Novel Functional Recombinant Human Follicle-Stimulating Hormone Acquired from Goat Milk.

An animal mammary bioreactor is regarded as an excellent biological system which is applied to produce large-scale recombinant proteins in milk. However, there are no effective methods to produce a large amount of some pharmaceutical proteins, such as human follicle-stimulating hormone (FSH), by large animal mammary gland bioreactors due to the fact that accumulation of excessive bioactive FSH might cause serious diseases in animals. Here, we report a novel strategy of preparing recombinant human FSH (rhFSH) from goat mammary glands, which could avoid the accumulation of bioactive FSH in goats. First, the single inactive FSHα and FSHß subunits expressed in goat mammary epithelial cells and goat mammary glands were performed to reassemble in vitro and were found to self-assemble into a complete heterodimer rhFSH at 4 °C and pH 7.4. Further, a cyclic adenosine monophosphate (cAMP) induction assay showed that the cAMP levels in cell lysate of HEK 293/FSHR cells were increased by about 8-fold in reassembled rhFSH groups than that in the control group (P < 0.01). Pharmacokinetic analysis indicated that the reassembled rhFSH from goat mammary glands was comparable to that of the commercially available Gonal-F (P > 0.05). In addition, the increasing dose of reassembled rhFSH significantly promoted ovulation of mouse and ovary weight gain of Sprague Dawley rat compared with the control groups and maximum values were up to 3-fold (P < 0.01) and 2.8-fold (P < 0.01), respectively. The reassembled rhFSH showed a similar effect to Gonal-F in inducing expression of FSH target genes in vivo and activating the PI3K pathway in granulosa cells. Our study developed a novel method to produce rhFSH and provided the basis for preparing FSH by the goat mammary gland bioreactor with less health problems on the producing animals.

Development and characterization of in vitro self-assembled recombinant human follicle stimulating hormone originated from goat mammary epithelial cells.

Follicle stimulating hormone (FSH), composed of FSHα and FSHß subunits, is essential for female follicle development and male spermatogenesis. The recombinant human FSH (rhFSH) products on the market are mainly generated from mammalian cells and are expensive. Large animal mammary gland bioreactors are urgently needed to produce large amounts of rhFSH. However, there are currently no effective methods to prepare rhFSH by large animals mainly due to the fact that excessive accumulation of FSH might cause many adverse effects in animals. We herein report the development and characterization of functional self-assembled rhFSH produced in goat mammary epithelial cells (GMECs). FSHα and FSHß stably expressed in Chinese hamster ovary (CHO) cell lines were secreted into culture medium and well glycosylated. Importantly, FSHα and FSHß expressed apart were able to assemble into functional FSH. We next inserted human FSHα or FSHß gene separately into goat ß-Lactoglobulin locus in GMECs by CRISPR/Cas9. Inactive FSHα and FSHß subunits expressed from GMECs assembled into rhFSH as analyzed by His-tag pull down assay. Functional assessment of rhFSH by cAMP induction assay, mouse ovulation induction and rat ovarian weight gain experiments showed that the bioactivity of self-assembled rhFSH expressed by GMECs was comparable to that of Gonal-F both in vitro and in vivo. Our study demonstrated that FSHα and FSHß can be separately expressed and assembled into functional rhFSH, and provided the basis for future preparing FSH by goat mammary gland bioreactor with less health problems on the producing animals.

Rutin protects boar sperm from cryodamage via enhancing the antioxidative defense.

This study was aimed to investigate whether and how Rutin protects boar sperm against cryoinjury during cryopreservation. Five concentrations of Rutin with 0.2, 0.4, 0.6, 1.0, and 2.0 mM were added to the freezing extender of boar sperm, respectively, and the effects on quality and function of boar sperm after freezing-thawing were assessed. The results showed that the sperm motility, mitochondrial activity, plasma membrane integrity, and acrosomal integrity were significantly improved in 0.4 mM and 0.6 mM Rutin groups (p < .05). Compared with ganoderma lucidum polysaccharide (GLP) or Tanshinone IIA, Rutin exhibited higher rates of mitochondrial activity and acrosome integrity (p < .05). Mechanistically, the addition of Rutin at the concentration of 0.6, 0.8, and 1.0 mM significantly attenuated ROS accumulation and MDA production by improving antioxidant enzymatic activity, including SOD, CAT, and GSH-Px (p < .05). Functionally, a higher penetration rate and the increased total efficiency of fertilization were observed in the 0.4, 0.6, and 1.0 mM Rutin groups than in the control group (p < .05). Moreover, the addition of Rutin (0.6 mM) significantly induced an increase in both the cleavage and blastocyst rates (p < .05). In summary, supplementation with Rutin in cryopreservation medium protects boar sperm against ROS attack by enhancing the antioxidative defense.

A novel role of SIRT2 in regulating gap junction communications via connexin-43 in bovine cumulus-oocyte complexes.

SIRT2, the predominantly cytosolic sirtuin, plays important role in multiple biological processes, including metabolism, stress response, and aging. However, the function of SIRT2 in gap junction intercellular communications (GJICs) of cumulus-oocyte complexes (COCs) is not yet known. The purpose of the present study was to evaluate the effect and underlining mechanism of SIRT2 on GJICs in COCs. Here, we found that treatment with SIRT2 inhibitors (SirReal2 or TM) inhibited bovine oocyte nuclear maturation. Further analysis revealed that SIRT2 inactivation disturbed the GJICs of COCs during in vitro maturation. Correspondingly, both the Cx43 phosphorylation levels and MEK/MER signaling pathways were induced by SIRT2 inhibition. Importantly, SIRT2-mediated Cx43 phosphorylation was completely abolished by treatment with MEK1/2 inhibitor (Trametinib). Furthermore, treatment with SIRT2 inhibitors resulted in the high levels of MEK1/2 acetylation. Functionally, downregulating the MER/ERK pathways with inhibitors (Trametinib or SCH772984) could attenuate the closure of GJICs caused by SIRT2 inactivation in partly. In addition, inhibition of SIRT2 activity significantly decreased the membrane and zona pellucida localization of Cx43 by upregulating the levels of Cx43 acetylation. Taken together, these results demonstrated a novel role that SIRT2 regulates GJICs via modulating the phosphorylation and deacetylation of Cx43 in COCs.

SIRT2 functions in aging, autophagy, and apoptosis in post-maturation bovine oocytes.

AIMS: Sirtuins have been implicated in the aging process, however, the functions of SIRT2 in post-maturation aging of oocytes are not fully understood. The purpose of the present investigation was to assess the roles of SIRT2 in aged oocytes and mechanisms involved. MAIN METHODS: The fresh MII oocytes were aging in vitro, and treated with SIRT2 inhibitor (SirReal2), autophagy activator (Rapamycin), and autophagy inhibitor (3-Ma) for 24 h, respectively. Oocyte activation, cytoplasmic fragmentation, and spindle defects, mitochondrial distribution, ROS levels, ATP production, mitochondrial membrane potential, and early apoptosis were investigated. Western blotting was performed to determine LC3-II accumulation, SQSTM1 degradation, and caspase-3 activity. KEY FINDINGS: SIRT2 expression gradually decreased in a time-dependent manner during oocyte aging. Treatment with SirReal2 significantly increased the rates of oocyte activation, cytoplasmic fragmentation, and spindle defects. In particular, the high ROS levels, abnormal mitochondrial distribution, low ATP production, and lost ΔΨm were observed in SirReal2-exposed oocytes. Further analysis revealed that LC3-II accumulation and SQSTM1 degradation were induced by SIRT2 inhibition. By performing early apoptosis analysis showed that oocyte aging was accompanied with cellular apoptosis, and SIRT2 inhibition increased apoptosis rates of aged oocytes. Importantly, upregulating autophagy with Rapamycin could mimic the effects of SIRT2 inhibition on apoptosis by increasing caspase-3 activation, whereas downregulating autophagy with 3-MA could abolish those effects by blocking caspase-3 activation. SIGNIFICANCE: Our results suggest that SIRT2 inactivation is a key mechanism underlying of cellular aging, and SIRT2 inhibition contributes to autophagy-dependent cellular apoptosis in post-maturation oocytes.

SIRT2 Inhibition Results in Meiotic Arrest, Mitochondrial Dysfunction, and Disturbance of Redox Homeostasis during Bovine Oocyte Maturation.

SIRT2, a member of the sirtuin family, has been recently shown to exert important effects on mitosis and/or metabolism. However, its roles in oocyte maturation have not been fully clarified. In this study, SIRT2, located in the cytoplasm and nucleus, was found in abundance in the meiotic stage, and its expression gradually decreased until the blastocyst stage. Treatment with SIRT2 inhibitors resulted in the prevention of oocyte maturation and the formation of poor-quality oocytes. By performing confocal scanning and quantitative analysis, the results showed that SIRT2 inhibition induced prominent defects in spindle/chromosome morphology, and led to the hyperacetylation of α-tubulin and H4K16. In particular, SIRT2 inhibition impeded cytoplasmic maturation by disturbing the normal distribution of cortical granules, endoplasmic reticulum, and mitochondria during oocyte meiosis. Meanwhile, exposure to SirReal2 led to elevated intracellular reactive oxygen species (ROS) accumulation, low ATP production, and reduced mitochondrial membrane potential in oocytes. Further analysis revealed that SIRT2 inhibition modulated mitochondrial biogenesis and dynamics via the downregulation of TFAM and Mfn2, and the upregulation of DRP1. Mechanistically, SIRT2 inhibition blocked the nuclear translocation of FoxO3a by increasing FoxO3a acetylation, thereby downregulating the expression of FoxO3a-dependent antioxidant genes SOD2 and Cat. These results provide insights into the potential mechanisms by which SIRT2-dependent deacetylation activity exerts its effects on oocyte quality.

Raf-ERK1/2 signalling pathways mediate steroid hormone synthesis in bovine ovarian granulosa cells.

Steroid hormones are required for normal reproductive function of female. The aim of this study was to investigate the role of Raf-ERK1/2 on steroid hormone synthesis in bovine ovarian granulosa cells. Immunohistochemistry assay showed that both B-Raf and C-Raf were expressed in granulosa cells, theca cells and Sertoli cells. The protein expression of Raf or ERK1/2 was clearly decreased by Raf inhibitor GSK2118436 or ERK1/2 inhibitor SCH772984, respectively (p < 0.05). In addition, western blotting was performed for investigating the crosstalk between Raf and ERK1/2, the data showed that Raf positively regulated ERK1/2, whereas ERK1/2 had a negative feedback effect on Raf. The biosynthesis of oestradiol or testosterone was significantly decreased by treatment with GSK2118436 or SCH772984 (p < 0.05). Conversely, the progesterone biosynthesis was clearly increased by treatment with those inhibitors (p < 0.05). Furthermore, the mRNA expression of STAR, aromatase and CYP17 was blocked by Raf-ERK1/2 signalling inhibition, which oppositely induced the mRNA expression of CYP11. Together, these findings suggested that Raf-ERK1/2 signalling pathways mediate steroid hormone synthesis via affecting the expression of steroidogenic enzymes.

Combined effect of apigenin and ferulic acid on frozen-thawed boar sperm quality.

The main aim of the present study was to evaluate the cryoprotective effect of apigenin (AP) and ferulic acid (FA) on boar sperm during cryopreservation. AP and FA were both demonstrated to be high-efficiency antioxidants and had not previously been used to protect sperm from cryodamage. As boar sperm is sensitive to oxidative stress, suitable antioxidants are still needed for improving frozen-thawed sperm quality. With this purpose, semen samples coming from five boars were used in this study. Ejaculates of five boars were mixed and split into 16 aliquots, in which different doses of AP and FA were added separately or together. The motility, the plasma membrane integrity, the mitochondrial activity, the acrosomal integrity, the antioxidase activities and the malondialdehyde concentration of the frozen-thawed boar sperm were assessed. The results suggested that both AP and FA significantly improved the frozen-thawed boar sperm quality in all these aspects when they were added to the freezing extender separately, while the highest improvement was recorded when the extender was supplemented with 0.1 mmol/L AP plus 0.15 mmol/L FA. These findings demonstrated that supplementation of freezing extender with both AP and FA had a combined, beneficial effect on frozen-thawed boar sperm.

Effects of alginate on frozen-thawed boar spermatozoa quality, lipid peroxidation and antioxidant enzymes activities.

Although alginate was reported to play an important role as free radical scavengers in vitro and could be used as sources of natural antioxidants, there was no study about the cryoprotective effects of alginate on boar spermatozoa freezing. The objective of this research was to evaluate the effects of different concentrations of alginate added to the freezing extenders on boar spermatozoa motility, plasma membrane integrity, acrosomal integrity, mitochondrial activities, lipid peroxidation and antioxidative enzymes activities (SOD and GSH-Px) after thawing. Alginate was added to the TCG extender to yield six different final concentrations: 0, 0.2, 0.4, 0.6, 0.8, and 1.0mg/mL. The semen extender supplemented with various doses of alginate increased (P<0.05) total motility. The spermatozoa plasma membrane integrity and mitochondrial activity were improved at four different concentrations: 0.4, 0.6, 0.8, 1.0mg/mL. The addition of alginate also provided significantly positive effect on post-thaw boar spermatozoa acrosomal integrity at concentrations of 0.6, 0.8, 1.0mg/mL, compared with that of the control (P<0.05). The freezing extenders with the presence of alginate led to higher SOD and GSH-Px activities and lower MDA levels, in comparison to the control (P<0.05). In summary, alginate exhibited a dose-related response on frozen-thawed boar spermatozoa motility, functional integrity and antioxidative capacity at appropriate concentrations. Therefore alginate could be employed as an effective cryoprotectant in boar spermatozoa cryopreservation.

Determination of swainsonine in the endophytic Undifilum fungi by high-performance liquid chromatography with evaporative light-scattering detector.

Endophytic Undifilum oxytropis found within toxic locoweeds (Astragalus and Oxytropis spp.) produces the indolizidine alkaloid swainsonine, which is responsible for locoism in grazing animals. The aim of the current study is to establish an easy and accurate method for the determination of swainsonine in the endophytic Undifilum fungi. High-performance liquid chromatography (HPLC) with evaporative light-scattering detector (ELSD) was used for the assay of swainsonine in this study for the first time. The HPLC conditions were Waters XBridge hydrophilic interaction liquid chromatography column using acetonitrile-5 mM ammonium acetate (1:1, vol/vol) containing 0.02% (vol/vol) aqueous ammonium hydroxide as mobile phase at a flow rate of 0.5 mL/min. ELSD conditions were optimized at nebulizer-gas flow rate of 25 psi and drift tube temperature of 55 °C. The method was validated to achieve the satisfactory precision and recovery, and the calibration range was 15.625-250 µg/mL. Application of the developed analytical procedure to determine swainsonine content in the endophytic Undifilum fungi samples ensured its suitability for the routine analysis of swainsonine.

Antitumor activity of polysaccharides isolated from Patrinia heterophylla.

The research investigated the effect of Patrinia heterophylla Bunge (Valerianaceae) polysaccharides (PHB-P1) on U14-bearing mice. The tumor weight of mice treated with PHB-P1 (30, 60 mg/kg body weight) was significantly lower than that of the control group, a decrease of serum lactate dehydrogenase (LDH) activity was observed, and the serum alkaline phosphatase (AKP) level was increased slightly. The number of apoptotic tumor cells was significantly increased in the mice by treatment of PHB-P1 (30, 60 mg/kgbw). Cell cycle analysis showed the accumulation of tumor cells in the G2/M phase and a relative decrease of the S phase. By the immunohistochemical analysis, PHB-P1 (30, 60 mg/kgbw) might up-regulate the expression of p53 and Bax, and significantly inhibited the expression of Bcl-2 in tumor tissues. In conclusion, PHB-P1 could inhibit tumor growth and induce tumor cell apoptosis.

Swainsonine-producing fungal endophytes from major locoweed species in China.

Locoweeds including the toxic species of Astragalus spp and Oxytropis spp. are widely distributed in the western region of China and result in a chronic neurological disease known as locoism in animals. To determine the presence of swainsonine-producing fungal endophyte of major locoweed species in China, endophytes were isolated from 8 locoweed species that including A. variabilis, A. strictus, O. glacialis, O. kansuensis, O. ochrocepala, O. sericopetala, O. glabra and O. latibracteata. Seven species of locoweed were confirmed contain substantial amounts of swainsonine and infect swainsonine-producing fungal endophyte. These endophytes were classified as Undifilim oxytropis according to the fungal morphology and phylogenetic analysis based on sufficient ITS sequences. PCR-RFLP analysis of IGS region showed that the interspecific or intraspecific variations were present among the endophytes from different locoweed species.

Anti-tumor activity of polysaccharides isolated from Patrinia scabra Bunge on U14 cervical carcinoma bearing mice.

The aim of our study was to investigate the effect of Patrinia scabra Bunge polysaccharide (PSB-P2) on cervical cancer cell (U14)-bearing mice. The tumor weight of mice treated with PSB-P2 (40, 80 mg/kg b.w.) was significantly lower than that of the control group and serum lactate dehydrogenase (LDH) activity was decreased, while serum alkaline phosphatase (AKP) level was only changed slightly. Meanwhile, the number of apoptotic tumor cells was significantly increased in the mice by the treatment of PSB-P2 (40, 80 mg/kg b.w.). At the same time, cell cycle analysis showed the accumulation of tumor cells in the G0/G1 phase and a relative decrease in the S phase. On the other hand, using the reverse transcription-polymerase chain reaction (RT-PCR) assay, PSB-P2 (40, 80 mg/kg b.w.) showed the up-regulation of p53 and Bax, and significant inhibition of Bcl-2 in tumor tissues. It suggests a possible mechanism of the inhibitory effect of PSB-P2 on tumor growth.

[Effect of Gleditsia sinensis stings on growth inhibition and expression of PCNA and p53 in mice bearing uterine cervical carcinoma (U14)].

OBJECTIVE: To study the antitumor effect of the stings of Gleditsia sinensis on mice bearing uterine cervical carcinoma (U14) and the expression of PCNA (proliferating cell nuclear antigen) and p53. METHOD: The effect of the ethanolic extract of G. sinensis stings on the inhibition rate of solid tumor and the life span of ascites tumor were calculated by the animal tumor model experiment in vivo. The positive cell numbers of PCNA and mutant p53 protein were measured by immunohistochemical SP method. RESULT: As compared with the control group, the ethanolic extract of G. sinensis stings (250, 500 and 1 000 mg x kg(-1) body weight, p.o.) and CTX (25 mg kg(-1) body weight, i.p.) administration significantly reduced the tumor weight of solid tumor and increased the life span of ascites tumor harboring mice (P < 0.01). The inhibition rate of solid tumor and the rate in life span were up to 47.44%, 59.49%, 63.92%, 73.42% and 52.21%, 67.26%, 78.76%, 95.58% respectively. Meanwhile,the expression of PCNA and mutant p53 protein also suppressed by ethanolic extract (P < 0.05, P < 0.01). CONCLUSION: The stings of G. sinensis showed antitumor activity and its possible mechanism might be related with the expression inhibition of PCNA and mutant p53 protein.