mechanisms by which melatonin improves oocyte quality during IVM.

Protective effects of melatonin on the in vitro developmental competence of bovine oocytes.

The present study investigated the effects of melatonin on bovine oocyte maturation and subsequent embryonic development in vitro. Results showed that the nuclear and cytoplasmic maturation, characterized by first polar body extrusion, normal distribution of cortical granules and mitochondria, as well as increased mitochondrial membrane potential, were significantly improved in 10-9  mol/L melatonin-treated oocytes. Melatonin supplementation reduced intracellular reactive oxygen species level and enhanced glutathione production. Meanwhile, the presence of melatonin (10-9  mol/L) during oocyte maturation resulted in a decreased early apoptotic rate in oocytes. After in vitro fertilization, oocytes receiving melatonin supplementation exhibited a significantly higher blastocyst formation rate and yielded a markedly lower number of apoptotic cells. Mechanistic explorations showed that addition of 10-9  mol/L melatonin to in vitro maturation media significantly attenuated the transcript level of caspase-3, while the expressions of BCL-2, XIAP, CAT and HSP70 were significantly reinforced in the resultant embryos. Taken together, melatonin ameliorates bovine oocyte maturation potential, and the beneficial effects can affect subsequent embryonic development. The protective role of melatonin may be due to its anti-apoptotic and anti-oxidative activities.

Splicing-related single nucleotide polymorphism of RAB, member of RAS oncogene family like 2B (RABL2B) jeopardises semen quality in Chinese Holstein bulls.

RAB, member of RAS oncogene family like 2B (RABL2B) is a member of a poorly characterised clade of the RAS GTPase superfamily, which plays an essential role in male fertility, sperm intraflagellar transport and tail assembly. In the present study, we identified a novel RABL2B splice variant in bovine testis and spermatozoa. This splice variant, designated RABL2B-TV, is characterised by exon 2 skipping. Moreover, a single nucleotide polymorphism (SNP), namely c.125G>A, was found within the exonic splicing enhancer (ESE) motif, indicating that the SNP caused the production of the RABL2B-TV aberrant splice variant. This was demonstrated by constructing a pSPL3 exon capturing vector with different genotypes and transfecting these vectors into murine Leydig tumour cell line (MLTC-1) cells. Expression of the RABL2B-TV transcript was lower in semen from high- versus low-performance bulls. Association analysis showed that sperm deformity rate was significantly lower in Chinese Holstein bulls with the GG or GA genotype than in bulls with the AA genotype (P<0.05). In addition, initial sperm motility was significantly higher in individuals with the GG or GA genotype than in individuals with the AA genotype (P<0.05). The findings of the present study suggest that the difference in semen quality in bulls with different RABL2B genotypes is generated via an alternative splicing mechanism caused by a functional SNP within the ESE motif.

Embryonic stem-like cells from rabbit blastocysts cultured with melatonin could differentiate into three germ layers in vitro and in vivo.

The rabbit is considered an important model animal from which to obtain embryonic stem cells because of the utility of this animal in physiology and reproductive research. Here, we derived rabbit ES-like (rES-like) cells from blastocysts of superovulated Japanese white rabbits using culture medium containing 10-7 M melatonin, 10 ng/mL basic fibroblast growth factor, and 1,000 IU/mL human leukemia inhibitory factor. This concentration of melatonin had the most significant positive effects on the proliferation inner cell mass-derived cells (improving rates from 19.97% to 34.57%) and the longevity of passaging rES-like cells. Melatonin also enhanced the expression of pluripotent genes-including alkaline phosphatase, Pou5f1, Sox2, Klf4, c-Myc, Nanog, Line28a, and surface marker proteins-in fifth-passage rES-like cells. In vitro, these rES-like cells could spontaneously differentiate into some somatic cells, such as beating cardiomyocytes; formed embryoid bodies; expressed markers of the three germ layers after differentiation; and formed teratomas after injection into non-obese diabetic-severe combined immune deficient (NOD-SCID) mice. Thus, melatonin helped coax ES-like cells from rabbit blastocysts, which raises intriguing questions about the relationship between pluripotency and proliferation in rabbit embryonic stem cells. Mol. Reprod. Dev. 83: 1003-1014, 2016 © 2016 Wiley Periodicals, Inc.

[Pulmonary surfactant homeostasis associated genetic abnormalities and lung diseases].

Pulmonary surfactant (PS) is synthesized and secreted by alveolar epithelial type II (AEII) cells, which is a complex compound formed by proteins and lipids. Surfactant participates in a range of physiological processes such as reducing the surface tension, keeping the balance of alveolar fluid, maintaining normal alveolar morphology and conducting host defense. Genetic disorders of the surfactant homeostasis genes may result in lack of surfactant or cytotoxicity, and lead to multiple lung diseases in neonates, children and adults, including neonatal respiratory distress syndrome, interstitial pneumonia, pulmonary alveolar proteinosis, and pulmonary fibrosis. This paper has provided a review for the functions and processes of pulmonary surfactant metabolism, as well as the connection between disorders of surfactant homeostasis genes and lung diseases.

Testicular Busulfan Injection in Mice to Prepare Recipients for Spermatogonial Stem Cell Transplantation Is Safe and Non-Toxic.

Current methods of administering busulfan to remove the endogenous germ cells cause hematopoietic toxicity, require special instruments and a narrow transplantation time. We use a direct testicular injection of busulfan method for preparing recipients for SSC transplantation. Male ICR mice (recipients) were divided into four groups, and two experimental groups were treated with a bilateral testicular injection of 4 or 6 mg/kg/side busulfan (n = 60 per concentration group). Mice received an intraperitoneal injection (i.p.) of 40 mg/kg busulfan (n = 60, positive control) and bilateral testicular injections of 50% DMSO (n = 60, negative control). Donor SSCs from RFP-transgenic C57BL/6J mice were introduced into the seminiferous tubules of each recipient testis via efferent duct injection on day 16-17 after busulfan treatment. Recipient mice mated with mature female ICR mice and the number of progeny was recorded. The index detected at day 14, 21, 28, 35 and 70 after busulfan treatment. Blood analysis shows that the toxicity of busulfan treated groups was much lower than i.p. injection groups. Fertility was restored in mice treated with busulfan and donor-derived offspring were obtained after SSC transplantation. Our study indicated that intratesticular injection busulfan for the preparation of recipients in mice is safe and feasible.

Melatonin inhibits paraquat-induced cell death in bovine preimplantation embryos.

Preimplantation embryos are sensitive to oxidative stress-induced damage that can be caused by reactive oxygen species (ROS) originating from normal embryonic metabolism and/or the external surroundings. Paraquat (PQ), a commonly used pesticide and potent ROS generator, can induce embryotoxicity. The present study aimed to investigate the effects of melatonin on PQ-induced damage during embryonic development in bovine preimplantation embryos. PQ treatment significantly reduced the ability of bovine embryos to develop to the blastocyst stage, and the addition of melatonin markedly reversed the developmental failure caused by PQ (20.9% versus 14.3%). Apoptotic assay showed that melatonin pretreatment did not change the total cell number in blastocysts, but the incidence of apoptotic nuclei and the release of cytochrome c were significantly decreased. Using real-time quantitative polymerase chain reaction analysis, we found that melatonin pre-incubation significantly altered the expression levels of genes associated with redox signaling, particularly by attenuating the transcript level of Txnip and reinforcing the expression of Trx. Furthermore, melatonin pretreatment significantly reduced the expression of the pro-apoptotic caspase-3 and Bax, while the expression of the anti-apoptotic Bcl-2 and XIAP was unaffected. Western blot analysis showed that melatonin protected bovine embryos from PQ-induced damage in a p38-dependent manner, but extracellular signal-regulated kinase (ERK) and c-JUN N-terminal kinase (JNK) did not appear to be involved. Together, these results identify an underlying mechanism by which melatonin enhances the developmental potential of bovine preimplantation embryos under oxidative stress conditions.

Testicular injection of busulfan for recipient preparation in transplantation of spermatogonial stem cells in mice.

Intraperitoneal busulfan injections are used to prepare recipients for spermatogonial stem cell (SSC) transplantation but they are associated with haematopoietic toxicity. Testicular injections of busulfan have been proposed to overcome this limitation. To date, testicular injections have not been studied in the mouse model. Therefore, in the present study we used ICR mice as recipients for SSC transplantation and prepared these mice by testicular injection of busulfan on both sides (2, 3, 4 or 6mgkg-1 per side). Following this, donor germ cells expressing red fluorescent protein (RFP) from transgenic C57BL/6J male mice were transplanted into recipients via the efferent duct on Days 16-17 after busulfan treatment. Positive control mice were prepared by intraperitoneal injection of 40mgkg-1 busulfan and negative control mice were treated with bilateral testicular injection of 50% dimethyl sulfoxide. On Day 49 after transplantation, recipient mice that were RFP-positive by in vivo imaging were mated with ICR female mice. Donor-derived germ cell colonies with red fluorescence were observed on Day 60 after transplantation, and donor-derived offspring were obtained. The results demonstrated that endogenous germ cells were successfully eliminated in the seminiferous tubules via testicular busulfan administration, and that exogenous SSCs successfully undergo spermatogenesis in the testes of recipient mice prepared by testicular injections of busulfan. In addition to its effects on recipient preparation, this method was safe in rodents and could possibly be adapted for use in other species.

Exogenous glutathione supplementation in culture medium improves the bovine embryo development after in vitro fertilization.

To determine the beneficial effects and mechanisms of exogenous glutathione (GSH) during IVC on the embryonic development, bovine IVF zygotes were cultured in medium containing different concentrations of GSH, and the rate of cleavage and blastocyst development, total cell number of blastocysts, the inner cell mass:total cell number ratio, and intracellular GSH and reactive oxygen species concentrations were investigated. Gene expressions associated with embryonic development and GSH metabolism were measured using quantitative real-time polymerase chain reaction. At the concentrations of 1, 3, and 5 mM, GSH significantly increased the blastocyst rate and embryo quality. The highest blastocyst rate (51%) and the best embryo quality appeared in the 3-mM GSH treatment. Intracellular content of GSH of embryos at the two- to four-cell stage significantly increased, and the reactive oxygen species level decreased accordingly with 3-mM GSH treatment, but no significant differences were found in the four- to eight-cell stage and blastocysts. Gene expression analysis of the embryo regulator genes (OCT4, SOX2, and KLF4), GSH synthesis genes (GCLM, GCLC, and GSS), and GSH utilization genes (GSTP, GSTM, and GPX) showed that the GSH had no significant impact on these genes. In conclusion, exogenous GSH during IVC improved developmental potential and quality of bovine IVF embryos, which was probably caused by the ability of GSH to maintain the redox balance.

Effect of mouse cumulus cells on the in vitro maturation and developmental potential of bovine denuded germinal vesicle oocytes.

We investigated the effect mouse cumulus cells (mCCs) on the in vitro maturation (IVM) and developmental potential of bovine denuded germinal vesicle oocytes (DOs). Cumulus-oocyte complexes (COCs), DOs and DOs cocultured with either mCCs (DOs + mCCs) or bovine cumulus cells (bCCs; DOs + bCCs) were subjected to IVM. The meiosis II (MII) rates of DOs, glutathione (GSH) contents, zona pellucida (ZP) hardening and parthenogenetic blastocyst rates of MII oocytes were determined. The relative expression levels of bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9) in MII oocytes were measured using quantitative real-time polymerase chain reaction (PCR). mCCs significantly increased the MII rate of DOs from 53.5 ± 3.58% to 69.67 ± 4.72% (p < 0.05) but had no effect on the GSH content (2.17 ± 0.31 pmol/oocyte with mCCs, 2.14 ± 0.53 pmol/oocyte without mCCs). For the DOs + mCCs group, the BMP-15 and GDF-9 expression levels were significantly higher and the ZP dissolution time was significantly lower (162.49 ± 12.51 s) than that of the DOs group (213.95 ± 18.87 s; p < 0.05). The blastocyst rate of the DOs + mCCs group (32.56 ± 4.94%) was similar to that of the DOs group (31.75 ± 3.65%) but was significantly lower than that of the COCs group (43.52 ± 5.37%; p < 0.05). In conclusion, mCCs increased the MII rate of DOs and expression of certain genes in MII oocytes, and decreased the ZP hardening of MII oocytes, but could not improve their GSH content or developmental potential.

[Relationship of epidermal growth factor receptor in lung development].

The epidermal growth factor receptor (EGFR), a transmembrane protein receptor, is a member of ErbB family with signal-transducing tyrosine kinase activity. After combined with the ligand, EGFR homologous or heterologous dimers are formed to induce intracellular signal transduction, activate downstream signal transduction pathways, and then produce a series of biological effects. RAF/MEK/RAS/ERK pathway is relevant to cell proliferation, differentiation and apoptosis; while PDK1/AKT /PI3K pathway is involved in cell migration and adhesion. EGFR can promote the maturity of pulmonary type II epithelial cells and the synthesis and secretion of pulmonary surfactant. EGFR shows the effect on mammal lungs in a time-space and dose-dependent manner. The down-regulated expression of it will lead to immature lung development, while the over-expression can promote the cell proliferation, invasion and metastasis of the lung cancer cells. This paper reviewed advances in the study for EGFR and its signal pathway, as well as the relationship among EGFR, atelectasis and lung cancer.