Spermatozoa have a highly complex RNA profile. Several of these transcripts are suggested as biomarkers for male infertility and contribute to early development. To analyze the differences between sperm RNA quantity and expression of protamine ( PRM1 and PRM2 ) and testis-specific histone 2B ( TH2B ) genes, spermatozoa from 33 patients who enrolled in assisted reproduction treatment (ART) program were analyzed. Sperm RNA of teratozoospermic (T), oligoteratozoospermic (OT), and normozoospermic (N) samples was extracted, and the differences in transcript levels among the study groups were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The correlations of total RNA per spermatozoon and the expression of the transcripts were evaluated in relation to sperm characteristics and preimplantation embryo development. The mean (±standard deviation) RNA amount per spermatozoon was 28.48 (±23.03) femtogram in the overall group and was significantly higher in the OT group than that in N and T groups. Total sperm RNA and gene expression of PRM1 and PRM2 genes were related to preimplantation embryo development and developmental arrest. Specific sperm characteristics were correlated with the expressions of PRM1 , PRM2 , or TH2B genes. We conclude that the sperm RNA amount and composition are important factors and might influence early embryonic development and also differ in different cases of male infertility.
Male ; Humans ; Protamines/metabolism* ; Spermatozoa/metabolism* ; Embryonic Development/genetics* ; Adult ; RNA/genetics* ; Histones/genetics* ; Infertility, Male/genetics* ; Teratozoospermia/genetics* ; Oligospermia/genetics*

OBJECTIVETo construct a single-chain human anti-EGFR antibody (scFv) and truncated protamine (tP) fusion protein, ScFv/tP, carrying small interfering (si)RNA directed against the heat shock protein Hsp47, a collagen-binding glycoprotein, in order to evaluate the role Hsp47 in apoptosis of hepatic stellate cells.

METHODSA single chain of the human variable fragment was obtained by phage display and fused with the tP gene and with or without (negative control) the Hsp47 siRNA sequences. Following expression and purification of the scFv/tP fusion protein and the scFv/tPHsp47 siRNA fusion protein, internalization capabilities were tested in isolated human hepatic stellate cells and the QSG-7701 human hepatocyte cells with visualization by immunofluorescent staining. The DNA binding ability of the fusion proteins were verified by gel shift assay.Following ScFv/tP-Hsp47 siRNA fusion protein transfection into the human hepatic stellate cells, the levels of Hsp47 mRNA and protein expression were tested by RT-PCR and Western blotting; in addition, effects of siRNA-mediated silencing of Hsp47 on cell proliferation and apoptosis were analyzed by the cell counting kit (CCK)-8, flow cytometry and Western blot detection of the apoptosis marker poly (ADP-ribose) polymerase (PARP).

RESULTSIndirect immunofluorescence revealed that the ScFv/tP fusion proteins were internalized into human hepatic stellate cells but not into the QSG-7701 cells.The ScFv/tP-Hsp47 siRNA fusion protein caused reduced expression of Hsp47 mRNA and protein expression in the human hepatic stellate cells, as well as increased the cells' apoptosis remarkably.

CONCLUSIONThe ScFv/tP fusion protein can be used as a transfection reagent to deliver Hsp47 siRNA into hepatic stellate cells and to mediate apoptosis via blockade of Hsp47 expression.

Apoptosis ; Cell Proliferation ; HSP47 Heat-Shock Proteins ; genetics ; Hepatic Stellate Cells ; cytology ; Humans ; Protamines ; metabolism ; RNA, Messenger ; RNA, Small Interfering ; Receptor, Epidermal Growth Factor ; immunology ; Single-Chain Antibodies ; Transfection

PURPOSE: Premixed insulin is effective to improve glycemic control; however, clinicians may be less likely to know which premixed insulin is appropriate for which patients. This study aimed to evaluate the effects of twice-daily injections of premixed insulin lispro on glycemic control in type 2 diabetic patients. MATERIALS AND METHODS: Forty type 2 diabetic patients, who had been treated with twice-daily injections of human protamine mixture 30/70 insulin for at least 12 months, were divided into two groups; one group whose blood glucose 2 hours after breakfast was greater than 200 mg/dL, was switched to lispro mix50, and the other group whose blood glucose 2 hours after breakfast < 200 was switched to lispro mix25. RESULTS: Glycated haemoglobin (HbA1c) significantly improved in the Mix50 group from 8.3% to 7.5% (at 12 weeks; p < 0.05), and to 7.5% (at 24 weeks; p < 0.05). On the other hand, HbA1c levels in the Mix25 group were slightly decreased from 8.1% to 7.7% at 12 weeks (p < 0.05), and to 7.9% at 24 weeks (not significant). Both postprandial plasma glucose and fasting plasma glucose levels were significantly improved in the Mix50 group, but not in the Mix25 group. Overall, 95% of subjects preferred premixed lispro insulin from human insulin in the viewpoint of the timing of insulin injection by questionnaire analysis. CONCLUSION: Switching from human protamine mixture 30/70 insulin to lispro mix50 twice-daily injection therapy in patients with high postprandial plasma glucose could improve their glycemic control and quality of life.
Aged ; Blood Glucose/*analysis ; Body Mass Index ; Body Weight ; Diabetes Mellitus, Type 2/*drug therapy ; Female ; Hemoglobin A, Glycosylated/metabolism ; Humans ; Insulin/administration & dosage/*analogs & derivatives ; Male ; Middle Aged ; Postprandial Period ; Protamines/administration & dosage ; Treatment Outcome

AIMTo simultaneously determine the localization of histones and protamines within human sperm nuclei.

METHODSImmunofluorescence of the core histones and protamines and fluorescence in situ hybridization of the telomere region of chromosome 16 was assessed in decondensed human sperm nuclei.

RESULTSImmunofluorescent localization of histones, protamine 1 (PRM1) and protamine 2 (PRM2) along with fluorescence in situ hybridization localization of chromosome 16 telomeric sequences revealed a discrete distribution in sperm nuclei. Histones localized to the posterior ring region (i.e. the sperm nuclear annulus), whereas PRM1 and PRM2 appeared to be dispersed throughout the entire nucleus.

CONCLUSIONThe co-localization of the human core sperm histones with the telomeric regions of chromosome 16 is consistent with the reorganization of specific non-protamine regions into a less compacted state.

Cell Nucleus ; metabolism ; Chromosomes, Human, Pair 16 ; metabolism ; Histones ; metabolism ; Humans ; Male ; Protamines ; metabolism ; Spermatozoa ; metabolism ; Telomere ; metabolism

After the preparation of cationic liposomes composed of DDAB/DOPE, cationic liposome-DNA complexes and lipid-polycation-DNA (LPD) complexes were formulated, respectively. Gel retardation assay was employed to select appropriate ratios of cationic liposomes to DNA of the liposome-DNA complexes. The morphology of LPD and liposome-DNA complexes was observed by transmission electron microscopy. The diameter and surface charge of LPD and liposome-DNA complexes were measured by photon correlation spectroscopy (PCS). Their transfection efficiencies in Chang cells and HepG2 cells were evaluated by beta-gal assay kit. It was found that LPD and liposome-DNA complexes had a regular spherical surface. However, compared with liposome-DNA complexes, LPD had rather smaller particle size and much higher transfection efficiency in Chang cells and HepG2 cells in vitro. LPD could be prepared easily with small particle sizes and high transfection activities. LPD may be a good non-viral gene delivery vehicle for applications in gene delivery.
Cations ; DNA ; genetics ; Genetic Therapy ; Genetic Vectors ; Humans ; Lipids ; chemistry ; Liposomes ; chemistry ; Liver Neoplasms ; metabolism ; pathology ; Protamines ; chemistry ; Transfection ; Tumor Cells, Cultured

A novel transferrin modified non-viral gene delivery system Tf-PLPD was developed and the related characteristics was investigated. Blank procationic liposomes were prepared by film dispersion-filteration method. PLPD was prepared as follows by first mixing the plasmid DNA and protamine together, then the resulted polyplexes were incubated for 10 min at room temperature, followed by addition of preformed blank procationic liposomes. Transferrin was adsorbed at the surface of PLPD via electrostatic interactions to form Tf-PLPD. Central composite design (CCD) was employed to optimize the formulation. The HepG2 cells were transfected using lacZ as reporter gene and characteristics such as the morphology, the mean particle size, the zeta potential and the transfection efficiency in HepG2 cells were further investigated by different methods. The resulting PLPD had a regular spherical surface with an average size of (228. 9 +/- 8. 0) nm (polydispersity index, PDI = 0. 122 +/- 0. 02, n = 3) , a zeta potential of ( - 25. 08 +/-2. 50) mV (n = 3) and a transfection efficiency of (12. 18 +/- 3. 80) mU x mg(-1) (protein). The Tf-PLPD had an average size of (240 +/- 12) nm (polydispersity index, PDI = 0. 150 +/- 0. 03, n = 3), a zeta potential of ( - 24. 10 +/- 2. 50) mV ( n = 3) and a transfection efficiency of (24. 26 +/- 2. 60) mU x mg(-1) (protein) , 20 times greater than that of the naked plasmid DNA. The presence of serum didn' t affect the tansfection activity of PLPD or Tf-PLPD. Compared to one kind of cationic liposomes (liposome-protamine-DNA, LPD), the PLPD and Tf-PLPD had much less cytotoxicity to three hepatic cell lines (including HepG2, SMMC7721 and Chang' s normal hepatocyte). The results indicated that the Tf-PLPD is a perspective non-viral vector for gene delivery systems.
Cations ; chemistry ; Cell Line ; Cell Line, Tumor ; Cell Survival ; DNA ; chemistry ; genetics ; Hepatocytes ; cytology ; metabolism ; Humans ; Liposomes ; chemistry ; Liver Neoplasms ; genetics ; pathology ; Particle Size ; Plasmids ; chemistry ; genetics ; Protamines ; chemistry ; Transfection ; methods ; Transferrin ; chemistry ; genetics

OBJECTIVETo study the clinical application and effects of Yuziwan in the treatment of male sterility patient with abnormal protamine.

METHODSThe changes of protamine, semen and sex hormones of 30 male sterility patients treated by Yuziwan were observed before and after the treatment.

RESULTSAfter a 3-month course of treatment, 9 cases were cured, 15 obviously improved and 6 failed to respond. The ratio of histone to protamine was decreased from (1.34 +/- 0.52) before the treatment to (0.72 +/- 0.32) after it, with significant difference (P < 0.01), the semen quality obviously improved (P < 0.05), and the LH and T levels markedly raised (P < 0.01). Yuziwan evidently improved the abnormal protamine, sperm quality and endocrine function of the sterility patients.

CONCLUSIONYuziwan has good curative effect on male sterility.

Adult ; Drugs, Chinese Herbal ; therapeutic use ; Histones ; metabolism ; Humans ; Infertility, Male ; drug therapy ; metabolism ; Male ; Phytotherapy ; Protamines ; metabolism

OBJECTIVETo investigate the relationship between the expression of protamine-2 (P-2) mRNA and the results of sperm extraction in the corresponding testis tissues of patients with nonobstructive azoospermia.

METHODSBased on pathological diagnosis, 38 cases of azoospermia at the mean age of 32.4 (ranging 24 - 42) years were divided into a nonobstructive (NOA) group and an obstructive (OA) group. Two specimens were taken from different positions of one testis, each divided into three portions for general pathological test, sperm separation and mRNA extraction, respectively. The expression of P-2 mRNA was determined by RT-PCR and image analysis assay.

RESULTSAmong the 38 cases, 27 were diagnosed as nonobstructive and 11 as obstructive azoospermia. No regularity was found as to the positions where sperm could or could not be successfully isolated. The expression of P-2 mRNA was 1.40 +/- 0.21 in the tissues where sperm was isolated and 0.51 +/- 0.23 (P < 0.05) in those where no sperm was isolated.

CONCLUSIONThe expression of P-2 mRNA in the testicular tissues from the patient with nonobstructive azoospermia could reveal the results of sperm extraction in the corresponding tissues.

Adult ; Azoospermia ; metabolism ; Cell Separation ; Humans ; Male ; Protamines ; genetics ; metabolism ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Spermatozoa ; cytology ; Testis ; metabolism

OBJECTIVETo study the significance of the change of protamine content in the management of male infertility.

METHODSSperm nuclear proteins extracted from 199 infertile patients were analyzed by polyacrylamide gel-electrophoresis and scanning microdensitometry.

RESULTSForty-two (21%) cases of the total number had normal sperm nuclear proteins and 157 (79%) had aberrant ones, which mainly presented the interruption of HPRR and abnormality of P2 protamine. Thirty patients were selected at random from 157 abnormal cases for clinical treatment. After the treatment, the sperm nuclear proteins were extracted and analyzed and the results demonstrated that 11 cases (36.6%) improved markedly, 5 (16.6%) restored to normal and the other 16 (46.6%) remained unchanged.

CONCLUSIONThere is a reduced level or selective absence or even complete selective absence of protamines in infertile patients. Protamines may act as a parameter for evaluating the treatment effect of infertile males and protamine content can be influenced by a certain or several factors.

Adult ; Electrophoresis, Polyacrylamide Gel ; Humans ; Infertility, Male ; metabolism ; Male ; Oligospermia ; metabolism ; Protamines ; analysis ; Spermatozoa ; chemistry

OBJECTIVETo develop an efficient non-viral gene delivery system in order to transfer CDKN1B gene efficiently into lung and liver carcinoma cells.

METHODSA recombinant plasmid composed of CDKN1B sequence and EYFP as reporter gene was constructed and identified. The recombinant DNA was then formulated the lipids-polycation-DNA complexes(LPDs) with protamine sulfate. Several kinds of lung and liver carcinoma cells were transfected by means of LPDs. The physicochemical properties of LPDs were investigated using PCS method and TEM, respectively. The expression of EYFP in A549 cells was observed under fluorescent microscope and evaluated by flow cytometry analysis. Finally, the production of CDKN1B protein in transfected LLC, Chang and 7721 cells was identified by Western blot analysis.

RESULTSThe average diameter of the LPDs were 167 nm with the polydispersity index of 0.35. The average zeta potential of LPDs was +32.6 mV. LPDs look like a sunken sphere. The fluoresent microscope picture clearly indicated the expression of EYFP in A549 cells. The flow cytometry result showed that the transfection efficiency of LPDs in A549 cells was comparable with that of LipofectAMINE, the positive control. Western blot analysis confirmed the production of CDKN1B protein in LLC, Chang and 7721 cells transfected with LPDs, while no CDKN1B protein was detected in cells transfected with naked DNA.

CONCLUSIONThe construction of the recombinant plasmid is successful. LPDs can deliver the recombinant plasmid to lung carcinoma cells and liver carcinoma cells with high efficiency. Therefore, this kind of gene delivery system has the potential uses for the treatment of lung and liver cancer.

Blotting, Western ; Cell Line ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; metabolism ; Flow Cytometry ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Liposomes ; chemistry ; Microscopy, Electron, Transmission ; Microscopy, Fluorescence ; Plasmids ; chemistry ; genetics ; Polyamines ; chemistry ; Protamines ; chemistry ; Transfection ; methods