Human nucleolar protein SURF6/RRP14 participates in early steps of pre-rRNA processing.

The biogenesis of ribosomes requires tightly controlled transcription and processing of pre-rRNA which comprises ribosomal RNAs forming the core of large and small ribosomal subunits. Early steps of the pre-rRNA processing and assembly of the ribosomal subunits require a large set of proteins that perform folding and nucleolytic cleavage of pre-rRNAs in the nucleoli. Structure and functions of proteins involved in the pre-rRNA processing have been extensively studied in the budding yeast S. cerevisiae. Functional characterization of their human homologues is complicated by the complexity of mammalian ribosomes and increased number of protein factors involved in the ribosomal biogenesis. Homologues of human nucleolar protein SURF6 from yeast and mouse, Rrp14 and Surf6, respectively, had been shown to be involved in the early steps of pre-rRNA processing. Rrp14 works as RNA chaperone in complex with proteins Ssf1 and Rrp15. Human SURF6 knockdown and overexpression were used to clarify a role of SURF6 in the early steps of pre-rRNA processing in human cell lines HeLa and HTC116. By analyzing the abundance of the rRNA precursors in cells with decreased level or overexpression of SURF6, we demonstrated that human SURF6 is involved in the maturation of rRNAs from both small and large ribosomal subunits. Changes in the SURF6 level caused by knockdown or overexpression of the protein do not result in the death of HeLa cells in contrast to murine embryonic fibroblasts, but significantly alter the distribution of cells among the phases of the cell cycle. SURF6 knockdown in both p53 sufficient and p53 deficient HCT116 human cancer cells results in elongation of G0/G1 and shortening of G2/M phase. This surprising result suggests p53 independence of SURF6 effects on the cell cycle and possible multiple functions of SURF6. Our data point to the shift from pathway 1 to pathway 2 of the rRNA biogenesis caused by the SURF6 knockdown and its likely association with p53 pathway.

Overexpression of ribosomal RNA in cumulus cells of patients with polycystic ovary syndrome.

OBJECTIVE: To evaluate the level of ribosomal RNA (rRNA) in cumulus cells (CCs) from women with polycystic ovary syndrome (PCOS) undergoing ICSI. MATERIALS AND METHODS: The study population included three healthy oocyte donors (control) and three patients with PCOS. RNA expression in CCs was assessed using quantitative real-time PCR assay to measure the pre-rRNA transcripts (5'ETS region), 18 S, 5.8 S and 28 S rRNA. RESULTS: The level of 18 S rRNA is 3.9 times higher (p = 0.036) and the level of 5.8 S rRNA is 2.9 times higher (p = 0.049) in CCs of PCOS patients than in CCs of healthy women. The fold change in expression of 28 S rRNA in CCs of PCOS patients also exceeds that in the control group, but did not reach a statistical significance (p = 0.342). CONCLUSION: Our observations support the idea that CCs of PCOS patients contain comparatively more ribosomes that CCs of healthy oocyte donors that may indicate a higher proliferation rate or up-regulated translation of protein factors in ССs of PCOS patients.

Purification Process of a Recombinant Human Follicle Stimulating Hormone Biosimilar (Primapur®) to Yield a Pharmaceutical Product with High Batch-to-Batch Consistency.

Recombinant human follicle stimulating hormone (r-hFSH) is widely used for infertility treatment and is subject to the development of biosimilars. There are different purification strategies that can yield r-hFSH of pharmaceutical quality from Chinese hamster ovary cell culture broth. We developed a purification process for r-hFSH centered on immunoaffinity chromatography with single-domain recombinant camelid antibodies. The resulting downstream process is simple and devoid of ultrafiltration operations. Studies on chromatography resin resource and ligand leakage showed that the immunoaffinity matrix employed was suitable for industrial use and stable for at least 40 full chromatography cycles, and the leaked single-domain antibody ligand was completely removed by subsequent purification steps. All chromatography resins employed withstood the same 40 cycles of use without significant changes in separation efficiency and product binding capacity. The resulting industrial purification process yielded batches of r-hFSH with consistent levels of purity and bioactivity.

Association of the Serum Folate and Total Calcium and Magnesium Levels Before Ovarian Stimulation With Outcomes of Fresh In Vitro Fertilization Cycles in Normogonadotropic Women.

Background: Women of reproductive age are recommended to consume folic acid and other supplements before conception and during pregnancy. We aimed to investigate the association of the serum folate and total magnesium (Mg) and calcium (Ca) levels before ovarian stimulation with the outcomes of assisted reproductive technology (ART) in normogonadotropic women. Methods: We used a subanalysis of data obtained from a multicentre, randomized prospective study (NCT03088137). A total of 110 normogonadotropic, non-advanced aged, non-obese women with tubal and/or male infertility factors were enrolled for the single fresh ovarian stimulation GnRH antagonist cycle. The main outcome measures were the total oocyte yield, mature oocytes, fertilization rate, biochemical, clinical pregnancy, and live birth. Multivariable generalized linear models adjusted for covariates were used with a Poisson distribution and the log link function for adjusted oocyte counts, and a binomial distribution and the log link function were used for adjusted clinical ART outcomes. Results: The medians (interquartile range (IQR)) were as follows: baseline serum folate, 20.55 ng/ml (10.8, 32.9); Mg, 19.4 mg/L (18.7, 20.7); Ca, 94 mg/L (91.2, 96.4); and Ca/Mg ratio, 4.78 (4.55, 5.02). Women with higher serum folate concentrations (Q4≥33.0 ng/ml) had significantly lower total numbers of oocytes retrieved (adjusted mean (95% CI) 9.2 (7.6-11.3) vs 12.9 (10.9-15.4, p-trend=0.006)) and lower odds ratios (ORs) (95% CI) of 0.12 (0.02, 0.79) for clinical pregnancy and 0.10 (0.01, 0.70) for live birth compared with women in the lowest quartile (<10.8 ng/ml), all p-trend<0.001. Women in the highest Ca/Mg ratio quartile (≥5.02) had ORs (95% CI) of 6.58 (1.31, 33.04) for biochemical pregnancy, 4.85 (1.02, 23.08) for clinical pregnancy and 4.07 (0.83, 19.9) for the live birth rate compared with women in the lowest quartile (<4.55), all p-trend<0.001. Conclusions: Using multivariable models, we suggested that a baseline elevated serum folate level (≥33.0 ng/ml) and a lower Ca/Mg ratio were associated with worse ART outcomes in normogonadotropic women. Our findings might be useful for choosing safe dosages of folate, calcium, magnesium and complex supplementation for both fertile women and women undergoing infertility treatment. Further preconception large-scale studies with known micro- and macronutrient statuses of both parents and serum folate, Ca, Mg, and hormone levels, are needed.

High-level expression of biologically active human follicle stimulating hormone in the Chinese hamster ovary cell line by a pair of tricistronic and monocistronic vectors.

Recombinant human follicle stimulating hormone (FSH), produced in Chinese hamster ovary (CHO) cells, is widely used for treatment of fertility disorders and is subject to biosimilars development. Cell lines with high specific productivities may simplify the FSH production process. Here, we used our previously established expression system based on vector p1.1 to create new cell lines secreting heterodimeric FSH protein. To this end, we linked open reading frames of both FSH subunits by the wild-type internal ribosome entry site from the encephalomyocarditis virus (EMCV IRES). Intact and double-negative for the dihydrofolate reductase CHO cells were stably transfected by the FSH-coding plasmids. Stably transfected intact cells showed higher level of the FSH secretion and were utilized for subsequent methotrexate-driven transgene amplification, which doubled their productivity. The excess of the free α-subunit was corrected by transfecting the cells by the additional p1.1-based plasmid encoding the ß-subunit of the FSH. Clonal cell lines obtained secreted mostly the heterodimeric FSH and possessed specific productivities up to 12.3±1.7 pg/cell/day. Candidate clonal cell line C-P1.3-FSH-G4 maintained a constant specific productivity for at least 2 months of culturing without the section pressure. The resulting FSH protein conformed to the international pharmaceutical quality criteria as evidenced by the receptor binding kinetics, distribution pattern of hormone isoforms and biological activity. In conclusion, our expression system offers a simple and cost-effective approach to production of FSH.

A multicenter, randomized, phase III study comparing the efficacy and safety of follitropin alpha biosimilar and the original follitropin alpha.

OBJECTIVE: The aim of the present study was to investigate the therapeutic equivalence between the follitropin alpha biosimilar and the reference medication in women undergoing assisted reproductive technologies (ART). STUDY DESIGN: This multicenter, randomized (1:1), embryologist-blinded, parallel-group, comparative phase III study involved 110 women aged 20-35 years old with tubal and/or male factors of infertility. All of the subjects underwent controlled ovarian hyperstimulation (COH) using a gonadotropin-releasing hormone antagonist (GnRH-ant) protocol. Over the 5-day fixed-dose regimen, the women received 150 IU/day of follitropin alpha biosimilar (n = 55) or original follitropin alpha (n = 55), followed by dose adaptation. The primary endpoint for assessing the therapeutic equivalence was the number of retrieved oocytes using a pre-determined clinical equivalence margin of ± 3.4 oocytes. RESULTS: Similar numbers of oocytes were retrieved in both groups: 12.16 ± 7.28 in the follitropin alpha biosimilar group and 11.62 ± 6.29 in the original follitropin alpha group, with mean difference of 0.546 ± 1.297 oocytes (95% confidence interval [CI]: -2.026, 3.116), p = 0.002 (intention-to-treat [ITT] population). Additionally, no statistically significant differences were found for secondary endpoints: the onset of biochemical (34.7% and 36.7%, p = 0.883), clinical pregnancy (26.5% and 32.7%, p = 0.507), delivery (26.5% and 24.5%, p = 0.817) and take-home baby rate (28.6% and 26.5%, p = 0.816) for the follitropin biosimilar and original follitropin groups (per-protocol [PP] population). Ovarian hyperstimulation syndrome was observed in subjects with a positive pregnancy test in 0% and 3.64% of cases and after triggering ovulation in 7.27% and 3.64% for the follitropin biosimilar and original follitropin groups, respectively. CONCLUSIONS: This study demonstrated similar therapeutic equivalence and safety profiles between the follitropin alpha biosimilar and the reference follitropin in women who underwent COH in GnRH-ant cycles. TRIAL REGISTRATION NUMBER: 1. Name of the registry: ClinicalTrials.gov. TRIAL REGISTRATION NUMBER: NCT03088137. Date of registration: 02.03.2017, retrospectively registered. Trial conducted between 08.02.2017 and 17.08.2018, the date of enrollment of the first participant - 08.02.2017. 2. Name of the registry: Russian Ministry of Health, grls.rosminzdrav.ru. TRIAL REGISTRATION NUMBER: RCT 754. Date of registration: 26.10.2016, prospectively registered.

Involvement of the specific nucleolar protein SURF6 in regulation of proliferation and ribosome biogenesis in mouse NIH/3T3 fibroblasts.

The nucleolar proteins which link cell proliferation to ribosome biogenesis are regarded to be potentially oncogenic. Here, in order to examine the involvement of an evolutionary conserved nucleolar protein SURF6/Rrp14 in proliferation and ribosome biogenesis in mammalian cells, we established stably transfected mouse NIH/3T3 fibroblasts capable of conditional overexpression of the protein. Cell proliferation was monitored in real-time, and various cell cycle parameters were quantified based on flow cytometry, Br-dU-labeling and conventional microscopy data. We show that overexpression of SURF6 accelerates cell proliferation and promotes transition through all cell cycle phases. The most prominent SURF6 pro-proliferative effects include a significant reduction of the population doubling time, from 19.8 ± 0.7 to 16.2 ± 0.5 hours (t-test, p < 0.001), and of the length of cell division cycle, from 17.6 ± 0.6 to 14.0 ± 0.4 hours (t-test, p < 0.001). The later was due to the shortening of all cell cycle phases but the length of G1 period was reduced most, from 5.7 ± 0.4 to 3.8 ± 0.3 hours, or by ∼30%, (t-test, p < 0.05). By Northern blots and qRT-PCR, we further showed that the acceleration of cell proliferation was concomitant with an accumulation of rRNA species along both ribosomal subunit maturation pathways. It is evident, therefore, that like the yeast homologue Rrp14, mammalian SURF6 is involved in various steps of rRNA processing during ribosome biogenesis. We concluded that SURF6 is a novel positive regulator of proliferation and G1/S transition in mammals, implicating that SURF6 is a potential oncogenic protein, which can be further studied as a putative target in anti-cancer therapy.

Expression of proton-pumping nicotinamide nucleotide transhydrogenase in mouse, human brain and C elegans.

Proton-translocating nicotinamide nucleotide transhydrogenase is located in the mitochondrial inner membrane and catalyzes the reduction of NADP(+) by NADH to NADPH and NAD(+). The present investigation describes the expression of the transhydrogenase gene in various mouse organs, subsections of the human brain and Caenorhabditis elegans. In the mouse, the expression was highest in heart tissue (100%) followed by kidney (64%), testis (52%), adrenal gland (41%), liver (35%), pancreas (34%), bladder (26%), lung (25%), ovary (21%) and brain (14%). The expression in brain tissue was further investigated in the human brain which showed a distribution that apparently varied as a function of neuronal density, a result that was supported by estimations of expression in C. elegans using Green Fluorescent Protein (GFP) controlled by the transhydrogenase promoter. GFP-expressing C. elegans lines showed a clear concentration of fluorescence to the gut, the pharyngeal-intestinal valve and certain neurons. It is concluded that the transhydrogenase gene is expressed to various extents in all cell types in mouse, human and C. elegans.

Development of novel mouse hybridomas producing monoclonal antibodies specific to human and mouse nucleolar protein SURF-6.

SURF-6 is an evolutionarily conserved nucleolar protein that is important for cell viability; however, its function in mammals still remains uncertain. The aim of this study is to generate monoclonal antibodies to human SURF-6 protein suitable for fundamental and biomedical research. The full-size human SURF-6 was expressed as a recombinant GST-fusion protein and used as an antigen to generate monoclonal antibodies, S79 and S148, specific for SURF-6. The monoclonal antibody produced by hybridoma clone S79 specifically recognizes endogenous SURF-6 by Western and immunofluorescence analyses in various cultured human cells, and by immunohistochemistry in paraffin-embedded sections of human breast cancer samples. Moreover, S79 immunoprecipitates protein complexes containing SURF-6 from HeLa cells extracts. The antibody S79 recognizes SURF-6 only in human cells; however, the antibody produced by hybridoma clone S148 can detect SURF-6 of human and mouse origin. Monoclonal antibodies to the nucleolar protein SURF-6 described in this work can be a useful tool for studies of ribosome biogenesis in normal and cancer cells.

The nucleolar protein SURF-6 is essential for viability in mouse NIH/3T3 cells.

SURF-6 is a bona fide nucleolar protein comprising an evolutionary conserved family that extends from human to yeast. The expression of the mammalian SURF-6 has been recently found to be regulated during the cell cycle. In order to determine the importance of SURF-6 in mammalian cells, we applied the Tet-On system to regulate conditionally, in response to tetracycline, the expression of an antisense RNA (asRNA) that targets Surf-6 mRNA in mouse NIH/3T3 cells. Induced Surf-6 asRNA caused an effective depletion of SURF-6 protein resulted in cell death and in an apparent arrest in the G1 phase of the cell cycle. These results provide for the first time evidence that expression of SURF-6 is essential for mammalian cell viability, and suggest that SURF-6 might participate in the progression of cell cycle.

Identification of an evolutionary conserved SURF-6 domain in a family of nucleolar proteins extending from human to yeast.

The mammalian SURF-6 protein is localized in the nucleolus, yet its function remains elusive in the recently characterized nucleolar proteome. We discovered by searching the Protein families database that a unique evolutionary conserved SURF-6 domain is present in the carboxy-terminal of a novel family of eukaryotic proteins extending from human to yeast. By using the enhanced green fluorescent protein as a fusion protein marker in mammalian cells, we show that proteins from distantly related taxonomic groups containing the SURF-6 domain are localized in the nucleolus. Deletion sequence analysis shows that multiple regions of the SURF-6 protein are capable of nucleolar targeting independently of the evolutionary conserved domain. We identified that the Saccharomyces cerevisiae member of the SURF-6 family, named rrp14 or ykl082c, has been categorized in yeast databases to interact with proteins involved in ribosomal biogenesis and cell polarity. These results classify SURF-6 as a new family of nucleolar proteins in the eukaryotic kingdom and point out that SURF-6 has a distinct domain within the known nucleolar proteome that may mediate complex protein-protein interactions for analogous processes between yeast and mammalian cells.