UItrastructural Observation on Rat Spermatozoa Treatedwith Tripchlorolide(T_4) from Tripterygium wilfordii

Adult male rats were treated orally with monomer T_4(Tripchlorolide) isolated from Tripterygium wilfordii, at the dose of 50μg/kg/day,6 days/week for6 weeks.Ultrathin section and freeze etching replica of seminiferous tubules and epididymal spermatozoa were examined with elec-tron microscope. The results showed that the spemiogenesis was inhibited T_4 in seminiferous tu-bules. However,the damage and disruption of the spermatozoa were more serious in the epididymis.Damage of the structure and function ot microtubule and microfilament may be the chief reason for sperm damage. Sperm membrane was also very sensitive to the treatiment of monomer T_4.

Research advances in the biological characteristics of the crustacean spermatozoa

The biological characteristics of crustacean spermatozoa is important for the artificial reproduction and genetic breeding. With reference to the latest studies and related materials, this paper reviewed the research progress in the biological characteristics of crustacean spermatozoa, such as morphological structure of sperm, spermatogenesis, sperm viability, preservation in vitro and acrosome reaction et al. The prospects of the research field have also been anticipated.

辅助生殖技术中精子DNA损伤的研究现状

精子DNA完整性对受精卵的形成至关重要,精子DNA损伤可能影响辅助生殖技术(assisted reproductive technology,ART)的成功率。精子DNA损伤主要包括单链和双链损伤,这些损伤可能在受精、胚胎质量和着床能力等多个方面影响ART的成功率。精子DNA损伤的因素主要包括氧化应激、年龄、激素以及环境等。同时,卵母细胞对精子DNA损伤的修复能力也至关重要。目前临床和实验室主要通过药物干预、优化精子选择来降低精子DNA碎片指数(DNA fragmentation index,DFI),从而改善ART的临床治疗结局。综述精子DNA损伤的研究现状,分析其对ART治疗结局的影响,并提出了可能的改进精液制备方法。

环境因素调节精子非编码RNA参与代际间遗传的研究进展

环境因素不仅会影响男性生育功能,还可编码精子表观遗传信息,通过代际间遗传或跨代遗传影响下一代的健康。近年研究发现,非编码RNA(non-coding RNA,ncRNA)作为调控基因表达的重要表观遗传分子,可参与精子介导的代际间遗传过程。多种环境因素可调节精子ncRNA水平,导致子代甚至第三代出现内分泌、精神、生殖和运动等方面的健康问题。药物干预和生活方式改善可调节精子ncRNA的表达,提高精子的质量,从而提高后代的健康水平。了解ncRNA的分类和功能,环境因素改变精子ncRNA水平的机制,以及该机制在跨代遗传,尤其是在精子成熟过程中所发挥的作用,可为探索提高男性精子质量的方法提供思路。

Expression of hepatitis B virus genes in early embryonic cells originated from hamster ova and human spermatozoa transfected with the complete viral genome

Aim： To detect the expression of hepatitis B virus （HBV） genes （HB S and C genes） in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fertilization （IVF） technique. Methods： Human sperm-mediated HBV genes were delivered into zona-free hamster oocytes by the IVF method. Polymerase chain reaction （PCR） was used to detect HB S and pre-Core/Core （pre-C/C） coding genes both in one- and two-cell embryos. Reverse transcription-PCR （RT-PCR） analysis was used to study the expression of the two genes. Fluorescence in situ hybridization （FISH） analysis using the full-length HBV DNA as the hybridization probe was performed to confirm the integration of viral DNA in the host embryonic genome. Results： Both HB S and pre-C/C coding genes are present and transcribed in one- and two-cell embryos originated from hamster ova IVF with human spermatozoa carrying HBV DNA sequences. Conclusion： Sperm-mediated HBV genes are able to replicate and express themselves in early embryonic cells. These results provide direct evidence that HBV DNA could transmit vertically to the next generation via the male germ line.

Apoptosis and DNA damage in human spermatozoa

DNA damage is frequently encountered in spermatozoa of subfertile males and is correlated with a range of adverse clinical outcomes including impaired fertilization, disrupted preimplantation embryonic development, increased rates of miscarriage and an enhanced risk of disease in the progeny. The etiology of DNA fragmentation in human spermatozoa is closely correlated with the appearance of oxidative base adducts and evidence of impaired spermiogenesis. We hypothesize that oxidative stress impedes spermiogenesis, resulting in the generation of spermatozoa with poorly remodelled chromatin. These defective cells have a tendency to default to an apeptotic pathway associated with motility loss, caspase activation, phosphatidylserine exteriorization and the activation of free radical generation by the mitochondria. The latter induces lipid peroxidation and oxidative DNA damage, which then leads to DNA fragmentation and cell death. The physical architecture of spermatozoa prevents any nucleases activated as a result of this apoptotic process from gaining access to the nuclear DNA and inducing its fragmentation. It is for this reason that a majority of the DNA damage encountered in human spermatozoa seems to be oxidative. Given the important role that oxidative stress seems to have in the etiology of DNA damage, there should be an important role for antioxidants in the treatment of this condition. If oxidative DNA damage in spermatozoa is providing a sensitive readout of systemic oxidative stress, the implications of these findings could stretch beyond our immediate goal of trying to minimize DNA damage in spermatozoa as a prelude to assisted conception therapy.

Relationship between acrosin activity of human spermatozoa and oxidative stress

Aim: To study the association between seminal oxidative stress and human sperm acrosin activity. Methods: It is a prospective study consisting of 30 infertile men and 12 fertile normozoospermic volunteers. A full history, clinical examination and scrotal ultrasound were done to exclude other related factors such as smoking and varicocele. Presence of white blood cells (WBCs) in semen samples was evaluated by peroxidase staining. Lipid peroxidation in spermatozoa was induced after incubating with ferrous sulphate (4 mmol/L) and sodium ascorbate (20 mmol/L). Induced peroxidation of spermatozoa was assessed by determining the production of thiobarbituric acid reactive substances (TBARS). Acrosin activity was measured using the gelatinolysis technique. The halo diameters around the sperm heads and the percentages of spermatozoa showing halo formation were evaluated. An acrosin activity index was calculated by multiplying the halo diameter by the halo formation rate. Results: A significant difference was observed in acrosin activity parameters and TBARS levels between samples with WBCs (>1×106/mL of ejaculate) and those without. This difference was also noted between the normozoospermic and the oligoasthenoteratozoospermic semen samples. The TBARS production by spermatozoa had a significant negative correlation with the acrosin activity index (r = -0.89, P <0.001). Conclusion: The presence of oxidative stress in an individual with leukocytospermia and/or abnormal semen parameters is associated with impaired sperm function as measured by its acrosin activity.

Differentiation of murine male germ cells to spermatozoa n a soft agar culture system

Establishment of an in vitro system that allows the development of testicular germ cells to sperm will be valuable for studies of spermatogenesis and future treatments for male infertility. In the present study, we developed in vitro culture conditions using three-dimensional agar culture system （SACS）, which has the capacity to induce testicular germ cells to reach the final stages of spermatogenesis, including spermatozoa generation. Seminiferous tubules from testes of 7-day-old mice were enzymatically dissociated, and intratubular cells were cultured in the upper layer of the SACS in RPMI medium supplemented with fetal calf serum （FCS）. The lower layer of the SACS contained only RPMI medium supplemented with FCS. Colonies in the upper layer were isolated after 14 and 28 days of culture and were classified according to their size. Immunofluorescence and real-time PCR were used to analyse specific markers expressed in undifferentiated and differentiated spermatogonia （Vasa, Dazl, OCT-4, C-Kit, GFR- a-l, CD9 and a-6-integrin）, meiotic cells （LDH, Crem-1 and Boule） and post-meiotic cells （Protamine-1, Acrosin and SP-IO）. Our results reveal that it is possible to induce mouse testicular pre-meiotic germ cell expansion and induce their differentiation to spermatozoa in SACS. The spermatozoa showed normal morphology and contained acrosomes. Thus, our results demonstrate that SACS could be used as a novel in vitro system for the maturation of pre-meiotic mouse germ cells to post-meiotic stages and morphologically-normal spermatozoa.

The expression and significance of CATSPER1 in human testis and ejaculated spermatozoa

Aim： To investigate the distribution of cation channel of sperm 1 （CATSPER1） protein and the presence of CATSPER1 mRNA in human testis and ejaculated spermatozoa. The influence of anti-human CATSPER1 antibody upon human sperm motility was used to evaluate the function of human CATSPER1 and to estimate its possible use as a target for immunocontraception. Methods： Human ejaculated sperm from normozoospermic donors （n = 12） and liquid nitrogen frozen human testis were used for the study of mRNA and protein expression of CATSPER1 by reverse transcription polymerase chain reaction （RT-PCR） and immunohistochemistry, respectively. Spermatozoa from normozoospermic donors （n = 12） were individually processed using a swim-up procedure and were then incubated with CATSPER1 antibody at final concentrations of 20, 4 and 0.8 μg/mL. After 1, 2 and 6 h incubation, progressive motility and fast progressive motility were measured by means of computer-assisted semen analysis. Results： CATSPER1 transcript was detected in both human testis and each human ejaculated semen sample. CATSPER1 protein expressed in the membrane of spermatid and was localized in the principal piece of the sperm tail. The application of CATSPER1 antibody at all concentrations significantly inhibited both progressive motility and fast progressive motility after 1, 2 and 6 h incubation, and significant dose-dependent changes were observed. Conclusion： CATSPER1 is meiotically and post-meiotically expressed in human testis tissue. CATSPER1 mRNA in human ejaculated spermatozoa could be a more feasible target for study and infertility screening than testis biopsy. In addition, our results suggest that human CATSPER1 could be a possible target for immunocontraception.

The usefulness and significance of assessing rapidly progressive spermatozoa

It is possible and clinically relevant to distinguish between slow and rapid progressive spermatozoa in basic semen analysis. This is discussed in light of the different purposes of semen analysis for the subfertile couple and the male patient. The two groups of progressive spermatozoa should be distinguished to help ensure that pertinent information available in the semen sample is not neglected.

Whither must spermatozoa wander？ The future of laboratory seminology

This commentary celebrates the publication of the 5th for the Examination and Processing of Human Semen edition of the World Health Organization Laboratory Manual This is the most complete text to date on the creation of a conventional semen profile and includes invaluable reference limits for specific aspects of semen quality based on the analysis of over 1 900 recent fathers. The new edition of the manual also includes detailed protocols for monitoring different aspects of sperm function and new chapters on the preparation of spermatozoa for assisted conception and cryopreservation. Given that this publication is the definitive statement on how to perform a descriptive semen analysis, we might speculate on the future of this field and the sorts of tests that might feature in future editions of the manual. Cell biologists are currently being empowered by the ＇omics revolution, which is placing at their disposal technologies of unprecedented power to examine the biochemical composition of cells such as spermatozoa. Indeed, spermatozoa are perfect vehicles for this kind of analysis because they can be obtained as extremely pure suspensions, exist naturally in isolation and can be induced to express their capacity for fertilization and the initiation of embryonic development in vitro. The application of ＇omics technologies to these cells, in concert with detailed assessments of their functional competence, should provide insights into the biochemical basis of defective semen quality. This information will then help us understand the causes of male infertility and to develop rational methods for its treatment and possible prevention.

Differential expression of VASA gene in ejaculated spermatozoa from normozoospermic men and patients with oligozoospermia

Aim： To detect the expression of VASA in human ejaculated spermatozoa, and to compare the expression of VASA between normozoospermic men and patients with oligozoospermia. Methods： Ejaculated spermatozoa were collected from normozoospermic men and patients with oligozoospermia by masturbation, and subsequently segregated through a discontinuous gradient of Percoll to obtain the spermatozoa. Reverse transcription polymerase chain reaction （RT- PCR）, quantitative RT-PCR （QRT-PCR）, immunoflurescence and Western blotting were used to detect the expression of VASA in mRNA and protein levels. Results： VASA mRNA was expressed in the ejaculated spermatozoa. QRT-PCR analysis showed that VASA mRNA level was approximately 5-fold higher in normozoospermic men than that in oligozoospermic men. Immunofluorescence and Western blotting analysis showed that VASA protein was located on the cytoplasmic membrane of heads and tails of spermatozoa, and its expression was significantly decreased in oligozoospermic men, which is similar to the result of QRT-PCR. Conclusion： The expression of VASA mRNA and protein was significantly decreased in the sperm of oligozoospermic men, which suggested the lower expression of the VASA gene might be associated with pathogenesis in some subtypes of male infertility and VASA could be used as a molecular marker for the diagnosis of male infertility.

Cryopreservation-induced decrease in heat-shock protein 90 in human spermatozoa and its mechanism

<abstract>Aim: To study the protein changes of spermatozoa associated with sperm motility during sperm cryopreservation and its mechanism. Methods: In 18 healthy men, the seminal sperm motility and HSP90 levels were studied before and after cryopreservation using SDS-PAGE, Western blotting and computerized image analysis. Results: The sperm motility declined significantly after cryopreservation (P<0.01). The average grey level and the integrated grey level of sperm HSP90 before cooling were 34.1±3.2 and 243.0±21.6, respectively, while those after thawing were 23.2±2.5 and 105.7±28.5, respectively. Both parameters were decreased significantly (P<0.01). No HSP90 was found in the seminal plasma before and after cryopreservation. Conclusion: HSP90 in human spermatozoa was decreased substantially after cryopreservation. This may result from protein degradation, rather than leakage into the seminal plasma.

Proteomic changes in mammalian spermatozoa during epididymal maturation

Epididymal maturation is associated with the activation of a cAMP-induced tyrosine phosphorylation cascade, which is ultimately associated with the expression of capacitation-dependent sperm functions, such as hyperactivated movement and acrosomal exocytosis. As spermatozoa progress through the epididymis they first acquire the capacity to phosphorylate tyrosine on targets on the principal piece, followed by the midpiece. By the time these cells have reached the cauda epididymidis they can phosphorylate the entire tail from neck to endpiece. This particular pattern of phosphorylation is associated with the ontogeny of fully functional spermatozoa that are capable of fertilizing the oocyte. Proteomic analyses indicate that this change is associated with the phosphorylation of several mitochondrial proteins, creation of a mitochondrial membrane potential and activation of mitochondrial free radical generation. At least in rodent species, activation of sperm mitochondria appears to be a particularly important part of epididymal maturation. （Asian J Androl 2007 July; 9： 554-564）

Comparative study on density gradients and swim-up preparation techniques utilizing neat and cryopreserved spermatozoa

Aim: To 1) compare post-wash and post-thaw parameters of sperm processed with PureSperm density gradient technique and swim-up method; and 2) test the efficacy of two commonly available density gradient media PureSperm and ISolate. Methods: This prospective study used semen specimens from 22 patients. Specimens from nine patients were processed by both PureSperm density gradient and swim-up method. These specimens were then cryopreserved. Thirteen specimens were processed by both PureSperm (40 % and 80 %) and Isolate (50 % and 90 %) double density gradient techniques. The two fractions processed by both PureSperm and swim-up were analyzed for post-wash sperm characteristics. Post-thaw analysis was done after 24 hours. Sperm fractions obtained after processing with PureSperm and ISolate were compared for post-wash sperm characteristics and ROS levels. Results: Specimens prepared with PureSperm had significantly higher median total motile sperm counts (TMSC) (32.2 x 10~6 vs. 17.6 x 10~6), recovery rates (69.2 % vs. 50.0 %), and longevity at 4 hours (83.0 % vs. 55.0 %) compared to specimen prepared by swim-up. Post-thaw specimens also had a higher recovery and longevity at 4 hours with PureSperm as compared to the swim-up. Semen specimens processed by PureSperm had significantly higher total sperm count, TMSC, and percentage recovery rates (30.0 % vs. 19.7 %) than ISolate. Conclusion: Semen quality is better preserved in fresh and cryopreserved semen prepared with PureSperm density gradient compared to swim-up. A significant enrichment of sperm is observed with PureSperm compared to ISolate. Higher recovery rates of mature motile sperm obtained after PureSperm sperm preparation may be beneficial for successful ART. i

Morphological characteristics of spermatozoa before and after renal transplantation

Aim: To investigate the changes of the spermatozoa ultrastructures before and after renal transplantation in uremic patients. Methods: The sperm of five uremic patients before and after transplantation and four healthy volunteers were collected and examined by scanning electron microscopy. Results: Abnormal spermatozoa were found in patients pre-transplantation; abnormalities included deletion of the acrosome, absence of the postacrosomal and postnuclear ring, dumbbell-like changes of the head, tail curling, and absence of the mitochondrial sheath in the mid-segment. After renal transplantation, most of the spermatozoa became normal. Conclusion: There are many abnormalities with regard to the appearance and structure of the head, acrosome, mitochondria and tail of the spermatozoa in uremic patients. The majority of the spermatozoa returned to normal after renal transplantation, but a few still presented some abnormalities possibly relating to the administration of immunosuppressants.

Preparation and incubation conditions affect the DNA integrity of ejaculated human spermatozoa

Appropriate semen processing and assessment are critical for successful infertility treatment. We investigated whether laboratory procedures including semen preparation and incubation affect sperm DNA integrity. A total of 153 infertile men were involved. Conventional semen parameters and sperm chromatin structure assay （SCSA） parameters, that is, DNA fragmentation index （%DFI） and high DNA stainability （%HDS）, were assessed on the flesh ejaculated semen samples, which were treated and incubated under different conditions. Negative correlations were identified between the %DFI and sperm concentration, motility, progressive motility and morphology. A lower percentage of DFI was detected in spermatozoa when density gradient centrifugation （DGC） was followed by swimup treatment in comparison with DGC alone （P 〈 0.01）. Although the %DFI increased in a time-dependent manner with incubation both at room temperature （RT） and at 37℃ in air, the %DFI after 24 h at RT was significantly lower than that at 37℃ （P 〈 0.05）. Incubation with 5% CO2 was effective in maintaining sperm motility （P 〈 0.01）; however, it induced further elevation of %DFI （P 〈 0.001）. Thus, sperm DNA damage was associated with longer incubation periods. Interestingly, common culture conditions, such as maintaining pH and temperature, compromised the sperm DNA integrity.

Clinical pregnancies and livebirths achieved by intracytoplasmic injection of round headed acrosomeless spermatozoa with and without oocyte activation in familial globozoospermia： case report

We report the successful outcome of intracytoplasmic sperm injection （ICSI） treatment in two siblings with familial globozoospermia. After controlled ovarian hyperstimulation and oocyte pick-up, retrieved oocytes were mechanically activated before ICSI and a fertilization rate of 33.3% was achieved in the first case. The second couple underwent ICSI without oocyte activation and a 9.1% fertilization rate was obtained. The transfer of two grade I embryos in the first couple and one grade I embryo in the second couple resulted in clinical pregnancies with healthy livebirths. It was concluded that the main problem of cases with globozoospermia is a low fertilization rate, and even though ICSI and oocyte activation can increase this rate it is not necessarily needed to achieve a pregnancy. （Asian J Androl 2008 Mar; 10： 332-336）

Quantitative (stereological) study on the spermatozoal storage capacity of epididymis in rats and monkeys

Aim: To investigate the spermatozoal production rate of the testis and the spermatozoal storage capacity of the epi-didymis in monkeys and rats. Methods: The number of the late spermatids (steps 13 - 14 in the monkey or steps15 - 19 in the rat) per testis and the number of spermatozoa per epididymis were estimated in 6 normal adult monkeys(Macaca fascicularis) and 6 normal adult SD rats on 25 um-thick methacrylate-embedded sections using a contempo-rary unbiased and efficient stereological method-the optical disector. The diameter and length of the efferent ductulesand ductus epididymidis and the volume of the epididymal fluid in the tubules were also estimated. Results: The totalnumber of the late spermatids per testis was 2902 ±749 (million, x ±s) in the monkey, or 179 ±31 in the rat; thenumber of spermatozoa per epididymis was 3235 ±1835 in the monkey, or 241±76 in the rat. Conclusion: A largenumber of spermatozoa was densely packed and stored in the ductus epididymidis; the epididymal transit time for sper-matozoa was around 5 days in monkeys or 11 days in rats.

Tests to measure the quality of spermatozoa at spermiation

This commentary is to critique the revised World Health Organization （WHO） semen analysis manual as it pertains to characteristics of a spermatozoon at spermiation. The aims of the revised WHO manual include improving the ＇quality of semen analysis＇ without any restriction to clinical use. Furthermore, the manual states that semen analysis may be useful for （a） ＇investigating male fertility status＇ and （b） ＇monitoring spermatogenesis during and following male fertility regula- tion.＇ However, if the analysis of ejaculated spermatozoa is intended for the purposes described in （b）, then cells that are abnormal at spermiation must be identified. This paper takes the position that the manual does not identify methods to estimate the quality of spermatozoa at spermiation. Instead, it uses a ＇gold standard＇ of sperm passing through the cervical mucus or arriving near the site of fertilization. Although this standard is appropriate for drawing conclusions regarding the probability that an individual could impregnate his partner, it is not appropriate for studying illness of the testes per se. Herein, the measures of sperm quality presented in the WHO manual are critiqued with respect to the detection of spermatozoa that were abnormal at spermiation vs. those that became abnormal subsequently. Quality assessments based on the percentage of motile or ＇viable＇ spermatozoa are meaningless. Alternative quality attributes defining spermatozoa at spermiation are presented in this paper. In conclusion, assessment of spermatozoal quality at spermiation, on the basis of quality attributes of individual ejaculated spermatozoa, is best achieved through application of （a） a new paradigm for the morphological evaluation of sperm quality and （b） modern analytical techniques to evaluate, in an adequate sample, several appropriate independent attributes in each spermatozoon in order to more accurately identify the proportion of abnormal spermatozoa.