解脲脲原体感染动物模型的研究进展

解脲脲原体(Uu)是女性泌尿生殖道黏膜中常见的微生物,其致病性仍存在争议。Uu感染下生殖道可表现为尿道炎或宫颈炎,而定植于新生儿下呼吸道可引起肺炎、支气管肺发育不良、慢性肺部疾病等。研究Uu感染的发病机制、宿主的免疫反应以及Uu感染的防治,动物模型是不可缺少的。目前国内、外已成功建立多种Uu感染动物模型,主要包括下生殖道感染模型及呼吸道感染模型。在Uu生殖道感染动物模型的建立中,雌二醇的预处理必不可少,实验动物首选BALB/c小鼠;而呼吸道感染模型不需雌二醇预处理,多位学者成功使用C3H/HeN小鼠建立了该模型。     

Gold nanoparticles disturb nuclear chromatin decondensation in mouse sperm in vitro

The effect of gold nanoparticles on mouse epididymal sperm has been studied using the model system of nuclear chromatin decondensation in vitro. It is shown that the treatment of gametes, preliminary membrane-freed by sodium dodecyl sulfate, in the mediums containing gold nanoparticles (with diameter ∼2.5 nm) in concentrations 1.0 × 10¹⁵ or 0.5 × 10¹⁵ particles/ml and following incubation in dithiothreitol solution (DTT) resulted in failure of chromatin decondensation process and nucleus structure. We conclude that gold nanoparticles possess spermatotoxicity. The mechanism of cytotoxic effect of gold nanoparticles may be related with their interaction with molecules of double-helix DNA. The model system studied in this research is applicable for further investigations of cytotoxic effects of nanoparticles of different origin and made of different metals.     

Serological evidence of Ureaplasma urealyticum infection in neonatal respiratory disease

Since up to 80 percent of pregnant women and 30 percent of neonates may be colonized with genital mycoplasmas, it is difficult to determine whether true infection occurs. The antibody responses to eight serotypes of U. urealyticum were assessed in mothers and infants in 21 cases of neonatal respiratory disease (RD) and 24 normal cases. Among the normal population of mothers and infants, a titer of greater than or equal to 1:32 occurred in 0.25 percent (1/394). In mother-infant paired titers, a fourfold difference occurred in 2.6 percent (5/192). Among 54 RD neonates, 55.6 percent had a titer of greater than or equal to 1:32 compared to only 4.2 percent of normal neonates (p less than .001). Fourfold elevations in antibody titers of greater than 1:32 were observed in the neonate in 52.4 percent of RD cases compared to 0 percent of 24 normal pairs (p less than .001) and in 28.6 percent of mothers of RD neonates compared to 0 percent in normal cases (p = .013). We observed that 43.3 percent of RD neonates with titers greater than or equal to 1:32 died compared to 16.6 percent of RD neonates exhibiting no elevation of antibody response over the maternal level. Among the six who died, 66.7 percent of neonates and 16.7 percent of their mothers had elevated titers, compared to 33.3 percent of 15 surviving infants and 40.0 percent of their mothers. These elevated antibody responses strongly support the concept that U. urealyticum causes infection in the perinatal period in association with neonatal respiratory disease. Since the elevation in titers was detected close to delivery in many cases, the infection may occur in utero.     

Evolution of sexually transmitted and sexually transmissible human herpesviruses

Herpesviruses occur in an impressively wide range of animals and are associated with various diseases. The numerous routes taken during hundreds of millions of years of evolution have contributed to their striking adaptability and success as pathogens. Herpesviruses share a distinct virion structure and are classified taxonomically into a single order, the Herpesvirales, which is divided into three families. The phylogenetic relationships among members of the most populous family, the Herpesviridae, which includes all nine human herpesviruses, are generally similar to those among their hosts, supporting the view that there has been a large degree of coevolution between virus and host. Three human herpesviruses (human cytomegalovirus, Kaposi's sarcoma-associated herpesvirus, and herpes simplex virus type 1) are classed as agents capable of sexually transmissible infection (StxI), and one (herpes simplex virus type 2) as an agent capable of sexually transmitted infection (STI). The evolutionary characteristics of these viruses are described.     

In vitro and in vivo fertility of ram semen cryopreserved in different extenders

Seminal traits of frozen-thawed (FT) ram semen and in vitro and field fertility in native Portuguese breeds were evaluated in 4 experiments. In exp. 1 and 2 the cryopreservation capacity of 2 extenders, E1 (15% egg yolk-EY) and E2 (4.5% EY and trehalose) was compared through morphological evaluation and in vitro fertilizability of FT ram semen. Exp. 3 aimed to determine the usefulness of in vitro homologous/heterologous fertilization tests as tools for predicting ram fertility. Exp. 4 was conducted to verify if the identified differences between the 2 extenders could be confirmed by field fertility. E1 showed a better cryoprotective action expressed by higher in vitro and field fertility results. In conclusion, EY is difficult to be replaced in ram semen extenders. Heterologous fertilization seems to be a useful tool for predicting fertility of FT ram semen.     

DNA integrity in sexed bull sperm assessed by neutral Comet assay and sperm chromatin structure assay

During the production of sex-sorted spermatozoa from bull semen, the cells are exposed to a number of potential hazards including: dilution, centrifugation, incubation, exposure to DNA stains and laser light. These factors may affect the survival capacity and fertilization potential of the sperm. The objective of this study was to determine whether sex-sorted bull spermatozoa have more DNA damage than sperm from conventional processed bull semen. Two methods were used to determine DNA integrity: the neutral Comet assay (NCA) and the sperm chromatin structure assay (SCSA). The NCA showed that the conventional samples had a higher tail moment (TM) (P < 0.017) than the sorted samples and that there was no difference between the samples in tail length (TL) (P = 0.36). The SCSA showed that the DNA fragmentation index (DFI) was higher for conventional than the sorted samples (P = 0.011), but the standard deviation of DFI (SD-DFI) was higher for the sorted samples (P < 0.001). We conclude that the NCA and SCSA can be used in assessing DNA integrity in bovine sperm and that cell sorting by flow cytometry improves the integrity of the sperm cell population. Additionally the results from the SCSA indicated that the sex-sorted sperm had less homogenous sperm chromatin. In the future assessment of sperm DNA integrity may be used to select bulls for sperm sex sorting and optimizing sperm sex sorting procedures.     

Evaluating human sperm DNA integrity: relationship between 8-hydroxydeoxyguanosine quantification and the sperm chromatin structure assay

In our work, we have used 8-hydroxy-deoxyguanosine (8-OH-dG), one of the major oxidative products of sperm DNA, in a population of patients consulting for infertility. We found an inverse relationship between sperm concentration and the log of the ratio of 8-OH-dG to dG (P < 0.01). On the same patients' sperm samples, the sperm chromatin structure assay (SCSA) was performed. An inverse relationship was observed between the DNA fragmentation index and sperm concentration (P < 0.001). There was also a positive relationship between SCSA and log 8-OH-dG/dG. This indicates that DNA fragmentation measured by the SCSA originates in part from oxidative products. In a few patients, antioxidant treatment decreased the DNA fragmentation index below the threshold of 30% that is crucial for subfertility.     

Ureaplasma urealyticum and Ureaplasma parvum in etiology of urogenital mixt-infections

The complex study of 104 vaginal samples from patients with urogenital uroplasmosis was carried out. U. parvum were detected in 67.3% patients, U. urealyticum--in 12.5% and in 20.1% cases--two species were registered at the same time. Isolation of clinical significant concentration of both ureaplasma (> 10(4) CFU/ ml) was detected in about 50% of cases. Expression of inflammation of vaginal mucus depended on the level of concentration of infection agents. U. parvum were associated with bacterial vaginosis, while in urogenital candidosis U. parvum was detected rarer than U. urealyticum. The dominant numbers of clinical ureaplasma were high sensitive to "new" macrolides and chinolons, however the high percent of isolates were resistant to erytromicin and doxiciclin.     

In vitro fertilization of hamster and human oocytes by microinjection of human sperm

In this study, swollen sperm heads were obtained after the injection of human sperm into the perivitelline space of hamster oocytes. The number of injected sperm and the sperm concentration in the preincubation medium were found to have an influcnce on the rate of penetrated hamster oocytes. The optimal injected sperm number was always between five to 12 to obtain 8, 37, and 36% penetration for donors A, B, and C, respectively. The optimal sperm concentration in preincubation medium was between 6 and 22 × 10⁶ sperm/ ml to obtain 16, 47, and 43% penetration for donors A, B, and C, respectively. The rate of polyspermic oocytes was related to the injected sperm number (0, 55, and 100% for one to four, five to 12, and more than 12 injected sperm respectively). Ten human mature oocytes were injected with the sperm from six normal donors. Five fertilized eggs were obtained, and of these four cleaved in in vitro culture.     

The integrity of sperm chromatin in young tropical composite bulls

Sperm chromatin fragmentation is associated with subfertility, but its relationship with age progression in young bulls is poorly understood. The objective was to assess sperm chromatin fragmentation during the early post-pubertal development of 20 tropical composite bulls, using a sperm chromatin structure assay (SCSA) and sperm-bos-halomax (SBH). Bulls were subjected to bull breeding soundness evaluation (BBSE) at mean ages of 13, 18, and 24 mo. Traits measured included liveweight (WT), body condition score (BCS) and scrotal circumference (SC). Semen samples were collected by electroejaculation and assessed for mass activity (MA), motility (Mot), concentration (conc), sperm morphology and chromatin fragmentation. Concentration (r = 0.34, P = 0.0076), Mot (r = 0.36, P = 0.0041) and percentage of morphologic normal sperm (percent normal sperm (PNS); r = 0.31, P = 0.0132) were positively correlated with age. The percentage of sperm with proximal droplets (PD) was negatively correlated with age (r = −0.28, P = 0.0348), whereas neither SCSA nor SBH results were significantly correlated with age. The percentage of sperm with chromatin fragmentation using SCSA was correlated with PNS (r = −0.53, P < 0.0001), the percentage of sperm with head abnormalities (r = 0.68, P < 0.0001) and the percentage of intact sperm (Int) with SBH (r = −0.26, P = 0.0456). In summary, for assessment of sperm chromatin fragmentation, samples could be equally collected at 13, 18 or 24 mo of age, as results did not vary with age.     

Normospermic versus teratospermic domestic cat sperm chromatin integrity evaluated by flow cytometry and intracytoplasmic sperm injection

Teratospermia (>60% of morphologically abnormal spermatozoa) is well documented in felids. Even morphologically normal spermatozoa from teratospermic ejaculates have reduced ability to undergo tyrosine phosphorylation, acrosome react, and bind and penetrate oocytes compared with normospermic (<40% abnormal spermatozoa) counterparts. However, it is unknown whether fertilization deficiencies originate at a nuclear level. This study examined whether fertilization failure also was attributable to abnormal sperm chromatin, using the sperm chromatin structure assay (SCSA), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). Aliquots of unprocessed and swim-up-processed (to isolate morphologically normal spermatozoa) spermatozoa from teratospermic and normospermic domestic cats were analyzed by the flow cytometric SCSA. Swim-up-processed sperm were incubated with in vivo-matured oocytes or used for ICSI. Teratospermic ejaculates expressed more (P < 0.05) chromatin heterogeneity (abnormal chromatin structure) than their normospermic counterparts, both in unprocessed and swim-up-processed samples. Fertilization success in vitro was higher (P < 0.05) from normo- compared with teratospermic inseminates. Similar (P > 0.05) proportions of oocytes fertilized after ICSI using spermatozoa from normo- and teratospermic cats. Results reveal that teratospermia in the cat is expressed at the nuclear level as increased sperm chromatin heterogeneity, but ICSI showed that this does not apparently affect fertilization rates if the zona pellucida and oolemma can be bypassed.     

In vivo impairment of neutrophil recruitment during lentivirus infection

Evidence indicates that the lentivirus, HIV, infection affects neutrophil response to bacteria and bacterial products in vitro. We used a novel model of rapid onset immunosuppression following infection with a similar lentivirus, feline immunodeficiency virus (FIV), in cats to examine neutrophil function within the microvasculature in vivo and to determine the steps that are impaired in the neutrophil recruitment cascade. In uninfected cats and cats infected neonatally with FIV, the mesentery was exteriorized, but remained autoperfused during intravital microscopy for 4 h. When the tissue was superfused with 10 micro g/ml of LPS for 4 h, intravital microscopy displayed a profound increase in neutrophil rolling at both 8 and 12 wk of age in uninfected cats. At 12 wk of age, FIV-infected animals showed a profound decrease in the number of rolling neutrophils. In vitro studies revealed that neutrophils from infected and uninfected animals rolled equally well on surrogate selectin substrata. In addition, in vivo neutrophil adhesion and emigration out of the vasculature were severely reduced, and in vitro neutrophil chemotaxis from FIV-infected animals was significantly impaired in response to fMLP or IL-8. However, FIV infection of neutrophils could not be detected. In summary, in vivo lentivirus infection with immunosuppression leads to a severe impairment in neutrophil rolling, adhesion, and emigration in response to bacterial stimulants potentially involving both endothelial and neutrophil dysfunction. These in vivo studies also indicate that neutrophil dysfunction should be taken into account when treating infections and tissue injury.     

In vitro evaluation of ram sperm frozen with glycerol, ethylene glycol or acetamide

The aim was to assess the in vitro effect of glycerol, ethylene glycol or acetamide on frozen-thawed ram spermatozoa. Aliquots of each sixteen ejaculates from four rams of the Morada Nova breed were diluted in Tris-egg yolk with glycerol (5%), ethylene glycol (3% or 5%) or acetamide (3% or 5%) and frozen at -196°C. After thawing, progressive sperm motility was greater (P<0.05) in cryopreservation with glycerol 5% and ethylene glycol (3% or 5%) than with acetamide (3% or 5%). Acrosome integrity was greater (P<0.05) with ethylene glycol 5% than acetamide (3% or 5%). The percentage of sperm without oxidative stress was greater (P<0.05) with ethylene glycol (3% or 5%) than with acetamide (3% or 5%). Plasma membrane integrity was greater with glycerol 5% (P<0.05) than with the other cryoprotectants. Thus, it is concluded that glycerol 5% and ethylene glycol 3% or 5% protect ram sperm against the harmful effects of freezing and that glycerol 5% offers greater protection to sperm plasma membrane.     

光动力抗微生物化学疗法对解脲脲原体体外活性的影响

解脲脲原体是一种重要的病原微生物,近年来其耐药形势十分严峻,因此寻找一种全新的有效替代治疗方案尤为重要。本研究旨在探索光动力抗微生物化学疗法对解脲脲原体体外活性的影响。选取解脲脲原体两种生物群（Parvo生物群及T960生物群）代表菌株,包括标准株及临床株,与系列稀释的2.5~0.039 062 5 mmol/L光敏剂甲苯胺蓝孵育20 min或60 min,再以（633±10）nm红光照射,设置48、102、204和408 mJ/cm²共4组能量密度,48 h后判读结果。观察不同解脲脲原体与甲苯胺蓝孵育时间、甲苯胺蓝浓度、光照能量密度对光动力抗微生物化学疗法灭活解脲脲原体效果的影响,并观察两种生物群对光动力抗微生物化学疗法敏感性的差异。结果显示,光动力抗微生物化学疗法在体外对解脲脲原体有明显灭活作用。在光照能量密度及解脲脲原体与甲苯胺蓝孵育时间固定的前提下,这种灭活作用随甲苯胺蓝浓度的增加而增强;单一633 nm红光光源在408 J/cm²及以下的能量密度对解脲脲原体的活性无明显影响。在甲苯胺蓝浓度及解脲脲原体与甲苯胺蓝孵育时间固定的条件下,光动力抗微生物化学疗法对解脲脲原体的灭活作用随光照能量密度（48~408 mJ/cm²）的增加而增强;随甲苯胺蓝孵育时间（30~60 min）延长,光动力抗微生物化学疗法对解脲脲原体的灭活作用有增强的趋势。结果提示,解脲脲原体两种生物群对光动力抗微生物化学疗法的敏感性相似。本研究证实,光动力抗微生物化学疗法在体外能有效灭活解脲脲原体,有望成为解脲脲原体感染的有效替代治疗方法。     

In vitro and in vivo impairment of alpha2-adrenergic receptor-dependent antilipolysis by fatty acids in human adipose tissue.

The aim of the present study was to study the influence of fatty acids on the adrenergic control of lipolysis both in vitro and in vivo. Human subcutaneous adipose tissue explants were cultured for 48 h in the presence of 100 microM bromopalmitate (BrPal), and lipolysis was measured in isolated adipocytes. In control conditions, beta-AR-dependent activation of lipolysis by epinephrine was almost undetectable, and could be fully restored by pharmacological blockade of alpha2-AR-dependent antilipolysis. After BrPal treatment, epinephrine became fully lipolytic and was no longer influenced by alpha2-AR-blockade. Radioligand binding analysis revealed that BrPal treatment led to a significant reduction in the coupling of alpha2-AR to G proteins. In parallel, a chronic and significant increase in plasma fatty acids resulting from a 4-day high-fat diet (HFD) was accompanied by an impairment of the amplifying effect of the alpha2-AR antagonist phentolamine on exercise-induced lipolysis (measured in the subcutaneous adipose tissue with the use of a microdialysis probe) normally observed after a low-fat diet. In conclusion, in vitro and in vivo studies showed that fatty acids impair alpha2-AR-dependent antilipolysis.     

In vitro decondensation of the sperm chromatin in Holothuria tubulosa (sea cucumber) not affecting proteolysis of basic nuclear proteins

Sea urchin and sea star oocyte extracts contain proteolytic activities that are active against sperm basic nuclear proteins (SNBP). This SNBP degradation has been related to the decondensation of sperm chromatin as a possible model to male pronuclei formation. We have studied the presence of this proteolytic activity in Holothuria tubulosa (sea cucumber) and its possible relationship with sperm nuclei decondensation. The mature oocyte extracts from H. tubulosa contain a proteolytic activity to SNBP located in the macromolecular fraction of the egg‐jelly layer. SNBP degradation occurred both on sperm nuclei and on purified SNBP, histones being more easily degraded than protein Ø_o (sperm‐specific protein). SNBP degradation was found to be dependent on concentration, incubation time, presence of Ca²⁺, pH, and this activity could be a serine‐proteinase. Thermal denaturalization of the oocyte extracts (80°C, 10–15 min) inactivates its proteolytic activity on SNBP but does not affect sperm nuclei decondensation. These results would suggest that sperm nuclei decondensation occurs by a mechanism different from SNBP degradation. Thus, the sperm nuclei decondensation occurs by a thermostable factor(s) and the removal of linker SNBP (H1 and protein Ø_o) will be a first condition in the process of sperm chromatin remodeling.     

In vitro activation of human sperm induced by amphibian egg extract

In this paper we characterize a system for the activation of human sperm using cell-free extracts from Xenopus laevis eggs. We characterize the kinetics of sperm activation in terms of morphological and DNA synthetic parameters. We have optimized some of the components which are necessary for chromatin decondensation and present data to demonstrate that the in vitro process is efficient over a wide range of salt concentrations and amounts of supplementary reducing agents.     

In vitro and in vivo quality of bovine embryos in vitro produced with sex-sorted sperm

In this work we analyzed the effects of three culture systems on developmental ability of bovine embryos in vitro produced with sexed sperm, the survival to vitrification (cryologic vitrification method) of such blastocysts, and their pregnancy rates after embryo transfer to recipients, both as fresh and after vitrification/warming. Finally, we measured the accuracy of the sorting protocol by a polymerase chain reaction-based method to validate the embryo sex at blastocyst stages. We confirmed an individual effect of the bull as well as development rates of embryos produced with sorted sperm lower than embryos with unsorted sperm, independent of the culture system used. The cryoresistance to vitrification of embryos produced with sexed sperm did not differ from that of conventionally produced embryos (re-expansion rates at 24 and 48 h: 74.6% vs. 75.5%, and 64.5% vs. 68.1% for embryos produced with conventional and sorted sperm, respectively; hatching rates at 48 h: 63.55% vs. 55.5% for embryos produced with conventional and sorted sperm, respectively). Finally, no significant differences were found in pregnancy rates after the embryo transfer of fresh and vitrified/warmed blastocysts (52.8% vs. 42.0%, respectively; P > 0.05). Male and female embryos produced with sorted sperm showed the same quality in terms of developmental ability, cryoresistance, and pregnancy rates after transfer. Our culture system, coupled with the vitrification in fiber plugs, provides good quality sex-known embryos which survive vitrification at similar rates than embryos produced with conventional unsorted sperm; also it produces good pregnancy rates after transfer of sexed embryos both fresh and after vitrification and warming.