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Objective: To investigate the correlation of the severity of teratospermia and the age of the patient with sperm DNA fragmentation index (DFI) and high DNA stainability (HDS) in male infertility patients., Methods: We collected semen samples from 1 393 infertile males from July to December 2021. Based on the WHO Laboratory Manual for the Examination and Processing of Human Semen (5th Ed), we performed sperm morphology analysis, examined perm DFI and HDS by flow cytometry, and analyzed the impacts of the severity of teratospermia and the age of the patients on sperm DFI and HDS., Results: Among the 1 393 male infertility patients, 124 (8.90%) were found with extremely severe, 214 (15.36%) with severe, 235 (16.87%) with moderate, 163 (11.70%) with mild teratospermia, and 657 (47.16%) with morphologically normal sperm (MNS), with statistically significant differences in sperm DFI and HDS among the five groups, and 822 (59.00%) were aged <35 years, 306 (21.97%) 35－<40 years, 223 (16.01%) 40－<45 years and 42 (3.02%) ≥45 years, with statistically significant differences in sperm DFI and HDS among different age groups (P < 0.05). Sperm DFI and HDS were correlated negatively with the percentage of MNS (P > 0.05), but positively with the age of the patients (P < 0.05)., Conclusion: Increased severity of teratospermia and age of the patient can increase sperm DFI and HDS, and sperm nuclear chromatin integrity and maturity are important indicators of male fertility.

Human seminal plasma is rich in potential biological markers for male infertility and male reproductive system diseases, which have an application value in the diagnosis and treatment of male infertility. The methods for the detection of semen biochemical markers have been developed from the manual, semi-automatic to the present automatic means. The automatic detection of semen biochemical markers is known for its advantages of simple reagent composition and small amount of reagents for each test, simple setting of parameters, whole automatic procedure with few errors, short detection time contributive to batch detection and reduction of manpower cost, simple calibration and quality control procedure to ensure accurate and reliable results, output of results in the order of the samples in favor of clinical diagnosis and treatment, and open reagents applicable to various automatic biochemistry analyzers. At present, the automatic method is applied in the detection of such semen biochemical markers as seminal plasma total and neutral alpha-glucosidase, acid phosphatase, fructose, γ-glutamyl transpeptidase, zinc, citric acid, uric acid, superoxide dismutase and carnitine, sperm acrosin and lactate dehydrogenase C4, and semen free elastase, which can be used to evaluate the secretory functions of the epididymis, seminal vesicle and prostate, sperm acrosome and energy metabolism function, seminal plasma antioxidative function, and infection or silent infection in the male genital tract.

Objective: To study the correlation of leukocyte subsets with sperm DNA damage in the semen of infertile men with asymptomatic genital tract infection (AGTI)., Methods: This study included 111 infertile males with AGTI. After routine semen analysis, we determined the concentration of CD45+ leukocytes in the semen by immunocytochemistry, measured the concentrations of CD14+ cells of the mononuclear / macrophagic system and activated macrophages and HLA-DR+ cells in the semen by flow cytometry, and examined the sperm DNA fragmentation index (DFI) and the expression of 8-hydroxy-2'-deoxyguanosine (8-OHdG) by TUNEL assay. Then we analyzed the correlation of seminal leukocyte subsets with sperm DNA damage and routine semen parameters., Results: The concentration of CD45+ leukocytes was correlated significantly with those of CD14+ and HLA-DR+ cells in the semen (P <0.01), but not that of leukocyte subsets with routine semen parameters, sperm DFI, or the percentage of 8-OHdG-positive cells. The percentage of 8-OHdG-positive sperm was correlated positively with the sperm DFI (r = 0.48, P<0.01) but negatively with sperm concentration (r = －0.44, P <0.01). After adjusted for age, abstinence time and cigarette smoking, the percentage of 8-OHdG-positive sperm was correlated independently with sperm concentration (β = －0.25, P = 0.008) and DFI (β = 0.23, P = 0.05)., Conclusions: Sperm DNA damage is associated with poor semen quality but not with seminal leukocyte subsets in infertile males with asymptomatic genital tract infection.

Objective: To investigate the application value of pre-implantation genetic screening (PGS) in intracytoplasmic sperm injection (ICSI) and embryo transfer (ET) for infertile patients with high sperm DNA fragmentation index (DFI)., Methods: We selected 90 infertile patients undergoing ICSI-ET in our center from April 2015 to October 2016, 60 with sperm DFI =≥15% and 30 with sperm DFI <15%. Of the 60 cases with sperm DFI =≥15%, 30 received PGS-ICSI (high DFI-PGS group) while the other 30 did not receive PGS (high DFI-ICSI group). The 30 cases with sperm DFI <15% were included in the low DFI-ICSI group. We made comparisons between the high and low DFI-ICSI groups in the rates of normal fertilization, good-quality embryos, blastocyst formation and embryo implantation as well as between the high DFI-PGS and high DFI-ICSI groups in the ages of the males and females, the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T) in the males, sperm concentration, the percentages of progressively motile sperm (PMS) and morphologically normal sperm (MNS), sperm DFI, and the rates of sperm nucleoprotein immaturity, normal fertilization, good-quality embryos, blastocyst formation and embryo implantation., Results: Statistically significant differences were found between the high and low DFI-ICSI groups in the rate of embryo implantation (31.25 vs 52.50%, P <0.01) but not in the rates of normal fertilization (［65.38 ± 24.62］ vs ［73.00 ± 17.00］%, P >0.05), good-quality embryos (［62.41 ± 25.97］ vs ［73.00 ± 22.10］%, P >0.05), or blastocyst formation (［62.55 ± 25.21］ vs ［64.30 ± 18.60］%, P >0.05). The rate of embryo implantation was markedly higher in the high DFI-PGS than in the high DFI-ICSI group (60.97% vs 31.25%, P <0.01), but there were no statistically significant differences between the two groups in the ages of the males and females, the levels of FSH, LH and T in the males, sperm concentration, the percentages of PMS and MNS, sperm DFI, sperm nucleoprotein immaturity, or the rates of normal fertilization (［69.76 ± 15.82］ vs ［65.38 ± 24.62］%, P >0.05), good-quality embryos (［64.42 ± 30.75］ vs ［62.41 ± 25.97］%, P >0.05) and blastocyst formation (［67.53 ± 19.24］ vs ［62.55 ± 25.21］%, P >0.05)., Conclusions: For the infertile patients with sperm DFI =≥15%, PGS-ICSI, rather than ICSI alone, can significantly increase the rate of embryo implantation.

To explore the association between single nucleotide polymorphisms (SNPs) in the promoter region of the inner centromere protein (INCENP) gene and bovine semen quality, the haplotypes in 250 Chinese Holstein bulls were detected using PCR-RFLP method in this study. Two SNPs (g.-556 G>T, rs 136823901 and g.-692 C>T, rs 211010999) and three haplotypes (CG, TT, TG) were identified in the promoter region of INCENP. The genotype frequency and allele frequency of these two SNPs as well as the correlation between different SNP haplotype combinations and bovine semen quality were then analyzed. Our results showed that fresh sperm motility of the GT genotype was significantly higher than that of the GG genotype (P<0.05) at the SNP site g.-556 G>T, while fresh and frozen-thawed sperm motilities of the haplotype combinations H1H1(CCGG), H1H3(CTGT), H2H3(TTGT) and H3H3(TTTT) were significantly higher than that of H1H2 (P<0.05). To further study the possible mechanisms by which g.-556 G>T and g.-692 C>T affect semen quality, three haplotype plasmids were respectively transfected into MLTC-1 cells. The TG haplotype demonstrated the highest luciferase activity, suggesting that g.-556 G>T and g.-692 C>T are functional mutations which could regulate INCENP gene expression by affecting promoter activity and thus affect semen quality.

The grasshoppers are ideal materials to study various meiotic stages of spermatogenesis due to their easy availability, fairly large chromosomes, and fewer numbers of chromosomes. It is easy to make temporary squash preparation of grasshopper testes; however, it is usually difficult for the beginners to differentiate between stages of meiosis. In view of this, we demonstrated the method of identification of meiotic stages by chromosome number and chromosome conformation, taking spermatogonial meiosis of Locusta migratoria manilensis as an example. We described briefly the mitosis of spermatogonia and the spermatogenesis of this species as well.

The role of layered double hydroxide (LDH) in the protection of herring testis DNA from heavy metals Cd2+ and Pb2+ was studied by X-ray diffraction ( XRD) spectra, Fourier transform infrared (FTIR) spectra, Scanning Electron Microscopy (SEM), Cyclic Voltammetry and Ultraviolet Spectrometry. Size expansion of the basal spacing (003) from 0. 76 nm in LDH to 2. 30 nm was observed in the resulting DNA-LDH nanohybrids and it gave peaks corresponding to C=O (1 534 cm(-1) and 1488 cm(-1)) in skeleton and bases, C-O stretching vibration (1228 cm(-1)), and P-O symmetrical stretching vibration (1096 cm(-1)) in functional groups of DNA, indicating that DNA were intercalated into the LDH by the ion exchange. However, the displacement of NO3(-) was not fully complete (partial intercalation of DNA). The DNA outside LDH interlayers was absorbed on the surface of LDH. The cyclic voltammetric curves showed that DNA in the composites exhibited a very similar peaks, which corresponded to the two reduction current peaks (E(P) = - 1.2 mV and E(P) = -2.4 mV) of free DNA. Also there was no cathode sag emerging in cyclic voltammetric curves, suggesting that both Cd2+ and Pb2+ cannot insert into the groove of DNA to associate with base pairs or other groups when DNA was bound on LDH. The results showed that, on the one hand, both Cd2+ and Pb2+ were absorbed on the external surface of LDH for immobilization, on the other hand, the layer of LDH provided ideal space for DNA by the action of protecting DNA molecules from Cd2+ and Pb2+.

Cryopreservation of sperm from Neolissochilus benasi was studied in 2011. The effects of various cryoprotectants of different concentrations, dilution ratios of milt to extender, storage volume and thawing temperature on motility of post-thawing of spermatozoa were examined to optimize cryopreservation procedures. Semen was stored in liquid nitrogen in 1.8 mL cryovial for 24 h, and the intensity of sperm motility was measured before and after cryopreservation. Post-thawing motility of frozen sperm obtained with cryoprotectants 10% MeOH or 15% EG were higher than for others. The most effective dilution ratio of milt to extender is 1:7. The maximal storage volume is 60 μL of 1.8 mL cryovial and the optimal sperm equilibration period in the extender D-15+10% MeOH was between 10-60 min. Thawing was optimal in a 37 Degrees Celsius water bath. When fresh sperm motility is (62.33±2.05)%, this cryopreservation protocol resulted in frozen-thawed semen with 20%-30% motile. The overall effect is not ideal, and cannot achieve extensive application. Different breeding management of different ground protection may have contributed to this result. Therefore, it is necessary to reduce stress capture induced in management of parent fish and provide suitable forming conditions. In the ex situ conservation of rare fish the broodstocks management of males is as important as that for females and the key to obtaining high quality larval fish.

Objective: To search for a simple, quick and accurate method for the sperm DNA integrity test by modifying the conventional sperm chromatin dispersion (SCD) test, and to investigate the reference value range of normal sperm DNA integrity by the improved SCD assay., Methods: We modified the conventional SCD test in its procedures, reagents preparing and utensils to be used, compared the improved method with the conventional one, and detected 293 normal semen samples for sperm DNA integrity by the modified SCD test., Results: An improved SCD test was established, and no statistical differences were found between the results of the modified and conventional methods. The reference value range was determined by the improved SCD assay for normal sperm DNA integrity, which was < 32.58% for abnormal sperm DNA integrity., Conclusion: The improved SCD test could detect sperm DNA integrity more quickly, and it reduced the cost in reagents.

Sexual reproduction is marked by the fusion of the sperm cell with the oocyte during fertilization to produce the diploid zygote, in which the lipids in the sperm plasma membrane play an important role. Due to the loss of most cell organelles and DNA transcription, spermatozoa lack protein expression and vesicular transport. However, the lipids of the sperm plasma membrane undergo complicated dynamic changes, which may facilitate the capacitation, binding with zona pellucida, acrosome reaction and fusion of the sperm cell with the oocyte. This paper summarizes the progress in the studies of the lipids in the sperm plasma membrane, their composition, structure, peroxidation, metabolism and role in fertilization.

Objective: To compare the effects of sperm chromatin dispersion (SCD) test and TdT-mediated dUTP nick end labeling (TUNEL) assay in assessing the DNA fragmentation in human sperm., Methods: Motile sperms were isolated from the semen samples obtained from 20 healthy fertile men and 32 clinically infertile patients by swim-up technique, and underwent SCD and TUNEL to analyze the DNA fragmentation., Results: The rate of sperm with DNA damage of the infertile patients was 12.8% +/- 5.8% tested by SCD, significantly higher than that of the healthy fertile men (7.6% +/- 3.3%, t = 3.576, P = 0.001), and the rate of sperm with DNA damage of the infertile patients was 11.1% +/- 5.1% tested by TUNEL assay, significantly higher than that of the healthy fertile men (6.8% +/- 2.8%, t = 3.467, P = 0.001). The proportion of sperm cell with abnormal DNA integrity measured by SCD test was correlated strongly with that determined by TUNEL for the infertile men (r = 0.841, P = 0.000) and for the fertile men too (r = 0.823, P = 0.000). The rate of sperm with DNA damage measured by SCD were not significantly different from those of TUNEL-positive sperm in fertile men (t = 1.996, P = 0.060). The rate of sperm with DNA damage measured by SCD was significantly higher than that measured by TUNEL among infertile patients (t = 3.023, P = 0.005)., Conclusion: The presence of sperm DNA damage may lead to male infertility. SCD is simpler, cheaper and more reliable than TUNEL in testing the sperm DNA damage.

Objective: To study the effect of the split of sperm nuclei on the yield of RNA from human sperm., Methods: Human sperm were purified by two sequential centrifugations through 40:80 discontinuous gradients of Percoll. Human leukocytes separated from peripheral blood were used as the control. Total RNAs from purified sperm and leukocytes were extracted with both TRIzol and RNeasy Kit. The RNAs from equal number of cells were reverse-transcribed, and quantified by the levels of beta-ACTIN mRNA, which were evaluated by real time polymerase chain reaction., Results: TRIzol failed to digest majority of sperm nuclei even the incubation time was prolonged to 1 h, while no sperm nucleus was found under the light microscope after 1 min digestion with RLT buffer of the RNeasy Kit. Both reagents can digest the nuclei of human leukocytes well. The amount of RNA per 10(6) sperms isolated with RNeasy Kit (149.8+/-24.5 ng) was 4-fold higher (P=0.01) than that extracted with TRIzol (35.5+/-4.0 ng per 10(6) spermatozoa; n=3). The similar yields of the leukocyte RNAs extracted with RNeasy Kit and TRIzol [(765.5+/-229.8) and (958.8+/-201.0) ng per 10(6) cells respectively; n=3, P=0.168] excluded the possibility of different efficacy of these two reagents in RNA isolation., Conclusion: The split of sperm nuclei is important to the yield of RNA in the human sperm RNA extraction. The nucleus may be the major area for human sperm RNA repositories.

Objective: To establish a method of preparing and analyzing DNA of sperm cells from sample mixtures., Methods: Sperm cells were captured by micromanipulation method, DNA template was prepared from these sperm cells, and then amplified by multiple displacement amplification (MDA)., Results: High-yield of highly integrated DNA was produced by MDA from very small amount of DNA template of 20 sperm cells. The DNA could be amplified at least 30,000-fold, and the length of most the DNA segments were greater than 15 kb, providing sufficient amount of short tandem repeat (STR) products for DNA genotyping., Conclusion: The micromanipulation method is efficient to isolate sperm cells from sample mixtures and multiple displacement amplification can provide sufficient amount of DNA products for STR genotyping. The method seems to be feasible in detecting DNA from rape cases.

Sptrx-1, 2 and 3 are a series of thioredoxins specifically expressed in the testis/sperm. They play a significant role structurally and functionally in the process of spermiogenesis. The genesis and mutation of sptrx-1, 2 and 3 are correlated to male reproduction. Taking sptrx-1, 2 and 3 as the target of study and treatment will open up a new field in the clinical study of male reproduction.

Objective: To investigate the progesterone-binding site on the normal fertile human sperm membrane after 2 hours of in vitro capacitation., Methods: Viable spermatozoa were selected by a swim-up method. After 2 hours of in vitro capacitation, multipoint saturation binding experiments were performed. Sperm suspension and increasing concentrations of progesterone-11alpha-glucuronide-[125I] iodotyramine (125I-P) were added to 7 total binding tubes respectively, and equal amounts of sperm suspension and 125I-P were added to another 7 corresponding non-specific binding tubes in the presence of 10 micromol/L progesterone. After incubation for 1 hour at 4 degrees C, the radioactivity of both the tubes and the pellets after centrifugation was measured respectively. The equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) were calculated using the mathematical model of single site multi-point saturation method of Scatchard function and least-squares regression., Results: Kd was (0.61 +/- 0.04) nmol/L and Bmax was (830 +/- 344) sites/cell. The significance test of the regression equation indicated that r = -0.980, P < 0.01., Conclusion: There is a high affinity and low capacity binding site for the progesterone (progesterone receptor) on the normal fertile human sperm membrane.

Two novel complexes of [Cu(HAsp)ImH2O]SO4 x 4 H2O and [Cu(Asp)Im(OH)] x 4 H2O (HAsp = Aspartic acid molecule, Asp = Aspartic acid ion, Im = Imidazole) were synthesized and characterized by elemental analysis, IR spectroscopy and TG-DTA. The interactions of the complexes with sperm DNA were studied by electronic absorption and fluorescence spectra. Results showed that the reactions of the two complexes with DNA are obviously different: for [Cu(HAsp)ImH2O]SO4 x 4 H2O, it is intercalation companied by electrostatic effect, while [Cu(Asp)Im(OH)] x 4 H2O mainly cooperates with the nitrogen atom of the DNA base pair, which induces the breakage of DNA double helix. The reasons for these differences in their DNA binding modes were also discussed.

Objectives: To evaluate the application of two-dimensional electrophoresis in the research of differentially expressed proteins in human spermatozoa., Methods: Two-dimensional gel electrophoresis was performed on 4 sperm samples from normal healthy men and another 4 from asthenospermia patients. After silver staining, the differential expression proteins were analyzed by PDQuest 2D analysis software., Results: Seven differential protein spots were identified, 2 expressed highly in the asthenospermia sperm but lowly in the normal spermatozoa, while the other 5 expressed just the opposite way., Conclusion: The protein profiles of differential expression between the normal spermatozoa and idiopathic asthenospermia were established and some differential proteins were found. The data obtained in this study may help prepare the ground for further studies on the isolation and identification of differentially expressed proteins in human asthenospermia sperm.

Oxidative stress, deficiencies in natural processes such as chromatin packaging and abortive apoptosis are the main factors that lead to sperm DNA damage. The DNA integrity of sperm is essential for fertilization, as well as for the normal development of the blastocyst, embryo and child. There is increasing evidence that sperm DNA anomalies may lead to infertility, abortion, stillbirth, fathead and chromosomal diseases. The routine examination of semen can not identify subtle defects in sperm DNA, so several detection techniques have been developed for estimating the DNA (nuclear DNA/mitochondria DNA) integrity of sperm, which is considered to be a better marker of male fertility potential than conventional semen parameters. This review attempts at a detailed description of these detection techniques, including their principles, methods, steps and clinical application.

The damage to sperm DNA is one of the most important causes of male infertility. Some sperm with damaged DNA may escape from the sperm surveillance mechanism and transmit the damage to the offspring. So research on the damage to sperm DNA has become one of the hot spots in reproductive medicine. The factors that would damage sperm DNA include oxidative stress, microelements, reproductive toxic substances, radioactive rays, and so on, while the body depends on the compressed sperm DNA and anti-oxidation system for the protection of the integrity of sperm DNA. Some drugs such as anti-oxidant, black tea extract, etc, may help to improve and rebuild these protective mechanisms.

Exogenous DNA localization and the frequency of spermatozoa carrying exogenous DNA after sperm/DNA co-culture are key to a successful sperm mediated-gene transfer (SMGT). In the study, the characteristics and influencing factors of exogenous DNA uptake by spermatozoa were tested using digoxigenin (DIG) labeled DNA as trace. Results showed that goat spermatozoa could spontaneously take up exogenous DNA. The exogenous DNA was initially bound to the outer sperm membrane at postacrosomal region; subsequently party of the bound DNA was internalized into nucleus. There were considerable differences in the capability of spermatozoa from different donors to bind and internalize exogenous DNA. In 35 samples, binding rates (before DNase I digestion) and internalization rates (the positive rate after DNase I digestion) varied between 4.6%-62.4% and 2.1%-53.8%, respectively. For the spermatozoa from the same goat, the binding and internalization capacities were mostly inhibited by the seminal fluid. Compared to ejaculate sperm, the binding rate and internalization rate were increased three and five times in washed sperm cells, respectively. At the same time, capacitated spermatozoa also had lower exogenous DNA uptake (P<0.01). Dead spermatozoa did not complete the internalization process. The highest positive rate (before DNase I digestion) was found in membrane-broken spermatozoa as a result of freeze-thawing and this was independent of the sperm donors. These results suggest that selection of appropriate sperm donors and optimization of sperm processing procedures are the key steps for successful SMGT.

Objective: To separate the low abundance protein and establish the 2-DE synthetic map of total protein of human normal spermatozoa by using the 2-DE technology., Methods: All the needed human spermatozoa were collected and mixed, and proteins were extracted at one time with the method of urea/thiourea and ultra-sound. 0.8 mg, 0.6 mg, 0.5 mg, 0.3 mg sperm protein extracts were separated with 2-DE. Analyzed with MALDI-TOF-MS, PI and MW of 2 spots were obtained. Then set the 2 spots as the referent spots, different maps were compared and analyzed. At last, a synthetic map enriched with low abundance protein was obtained., Results: 1,080 +/- 23 protein spots have been separated on the 2-DE map with standard 0.5 mg loading amount and a synthetic map A was constructed which consist of 889 matched protein spots on the all maps with 0.5 mg loading amount. 381, 50 and 32 new spots were detected individually on the maps with 0.8 mg, 0.6 mg and 0.3 mg protein loading amount. A synthetic map with 1,352 protein spots was obtained., Conclusion: Low abundance protein was separated and a synthetic map enriched with low abundant protein was obtained by changing the protein loading amount.

The interaction of adriamycin (ADM) and DNA results in strongly enhanced resonance light scattering (RLS) spectra characterized by two peaks at 322 and 564 nm, respectively. The interaction of ADM and DNA was investigated by using absorption spectra, fluorescence spectra, and RLS spectra. The results indicate that RLS spectra have the highest sensitivity. It was found that the enhanced RLS intensity at 322 nm was proportional to the concentration of DNA in the range of 0-8.0 microg x mL(-1). An RLS method for the determination of DNA was accordingly described. The limit of determination was 36.8 and 40.1 ng x mL(-1) for calf thymus DNA and fish sperm DNA, respectively. The proposed method has a high sensitivity and good selectivity due to the specificity of the interaction of ADM and DNA.

Lead and Cadium as the key elements influencing human genetic health were selectively determined in the present paper. In the area of metal elements determination in body fluid, most samples used in the study are blood, plasma and saliva. No literature about genetic fluid determination has been found so far. In the present paper the preparation of samples, volume of samples, and determination parameters were investigated. An easy reliable method of determining lead and cadium in semen and liquor follicle is established. The recoveries of lead are 99.0%-118% and the recoveries of cadium are 96.3%-109%. The determination limit of lead is 0.8 microg x L(-1) and the determination limit of cadium is 0.05 microg x L(-1). The relative standard deviations (RSD)(n = 5) are 3.74%-8.54%.

Objective: To detect the zinc content in serum, seminal plasma and spermatozoa of asthenozoospermic and oligoasthenozoospermic patients, and analyse the relationship between zinc changes and sperm density as well as sperm motility., Methods: Sperm quality analysis was made according to the WHO standard of Laboratory Manual for the Examination of Human Semen and Sperm-cervical Mucus Interaction (4th edition), and then 90 asthenozoospermic patients, 60 oligoasthenozoospermic patients and 20 fertile men with normal sperm quality were screened out as the objects to be researched. The zinc content in their blood, seminal plasma and spermatozoa was detected by flame atomic absorption spectroscopy. And then a statistical analysis was made on the test results., Results: The blood zinc content among the 3 groups had no significant difference; the seminal plasma zinc of asthenozoospermic and oligoasthenozoospermic patients were significantly lower than that of fertile men (P < 0.05); the spermatozoa zinc content of oligoasthenozoospermic patients was significantly higher than that of asthenozoospermic patients and fertile men (P < 0.01), respectively., Conclusion: There may be a positive relationship between the feebleness of sperm production and motility of asthenozoospermic and oligoasthenozoospermic patients and the lower content of seminal plasma zinc; however, the relationship between the higher spermatozoa zinc content and the function of sperm production and sperm motility is still not clear, on which a further study should be made.

As an advanced technology, flow cytometry has its unique advantages in detecting single cells quantitatively and rapidly. So it has been used more and more widely in clinical examination. FCM offers a powerful tool and exciting opportunities for accurately evaluation of semen quality and sperm functions. And it's a new idea to apply this technology in andrological studies. This review introduces its basic principles, summaries its present and potential application in andrology studies, including separating and identifying spermatozoa, analyzing spermids structure and function, examining the biological active substances in semen and on the surface of spermids.

Objective: To study the significance of the change of protamine content in the management of male infertility., Methods: Sperm nuclear proteins extracted from 199 infertile patients were analyzed by polyacrylamide gel-electrophoresis and scanning microdensitometry., Results: Forty-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., Conclusion: There 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.

Our understanding of the molecular complexities of human spermatozoa continues to be rather rudimentary. This includes the unique packaging and structure of the sperm genome along with its paternally derived RNAs in preparation for their delivery to the egg. Both highlight the inadequacy in our understanding of how these genetic factors contribute to a healthy child. As a means to address this deficiency it was recently shown that human spermatozoal RNA isolated from the ejaculate can be used to transcriptionally profile the male gamete. It is fully expected that a genetic fingerprint characteristic of the normal fertile spermatozoon can be identified and that deviations from this profile can be detected.

Objective: To identify the resource suspected sperm of donor in human sperm bank and apply the parentage testing between the donor and his offspring., Methods: We took the 6 semen specimen of the donor involved and correspondently suspected semen as well as the semen of one volunteer and peripheral blood of his offspring. All specimens were amplified by PCR, and DNA fingerprint was detected by PAGE electrophoresis., Results: By DNA fingerprinting we discovered that the 5 suspected sperm samples came from corresponding donors and the other sample from another. The volunteer and his offspring were identified as consanguinity., Conclusion: We can identify the difference between sperm of donors and the suspected sperm of donors, and sperm of donors and the peripheral blood of their offspring exactly by DNA fingerprinting.

It is well known that mycoplasma can cause infection in the male reproductive tract. Some studies indicate that Ureaplasma urealyticum (Uu), a species of mycoplasma, is associated with male infertility. Sulfogalactosylglycerolipid(SGG) is the major mammalian male germ cell glycolipid, synthesized via sulfation of galactosylglycerolipid in early primary spermatocytes. Some experiments have proved that SGG is implicated in sperm-egg binding by linking arylsulfatase A (AS-A), SGG's ligand on the egg. SGG can be desulfated by binding mycoplasmas and transformed galactosylglycerolipid, which doesn't bind AS-A. So the binding and degradation of the sperm SGG by mycoplasmas may play a role in the induction of male infertility. As a kind of mycoplasma, Uu can also bind SGG, which offers another explantion for the association of Uu infection with male infertility caused by Uu infection.

Objective: To establish an effective method for testing DNA from the sperm smear., Methods: We did exploratory research for ninety-one cases of the sperm smear which were accumulated by our laboratory during working hours. Chelex extraction method was used to extract DNA. The digested solution of sperm was purified and concentrated by using Silicon bead. STR loci were typed after PCR amplification by Profile Plus kit., Results: Although there were a few sperm cells on the slide, it is easy to obtain good DNA typing result from it because the amalgamative method we used was high effective for DNA extraction., Conclusion: The DNA analysis of sperm smear could offer satisfactory typing results which would be useful in case.

Objective: To study the genotoxicity of carbon disulfide by detecting DNA damage in mice sperm with single-cell gel electrophoresis assay (SCGE)., Methods: SCGE was used to detect sperm DNA damage. The index of DNA damage, tail length and tail moment were used to evaluate the extent of DNA damage., Results: In three dosage groups, the rate of DNA damage (67.14%, 84.29% and 91.00%, respectively), index of DNA damage intensity (507, 656 and 745, respectively), tail length (5.87, 8.81 and 13.49 microm, respectively) and tail moment (1.30, 1.63, 2.66 microm, respectively) were significantly increased, while the percentage of head of the comet was significantly decreased (84.55%, 73.84% and 55.71%, respectively). A significant changes were clearly observed in all dosage groups compared to those of the control group (P<0.05)., Conclusion: SCGE which is a quick and sensitive method to detect DNA damage induced by CS2 may be used to monitor carcinogen and mutagen.

Objectives: To analyze human spermatozoa membrane proteins by two-dimensional gel electrophoresis and to provide a basis for drawing the protein map of normal human spermatozoa membrane proteins., Methods: Spermatozoa were purified by Percoll density centrifugation, and spermatozoa membrane proteins were analyzed by two-dimensional gel electrophoresis using isoelectric focusing and polyacrylamide gel electrophoresis., Results: About 800 protein spots could be identified by the imaging analysis system. The molecular weight and isoelectric point of most proteins were 20,000 to 100,000 and 3.0 to 7.0 respectively., Conclusions: There are more than 800 types of proteins in the human spermatozoa membrane. The spermatozoa membrane protein can be identified with a certain precision by two-dimensional gel electrophoresis.

Objective: To explore the protein factors that could interact with the testis-specific protein encoded by HSD-3.8 gene (GenBank Accession Number AF311312) related with female fertilization., Methods: Yeast two-hybrid system was used to screen the human ovary MATCHMAKER cDNA library with constructed "bait plasmid" containing the 0.7 kb fragment (HSD-0.7) of HSD-3.8. The interaction with the positive fragments using a series of truncated bait plasmids was investigated., Results: One positive gene fragment was obtained, which coded for 144 amino acids of the C-terminus of human G protein beta subunit 1. Truncated bait plasmids couldn't interact with the fish protein fragment in yeast., Conclusions: The protein encoded by HSD-3.8 gene may function through G protein signal transduction pathway and the interaction depends on the integration of the bait protein.

Objective: To investigate the immunocontraceptive ability of a new synthetic chimeric peptide with 35 amino acid residues., Methods: A chimeric peptide with 35 amino acid residues was synthesized based on the amino acid sequence of SP17, a protein specific to murine testis/sperm, and cyritestin. The purified product, with keyhole limpet hemocyanin (KLH), was injected subcutaneously into the pad of female and male Balh/C mice. Blood was extracted from the ophthalmic arteries of the mice to identify the corresponding proteins from organs of mice and/or rat and human being by Western blotting. The chimeric peptide was injected into other female mice that were then raised together with male mice in the same cages to observe their status of pregnancy. Freund' adjuvant or normal saline were injected into the controls., Results: The purity of the new chemiric peptide was 95%. The highest specific IgG titre in mouse serum was 1:6000 and the highest titre of specific IgA in the washing of vaginal mucous membrane of female mice was 1:300. The specific antiserum from the mice immunized by the chimeric peptide and KLH could identify two proteins with the molecular weights of 22 000 and 55 000 from testes of mice, rats, and human being. Western blotting showed no band for the extracts of mouse liver, spleen, and kidney. The pregnancy rate of female immunized mice was 61.11%, significantly lower that those in the controls (P < 0.05). The average number of cubs in a litter was less in the experimental group than in the controls (P < 0.05)., Conclusion: The new chimeric peptide with 35 amino acid residues stimulates powerfully humoral and mucous immunity in mice and obviously decreases the pregnancy rate and the number of cubs in each litter in mice.

Objectives: To investigate the localization and positive percentage of progesterone receptor (PR) on the human sperm surface., Methods: After in vitro capacitation, progesterone binding sites on the sperm were quantitatively analyzed by fluorescence microscopy and flow cytometry using fluorescein isothiocyanate-labeled bovine serum albumin-progesterone complex (P-BSA-FITC)., Results: The spermatozoa stained by P-BSA-FITC mainly showed two labeling patterns, with the green fluorescence on the whole acrosomal region or the equatorial acrosomal region only and the stainless postacrosomal and tail regions. The percentage of progesterone-binding sperm was (30.2 +/- 2.4)%., Conclusions: There is selective expression of PR on the human sperm acrosome surface.

Objectives: To assess the effect of freezing on the sperm DNA., Methods: To assess the sperm DNA preserved at -80 degrees C by using the single cell gel electrophoresis (SCGE)., Results: There was no statistical difference on the time factor by analysis of variance (ANOVA)., Conclusions: Integrity of sperm DNA could not be devastated in frozen states.

Objective: To explore the associations between concentrations of selenium (Se) in serum and semen and sperm parameters as well as oxidative DNA damage in human sperm., Methods: Concentrations of selenium in serum and semen were determined using atomic absorption spectrophotometer (AAS). The sperm parameters were detected according to WHO laboratory manual for the examination of human sperm. The levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and deoxyguanosine (dG) in human sperm DNA were measured using HPLC-ECD and HPLC-UV., Results: Mean semen concentration of selenium was significantly higher in fertile subjects (0.74 mumol/L) than that in infertile subjects (0.56 mumol/L), while there was no significant difference in serum concentrations of selenium between the two groups. Semen volume (3.91 ml vs. 2.79 ml), sperm density (46.4 x 10(9)/L vs. 36.5 x 10(9)/L) and sperm count (168.0 x 10(6) vs. 102.0 x 10(6)) were significantly higher in fertile subjects than those in infertile subjects. There was no significant difference in motility and viability of sperm, and proportion of deformity sperm between the two groups, Significantly positive correlation was observed between semen concentration of Se and sperm density (r = 0.48, P < 0.01 for fertile subjects and r = 0.32, P < 0.05 for infertile subjects) as well as sperm count (r = 0.32, P < 0.05 for fertile subjects and r = 0.26, P < 0.05 for infertile subjects), sperm motility (r = 0.46, P < 0.01 for fertile subjects and r = 0.65, P < 0.01 for infertile subjects) and viability (r = 0.33, P < 0.05 for fertile subjects and r = 0.64, P < 0.01 for infertile subjects). There was no correlation between serum Se concentrations and sperm parameters. Moreover, 8-OHdG levels in sperm DNA inversely correlated with semen Se concentrations in fertile (r = -0.21, P < 0.05) and infertile (r = -0.28, P < 0.05) subjects. However, no significant correlation was found between 8-OHdG levels in sperm DNA and serum Se concentrations in all subjects., Conclusions: Semen concentrations of Se were associated with sperm quality and 8-OHdG levels in human sperm. Serum concentrations of Se did not affect sperm quality and oxidative DNA damage in human sperm.

Objective: To establish the method of 2-dimensional electrophoresis(2-DE) for proteins of human spermatozoa and to construct a protein map of human spermatozoa., Methods: The sperm pellet was prepared with simple Percoll layer protocol. We studied the effects of various sample preparation methods, loading quantities and isoelectric-focusing protocols on the quality of silver-stained 2-DE map, and constructed a primary protein map of human spermatozoa., Result: Up to 703 protein spots were acquired with sample preparation Method I while only 194-210 spots with Method II. With immobilized pH gradients and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(IPG-DALT) we could acquire over 700 spots while only 280-300 with isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(ISO-DALT)., Conclusion: It is satisfactory to lyse sperm with sample preparation Method I and to separate sperm proteins by IPG-DALT for establishing 2-D map of human sperm.

Objective: To establish the single-cell gel electropherosis (SCGE) protocol for detection of DNA strand breakage in human spermatozoa., Methods: The slides on which sperm cells and agarose were layered were immersed in a cold lysing solution of pH10 to lyse the sperm cells. Sperm nuclei were then pretreated with 10 mmol/L of DTT for 1 h, 10 microg/ml of RNase A for 4 h and 200 microg/ml of proteinase K for 15 h. Lastly, electrophoresis was performed in electrophoresis running buffer(pH10) at 12V (0.46V/cm) and 100 mA for 1 h. Sperm nuclei were stained with 15 microg/ml of EtBr for 5 min. The percentage of comet cells was counted. The in vitro hydrogen peroxide-induced DNA damage in human spermatozoa was measured with SCGE established by this laboratory., Results: The comet sperm cells in human spermatozoa ranged from 2% to 38%. There was a significant variance on the percentage of comet cells between different subjects. Hydrogen peroxide increased the percentage of comet sperm in a dose- and time-dependent manner., Conclusion: SCGE may be used to detect DNA strand breakage in human spermatozoa. Hydrogen peroxide-induced DNA damage in human sperm cells was detected successfully using SCGE protocol established by this laboratory.

Using isotope incorporate amplification technique of special 31A, 31A-A, 31A-G primers and alpha-32PdCTP, the minisatellite MS31A (located at D7S21 loci) was studied. A rapid, simple, and accurate MVR-PCR technique was successfully established. The technique can be applied in individual identification minutes amples, such as blood stains, semen stains contaminated by vaginal fluid, hair and bones. The sensitivity analysis revealed that this technique could detect 1 ng genoma DNA. It is also discribed about the application of the method in 40 criminal cases of rape and murder.

Objective: To investigate the tissue specificity and expression in spermatogenic stage of BSD-2.4 cDNA which might be related with fertility and its sequence was received by GenBank., Methods: Dot blot, Northern blot, and in situ hybridization were used., Results: Northern blot analysis of sixteen human tissues showed that BSD-2.4 cDNA had one transcript only with testis. BSD-2.4 mRNA was detected in germ cells of human testis., Conclusions: BSD-2.4 is a testis-specific protein. The cDNA was expressed throughout the stages of the spermatogenic cycle.