Accumulative gene integration into a pre-determined site using Cre/loxP

Site-specific gene recombination systems, such as Cre/loxP, have been used for genetic modification of cells and organisms in both basic and applied research. We previously developed an accumulative gene integration system (AGIS), in which target gene cassettes could be repeatedly integrated into a pre-determined site on a plasmid or cellular genome by recombinase-mediated cassette exchange (RMCE), using Cre and mutated loxPs. In the present study, we designed a simplified AGIS. For gene integration into a target site, the previous system used two loxP sites in the acceptor DNA, whereas the new system uses a single loxP site. The gene integration reactions were repeated four times in vitro using Cre protein and specific plasmids. The expected integration reactions mediated by Cre occurred at the loxP sites, resulting in integration of four target genes. The system was also used for genomic integration of reporter genes using Chinese hamster ovary (CHO) cells. The reporter genes were efficiently introduced into the CHO genome in a Cre-dependent manner, and transgene expression was detected after the integration reaction. The expression levels of the reporter genes were enhanced, corresponding to the increase of transgene copy number. Recombinase-mediated AGIS provides a useful tool for the modification of cellular genomes.     

人丙酮酸羧化酶异位表达提高中国仓鼠卵巢细胞补料分批培养后期活率

构建了人丙酮酸羧化酶(hPC)异位表达的重组CHO细胞,并检测了重组细胞生长情况的改变。利用分子生物学实验技术构建hPC-pcDNA 3.0表达载体,将其转染CHO-K1细胞;转染细胞经过G418抗性筛选后,通过荧光定量PCR检测目的基因表达,并挑选表达量最高的克隆进行补料分批培养。电泳及测序结果显示,构建的hPC-pcDNA3.0表达载体序列与预期一致;在mRNA水平鉴定目的基因显示,4#克隆的mRNA表达水平最高;选其进行补料分批培养,生长曲线显示在培养后期,其活细胞密度和细胞活率均高于对照。成功构建了细胞生长和活率改善的重组CHO细胞,获得了生长改善的细胞株,为后续的重组蛋白表达研究与细胞培养工艺优化奠定了基础。     

Cysteine–Cysteine Motif Chemokine Receptor 5 Expression in Letrozole-Induced Polycystic Ovary Syndrome Mice

Polycystic ovary syndrome (PCOS), which affects 5–10% of women of reproductive age, is associated with reproductive and metabolic disorders, such as chronic anovulation, infertility, insulin resistance, and type 2 diabetes. However, the mechanism of PCOS is still unknown. Therefore, this study used a letrozole-exposed mouse model in which mice were orally fed letrozole for 20 weeks to investigate the effects of letrozole on the severity of reproductive and metabolic consequences and the expression of cysteine–cysteine motif chemokine receptor 5 (CCR5) in letrozole-induced PCOS mice. The letrozole-treated mice showed a disrupted estrous cycle and were arrested in the diestrus phase. Letrozole treatment also increased plasma testosterone levels, decreased estradiol levels, and caused multicystic follicle formation. Furthermore, histological analysis of the perigonadal white adipose tissue (pgWAT) showed no significant difference in the size and number of adipocytes between the letrozole-treated mice and the control group. Further, the letrozole-treated mice demonstrated glucose intolerance and insulin resistance during oral glucose and insulin tolerance testing. Additionally, the expression of CCR5 and cysteine-cysteine motif ligand 5 (CCL5) were significantly higher in the pgWAT of the letrozole-treated mice compared with the control group. CCR5 and CCL5 were also significantly correlated with the homeostasis model assessment of insulin resistance (HOMA-IR). Finally, the mechanisms of insulin resistance in PCOS may be caused by an increase in serine phosphorylation and a decrease in Akt phosphorylation.     

Impact of SGLT-2 Inhibition on Cardiometabolic Abnormalities in a Rat Model of Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women. PCOS is characterized by hyperandrogenism and ovulatory dysfunction. Women with PCOS have a high prevalence of obesity, insulin resistance (IR), increased blood pressure (BP), and activation of the renin angiotensin system (RAS). Effective evidence-based therapeutics to ameliorate the cardiometabolic complications in PCOS are lacking. The sodium-glucose cotransporter-2 (SGLT2) inhibitor Empagliflozin (EMPA) reduces BP and hyperglycemia in type 2 diabetes mellitus. We hypothesized that hyperandrogenemia upregulates renal SGLT2 expression and that EMPA ameliorates cardiometabolic complications in a hyperandrogenemic PCOS model. Four-week-old female Sprague Dawley rats were treated with dihydrotestosterone (DHT) for 90 days, and EMPA was co-administered for the last three weeks. DHT upregulated renal SGLT2, SGLT4, and GLUT2, but downregulated SGLT3 mRNA expression. EMPA decreased DHT-mediated increases in fat mass, plasma leptin, and BP, but failed to decrease plasma insulin, HbA1c, or albuminuria. EMPA decreased DHT-mediated increase in renal angiotensin converting enzyme (ACE), angiotensin converting enzyme 2 (ACE2), and angiotensin II type 1 receptor (AGT1R) mRNA and protein expression. In summary, SGLT2 inhibition proved beneficial in adiposity and BP reduction in a hyperandrogenemic PCOS model; however, additional therapies may be needed to improve IR and renal injury.     

Therapeutic Potentials of Low-Dose Tacrolimus for Aberrant Endometrial Features in Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS) is a major anovulatory infertility affecting a great proportion of women of childbearing age and is associated with obesity, insulin resistance and chronic inflammation. Poor endometrial receptivity and recurrent implantation failure are major hurdles to the establishment of pregnancy in women with PCOS. The accumulating body of evidence obtained from experimental and clinical studies suggests a link between inherent adaptive and innate immune irregularities and aberrant endometrial features in PCOS. The use of conventional therapeutic interventions such as lifestyle modification, metformin and ovarian stimulation has achieved limited clinical success in restoring ovulation and endometrial receptivity in women with PCOS. Unlike other immunosuppressive drugs prescribed in the clinical management of autoimmune and inflammatory disorders that may have deleterious effects on fertility and fetal development, preclinical studies in mice and in women without PCOS but with repeated implantation failure revealed potential therapeutic benefits for the use of low-dose tacrolimus in treating female infertility. Improved systemic and ovarian immune functions, endometrial progesterone receptor and coreceptor expressions and uterine vascular adaptation to pregnancy were among features of enhanced progesterone-receptor sensitivity in the low-dose tacrolimus-treated mouse model of the disease. In this review, we have compiled available experimental and clinical data in literature on endometrial progesterone resistance and current therapeutic options, as well as mechanisms of actions and reported outcomes relevant to the potential therapeutic benefits for the use of low-dose tacrolimus in treating PCOS-associated female infertility.     

Impacts of chronic and increasing lipopolysaccharide exposure on production and reproductive parameters in lactating Holstein dairy cows

Lipopolysaccharide (LPS) administration causes immunoactivation, which negatively affects production and fertility, but experimental exposure via an acute bolus is unlikely to resemble natural infections. Thus, the objectives were to characterize effects of chronic endotoxemia on production parameters and follicular development in estrous-synchronized lactating cows. Eleven Holstein cows (169 ± 20 d in milk; 681 ± 16 kg of body weight) were acclimated to their environmental surroundings for 3 d and then enrolled in 2 experimental periods (P). During P1 (3 d) cows consumed feed ad libitum and baseline samples were obtained. During P2 (7 d), cows were assigned to continuous infusion of either (1) saline-infused and pair-fed (CON-PF; 40 mL/h of saline i.v.; n = 5) or (2) LPS infused and ad libitum fed (LPS-AL; Escherichia coli O55:B5; 0.017, 0.020, 0.026, 0.036, 0.055, 0.088, and 0.148 μg/kg of body weight/h i.v. on d 1 to 7, respectively; n = 6). Controls were pair-fed to the LPS-AL group to eliminate confounding effects of dissimilar nutrient intake. Infusing LPS temporally caused mild hyperthermia on d 1 to 3 (+0.49°C) relative to baseline. Dry matter intake of LPS-AL cows decreased (28%) on d 1 of P2, then progressively returned to baseline. Relative to baseline, milk yield from LPS-AL cows was decreased on d 1 of P2 (12%). No treatment differences were observed in milk yield during P2. Follicular growth, dominant follicle size, serum progesterone (P4), and follicular P4 and 17β-estradiol concentrations were similar between treatments. Serum 17β-estradiol tended to increase (115%) and serum amyloid A and LPS-binding protein were increased (118 and 40%, respectively) in LPS-AL relative to CON-PF cows. Compared with CON-PF, neutrophils in LPS-AL cows were initially increased (45%), then gradually decreased. In contrast, monocytes were initially decreased (40%) and progressively increased with time in the LPS-AL cows. Hepatic mRNA abundance of cytochrome P450 family 2 subfamily C (CYP2C) or CYP3A was not affected by LPS, nor was there a treatment effect on toll-like receptor 4 or LBP; however, acyloxyacyl hydrolase and RELA subunit of nuclear factor kappa B tended to be increased in LPS-AL cows. These data suggest lactating dairy cows become tolerant to chronic and exponentially increasing LPS infusion in terms of production and reproductive parameters.     

Fibroinflammatory Signatures Increase with Age in the Human Ovary and Follicular Fluid

The female reproductive system ages before any other organ system in the body. This phenomenon can have tangible clinical implications leading to infertility, miscarriages, birth defects and systemic deterioration due to estrogen loss. “Fibroinflammation” is a hallmark of aging tissues; there is an increase in inflammatory cytokines and fibrotic tissue in the aging ovarian stroma. We systematically evaluated immunomodulatory factors in human follicular fluid, which, like the stroma, is a critical ovarian microenvironment directly influencing the oocyte. Using a cytokine antibody array, we identified a unique fibroinflammatory cytokine signature in follicular fluid across an aging series of women (27.7–44.8 years). This signature (IL-3, IL-7, IL-15, TGFβ1, TGFβ3 and MIP-1) increased with chronologic age, was inversely correlated to anti-Müllerian hormone (AMH) levels, and was independent of body mass index (BMI). We focused on one specific protein, TGFβ3, for further validation. By investigating this cytokine in human cumulus cells and ovarian tissue, we found that the age-dependent increase in TGFβ3 expression was unique to the ovarian stroma but not other ovarian sub-compartments. This study broadens our understanding of inflammaging in the female reproductive system and provides a defined fibroinflammatory aging signature in follicular fluid and molecular targets in the ovary with potential clinical utility.     

Changes in Fecundity and Egg Lipid Content of Lake Whitefish (Coregonus clupeaformis) in the Upper Laurentian Great Lakes between 1986–87 and 2003–05

Lake whitefish (Coregonus clupeaformis Mitchill), an important commercial species in the Laurentian Great Lakes, have experienced decreased growth and condition in regions of the upper Great Lakes over the past 20 years. Increases in lake whitefish density and decreases in the density of Diporeia spp., an energy rich and historically important part of the lake whitefish diet, have been implicated in the recent declines in lake whitefish growth and condition. The goal of this study was to describe lake whitefish fecundity, egg lipid content, and total ovary lipid content in selected regions of Lakes Huron, Michigan, and Superior in 1986–87 and 2003–05, two time periods with different lake whitefish and Diporeia densities. Under conditions of high lake whitefish density and low Diporeia density, female lake whitefish in the upper Laurentian Great Lakes generally produced fewer eggs. Egg lipid content was higher in 2003–05 than in 1986–87 at all sites, regardless of changes in lake whitefish or Diporeia densities. Total ovary lipid content and lake whitefish abundance were inversely related, while there was no significant relationship between total ovary lipid content and Diporeia density. The amount of energy that lake whitefish invested in egg production was more closely associated with lake whitefish abundance than with Diporeia density. This study provides evidence that recent changes in production dynamics of Great Lakes lake whitefish have not been driven solely by declines in Diporeia but have been significantly influenced by lake whitefish abundance.     

Intrauterine high androgen promotes obesity of the offspring of rats with polycystic ovarian syndrome via activating macrophage-angiogenesis-related androgen signaling

The development of polycystic ovary syndrome (PCOS) is closely related to the chronic inflammatory and obese. Recent studies have found macrophages regulate the chronic inflammation and adipose tissue remodelling, but the underlying mechanisms have not been clarified. In this study, we established a model of PCOS in the offspring rats by high androgen exposure during late pregnancy in parental and established a female rat macrophage eliminating model by rejection of clodronate liposome. Then, the offspring rat macrophage phenotype in offspring female rat adipose tissue, and levels of testosterone, angiogenic factors (PDGF and VEGF) and inflammatory factors (TNF-α and MCP-1) were investigated. By coculture of RAW264.7 macrophage with adipocytes or C166 endothelial cells (ECs), the mobility of adipocytes, and the ECs function with associated signalling pathway were detected by using of androgen inhibitor Apalutamide, NF-κB inhibitor JSH-23 and ERK1/2 inhibitor LY3214996. It was found that high androgen exposure during late pregnancy led to increased testosterone levels and overweight and obesity, increased size and reduced number of subcutaneous and intra-abdominal adipocytes, and increased secretion of TNF-α and MCP-1 in female rats in the offspring. Eliminating macrophages significantly increased adipocytes and angiogenesis in offspring of rats with intrauterine high androgen, and reduced TNF-α and MCP-1. Macrophages promoted mobility of adipocytes, and inhibited proliferation, migration, tube formation of ECs under hyperandrogenic condition, which were significantly inhibited by Apalutamide, JSH-23 and LY3214996. Thus, intrauterine high androgen promotes obesity of the offspring of rats with polycystic ovarian syndrome through increasing M1 differentiation of pro-inflammatory macrophages and activating VEGF-related angiogenesis via androgen/NF-κB/ERK1/2 signalling pathway.