Protein analysis of herpes simplex virus latency in vitro established with cycloheximide

Herpes simplex virus (HSV)-specific protein synthesis was examined during establishment of HSV latency and reactivation of virus in human embryonic lung cells treated with cycloheximide and incubated at 40.5 degrees. Eight viral proteins, identified during the first two days of establishment of latency at 40.5 degrees, were undetectable by Day 3. At least two synthesized proteins were present during the maintenance phase of latency. Reactivation of HSV (viral protein 135K) was first detected in latently infected cultures between 2 and 3 hr after superinfection with human cytomegalovirus (HCMV). During this period an 82K protein with the same molecular weight as one of the HCMV immediate-early proteins (82 and 75K) was detected in the immunoprecipitates of latently infected cultures with anti-HSV serum. Thus, this HSV latency system can be used to analyze protein synthesis and clarify reactivation of HSV by HCMV superinfection.     

Transmission risk of hepatitis C virus in assisted reproductive techniques

Medical assistance for procreation in a couple where one or both parents has hepatitis C viral infection (HCV) raises the issue of the transmission of the infection to the baby and/or of possible contamination of both the technicians and the gametes or embryos from virus-free parents in the laboratory. It becomes essential to assess transmission risk in assisted reproductive techniques in order to define clearly the management of couples according to their viral status. To define the HCV transmissibility risk in assisted reproduction related to the presence of virus in semen from infected infertile men, HCV RNA detection was performed in sera, and semen and sperm fractions obtained after Percoll gradient centrifugation. HCV RNA was detected in 5% (2/39) of the semen samples tested: in the raw semen, in the seminal fluid and in the cell pellet but never after Percoll selection. According to these results, we suggest a strategy for HCV-infected infertile men who need assistance for procreation.     

Endeometriosis: The influence of peritoneal fluid from patients with minimal stage or treated endometriosis on sperm motility parameters using computer-assisted semen analysis

The aim of this study was to determine the influence of peritonealfluid from patients with minimal stage or treated endometriosison sperm motility parameters. Peritoneal fluid aspirated atdiagnostic laparoscopy for unexplained infertility from womenduring the luteal phase of the menstrual cycle (days 20–23)was incubated for 5 h with fresh semen samples obtained frommen of recently proven fertility. Spermatozoa were preparedby a swim-up technique from unprocessed semen. Using computer-assistedsemen analysis (Hamilton-Thorn Research, MA, USA), sperm motilityand motion parameters were observed at 0, 120, 180 and 300 min.Compared with spermatozoa incubated in Earle's balanced saltsolution/human serum albumin, the percentage motility, percentageprogressive motility and progressive velocity of spermatozoaincubated in peritoneal fluid from patients without visibleendometriosis were significantly higher (P< 0.05). Maximaleffect was observed at 3 h and maintained until 5 h. We concludethat in an in-vitro study, in contrast to peritoneal fluid frompatients with minimal stage endometriosis, peritoneal fluidfrom patients with unexplained infertility and no visible endometriosiscan improve sperm motility when compared with culture medium.     

Chemical composition and protein source in the capacitation medium significantly affect the ability of human spermatozoa to undergo follicular fluid induced acrosome reaction

We studied the effect of media composition on sperm capacitation,using Biggers—Whitten—Whittingham (BWW) medium,Ham's-F10 and a modified Tyrode's medium (HSM) supplementedwith bovine serum albumin (BSA) or fetal cord serum (FCS). Weevaluated the effect of chemical environment and protein supplementationon the sperm motion parameters of curvilinear velocity and linearity,and on the ability of incubated spermatozoa to undergo follicularfluid induced acrosome reaction. Neither chemical compositionnor protein supplementation of capacitation media greatly affectedmotion parameters after 2 h incubation. Furthermore, chemicalcomposition had only a small effect on the ability of spermatozoato undergo the acrosome reaction upon exposure to follicularfluid. A higher proportion of spermatozoa underwent acrosomereaction after incubation in HSM (8% control (C); 28% follicularfluid) than in BWW (8% C, 17% follicular fluid) or Ham‘sF-10 (6% C, 19% follicular fluid). By contrast, protein sourceproved critical in determining acrosome reaction inducibility.Spermatozoa incubated in BSA-supplemented media showed a 4-foldincrease in acrosomal discharge when exposed to follicular fluid(6% C, 22% follicular fluid) compared to controls while spermatozoaincubated in FCS were unable to undergo acrosome reaction (6%C, 6% follicular fluid). Simultaneous addition of FCS to BSAcapacitation medium blocked acrosome reaction inducibility andthe late addition of BSA, after sperm incubation in FCS, didnot facilitate acrosome reaction. We propose that an inhibitorof sperm capacitation is present in FCS and therefore, the selectionof optimum incubation conditions for spermatozoa may be of criticalimportance when evaluating or treating infertile patients.     

Viral etiology of aseptic meningitis among children in southern Iran

Aseptic meningitis refers to a clinical syndrome of meningeal inflammation in which bacteria cannot be identified in the cerebrospinal fluid (CSF). The viral etiology and the epidemiological, clinical, and laboratory characteristics of aseptic meningitis among children aged 2 months to 15 years in Shiraz, southern Iran were determined. From May 2007 to April 2008, 65 patients were admitted to the hospital with aseptic meningitis. Seven viruses, non-polio human enteroviruses, mumps virus, herpes simplex virus (HSV), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), human herpes virus type 6 (HHV-6), and Epstein-Barr virus (EBV) were investigated by polymerase chain reaction (PCR) method. Viruses were detected in 30 (46.2%) patients in whom non-polio human enterovirus and mumps virus were detected in 13 (43.3%) and 11 (36.7%), respectively. The remaining 6 (20%) of the cases were caused by HSV, VZV, HCMV, and HHV-6. Haemophilus influenzae and non-polio human enterovirus were detected in one patient simultaneously. Viral meningitis was found to be more frequent during spring and summer. The majority (66.6%) of the patients were treated in the hospital for 10 days and had received antibiotics in the case of bacterial meningitis. Rapid diagnosis of viral meningitis using PCR testing of CSF can help shorten hospitalization, and avoid the unnecessary use of antibiotics.     

Human sperm responses to calcitonin, angiotensin II and fertilization-promoting peptide in prepared semen samples from normal donors and infertility patients

BACKGROUND: Fertilization-promoting peptide (FPP), angiotensin II (AII) and calcitonin, present in seminal plasma, have significant effects on mouse sperm function in vitro. This study investigated responses of uncapacitated and capacitated human sperm to these peptides, initially using samples from donors with normal semen parameters and then samples from men attending infertility clinics. METHODS: Prepared suspensions were incubated in the presence/absence of a range of peptide concentrations and assessed using chlortetracycline (CTC) analysis and the hamster oocyte penetration test. RESULTS: In uncapacitated suspensions, maximal stimulatory responses (CTC) were obtained with calcitonin at 0.5-15 nmol/l and AII at 0.3-100 nmol/l; FPP is known to be most effective at 100 nmol/l. All peptides also significantly stimulated sperm penetrating ability. Combinations of peptides at low concentrations, having no detectable effect when used singly, elicited significant responses, suggesting that they work via the same signalling pathway. In suspensions incubated in the presence of fucose to accelerate capacitation and acrosome reactions, both FPP and calcitonin, but not AII, inhibited acrosome loss; however, AII did not interfere with responses to FPP and calcitonin. Unlike samples from 15 donors, some samples from >70 patients had high proportions of capacitated and/or acrosome-reacted cells when assessed immediately following preparation. Even so, the peptides usually elicited responses similar to those obtained with donor samples and combinations of peptides inhibited spontaneous acrosome loss for at least 3 h. CONCLUSIONS: The responses obtained in vitro suggest that these peptides could have significant effects on human sperm function in vivo and could also be used effectively in infertility clinics.     

Immunofluorescence study of actin, acrosin, dynein, tubulin and hyaluronidase and their impact on in-vitro fertilization.

Using polyclonal antibodies, the distribution of actin, acrosin, dynein, tubulin and hyaluronidase has been examined by indirect immunofluorescence in sperm preparations from fertile donors and in-vitro fertilization (IVF) patients. After recording sperm parameters in native semen, spermatozoa were washed free of seminal plasma using either the swim-up or the Percoll filtration technique. Prior to insemination, aliquots of the washed sperm suspensions were prepared for antibody staining. Spermatozoa from fertile donors were analysed in order to establish the specific fluorescence patterns of each antibody and the threshold scores of normality. Immunofluorescence scores obtained from IVF patients were then analysed with respect to IVF outcome. For each tested protein, the number of normal samples were significantly lower in the group which did not fertilize and fertilization rates were significantly reduced when any of the tested proteins were scored as pathological. Normal fluorescence scores were correlated with morphology, motility, velocity and to a lesser extent with sperm concentration in native semen. On the basis of receiver-operating characteristic curves, likelihood ratios and Cohen's kappa values, the presence of acrosin and tubulin yields the most useful information on sperm functional and structural status and on its fertilizing ability.     

Human spermatozoa selected by Percoll gradient or swim-up are equally capable of binding to the human zona pellucida and undergoing the acrosome reaction.

Several techniques have been used for selecting motile spermatozoa including Percoll and albumin gradients, swim-up, and glass wool filtration. A high yield of motile spermatozoa as well as an enhancement of motility are the most desirable features of a practical method. An equally important consideration is whether or not these techniques select functionally normal spermatozoa. In this study we have compared two methods for separation of motile cells, swim-up and Percoll gradient. Normal semen samples from 12 different men were used in this study. Each sample was simultaneously processed by swim-up and Percoll gradient using modified Tyrode's medium. After the sperm concentration was adjusted to 1 x 10(7) spermatozoa/ml, the suspensions were incubated at 37 degrees C, 5% CO2 in air. In each suspension the percentage of sperm recovery, percentage of motile spermatozoa, percentage of acrosome reacted spermatozoa (either spontaneously or stimulated with human follicular fluid), percentage of zona-free hamster oocytes penetrated, and number of spermatozoa bound to the human zona pellucida were determined. The results obtained indicated that the percentage of sperm recovery was higher with the Percoll gradient than with the swim-up procedure (P less than 0.001). However, no significant differences were found between these two sperm populations in the percentage of motile cells, in the percentage of acrosome reacted spermatozoa, and in the percentage of zona-free hamster oocytes penetrated. In addition, the number of spermatozoa bound per zona pellucida was similar for spermatozoa selected by Percoll or swim-up. We conclude that there were no functional differences between the spermatozoa selected by either method.     

Detection of human immunodeficiency virus-1 RNA and DNA by extractive and in situ PCR in unprocessed semen and seminal fractions isolated by semen-washing procedure

BACKGROUND: To determine the presence of human immunodeficiency virus-1 (HIV-1) viral RNA/DNA in whole semen, in properly isolated seminal fractions and in spermatozoa after swim-up, by extractive nested PCR and to compare the detection of HIV DNA by in situ PCR (IS-PCR) with the results of nested PCR. METHODS: We tested HIV-1 RNA and DNA by nested PCR in semen and in seminal fractions from 55 patients. Non-spermatic cells and spermatozoa pellet fractions from 10 HIV-1-positive and five HIV-1-negative men were tested for proviral DNA by IS-PCR. RESULTS: All samples of spermatozoa recovered after sperm washing were free of HIV RNA. HIV RNA tested positive in seven (13%) seminal plasma samples and only in two (4.2%) whole semen of these same samples. Of the seven seminal plasma samples testing positive for HIV RNA, four men had elevated blood viral load and three an undetectable viraemia. HIV DNA by IS-PCR turned positive in three of five samples in semen of HIV-noninfected men. CONCLUSION: HIV RNA/DNA detection in the semen of HIV-infected men proves the efficacy of sperm washing with swim-up of spermatozoa. It is recommended that nested PCR be conducted on purified seminal compartments. IS-PCR is inadequate for detecting HIV in semen.     

Relationships between sex steroids of the seminal fluid and milt quality parameters of Persian sturgeon, Acipenser persicus Borodin, 1897

To investigate the role of sex steroids of the seminal fluid on sperm quality, the relationships between sex steroids and milt quality parameters (sperm motility and sperm production) were investigated in the Persian sturgeon. The seminal fluid levels of 17α,20β,21-trihydroxy-4-pregnen-3-one (20βs), and 11-ketotestosterone (11-KT) had positive relationships with sperm motility characteristics (percentage and duration of motility) and sperm density. Also, no relationships were found between other sex steroids including: Testosterone (T), progesterone (P), 17α-hydroxyprogesterone (OHP), and milt quality parameters. The good correlation of 20βs and 11-KT of the seminal fluid with sperm motility and sperm density suggests that these steroids may be important hormones involving in final maturation of the Persian sturgeon spermatozoa.     

Distribution of the receptor for advanced glycation end products in the human male reproductive tract: prevalence in men with diabetes mellitus

BACKGROUND: Diabetics have a significantly higher percentage of sperm with nuclear DNA (nDNA) fragmentation and increased levels of advanced glycation end products (AGEs), in their testis, epididymis and sperm. As the receptor for AGEs (RAGE) is important to oxidative stress and cell dysfunction, we hypothesise, that it may be involved in sperm nDNA damage. METHODS: Immunohistochemistry was performed to determine the presence of RAGE in the human testis and epididymis. A comparison of the receptor's incidence and localization on sperm from 10 diabetic and 11 non-diabetic men was conducted by blind semi-quantitative assessment of the immunostaining. Enzyme-linked immunosorbent assay analysis ascertained RAGE levels in seminal plasma and sperm from 21 diabetic and 31 non-diabetic subjects. Dual labelling immunolocalization was employed to evaluate RAGE's precise location on the sperm head. RESULTS: RAGE was found throughout the testis, caput epididymis, particularly the principle cells apical region, and on sperm acrosomes. The number of sperm displaying RAGE and the overall protein amount found in sperm and seminal plasma were significantly higher in samples from diabetic men (P < 0.01, P < 0.0001 and P < 0.0001, respectively). CONCLUSIONS: The presence of RAGE implies that it may play a central role in sperm nDNA damage particularly in diabetic men where the levels are elevated.     

Retrieval of human cytomegalovirus glycoprotein B from the infected cell surface for virus envelopment

Summary Surface biotinylation of human cytomegalovirus (HCMV)-infected fibroblasts under pulse-chase conditions was used to define the cellular route of the dominant viral envelope glycoprotein gB into the cytoplasmic compartment of viral maturational envelopment. The results showed that a major fraction of gB was re-internalized from the infected cell surface prior to incorporation into the viral envelope. Viral particles carrying biotinylated gB were subsequently released into the culture medium. Viral release appeared to be inhibited in the presence of gB-specific antibody or when infected cultures were incubated at room temperature, but was not reduced by inhibitors of cellular glycoprotein transport. To our knowledge this is the first report describing that HCMV gB is retrieved from the infected cell surface prior to viral envelopment.     

Effect of fibronectin on proteasome activity, acrosome reaction, tyrosine phosphorylation and intracellular calcium concentrations of human sperm

BACKGROUND: Previously we showed that the human sperm proteasome plays significant roles during mammalian fertilization. Here we studied the effect of fibronectin (Fn), an extracellular matrix protein present in the cumulus oophorus of the oocyte, on proteasome activity, acrosome reaction, intracellular calcium concentration ([Ca(2+)](i)) and protein tyrosine phosphorylation of human sperm. METHODS: Aliquots of motile sperm were incubated for 15 min (T0), 5 h (T5) and 18 h (T18), at 37 degrees C, 5% CO(2) and 95% air with Fn (0-100 microg/ml). The chymotrypsin- and trypsin-like activity of the proteasome was measured using the fluorogenic substrates, Suc-Leu-Leu-Val-Tyr-AMC and Boc-Gln-Ala-Arg-AMC, respectively. At T18, sperm aliquots were incubated for 15 min with Fn and/or progesterone in the presence or absence of epoxomicin (a proteasome inhibitor). The percentage of viable acrosome reacted sperm was evaluated using the Fluorescein isothiocyanate (FITC)-labeled Pisum sativum agglutinin. Tyrosine phosphorylation was evaluated by western blot and [Ca(2+)](i) using fura 2. RESULTS: Fn stimulated both enzymatic activities of the proteasome and the acrosome reaction of human sperm. Progesterone enhanced and epoxomicin drastically inhibited the effect of Fn. Fn treatment also increased the [Ca(2+)](i). Western blot analysis revealed that Fn increased tyrosine protein phosphorylation and that some proteasome subunits became tyrosine phosphorylated upon Fn treatment. CONCLUSIONS: These results suggest that Fn activates the proteasome and induces the acrosome reaction in human sperm. This effect may involve binding with specific receptors (integrins) on the sperm surface and the activation of tyrosine kinases.     

兔感染人巨细胞病毒AD169的初步探讨

目的 探讨兔对人巨细胞病毒（Human cytomegalovirus,HCMV）AD169毒株（静脉途径接种）的敏感性。方法 将HCMVAD169接种于体外培养的兔胚肺（Rabbit embryo lung,REL）细胞,观察病变,免疫组化检测病毒抗原,电镜观查细胞病毒颗粒。30只新西兰兔分别静脉途径接种1万个TCID50、100个TCID50、灭活HCMVAD169,观察动物发病情况、组织病理学变化和病毒DNA。结果 REL细胞在接种病毒后16h与HEL细胞同步开始出现细胞病变,免疫组化在接种病毒16h的细胞内查到HCMV特异抗原,在接种病毒36h的细胞核、浆内查到病毒颗粒。动物在接种病毒后7d出现病态表现,病情与接种量呈正相关。尸体解剖发现受累器官广泛,在多种组织细胞内可以查到HCMV基因。结论 HCMVAD169株在体外REL细胞中进行病毒复制。静脉途径接种HCVMAD169株可以感染家兔,通过控制病毒接种量可获得不同感染程度的模型。     

Semen characteristics in human immunodeficiency virus (HIV)- and hepatitis C (HCV)-seropositive males: predictors of the success of viral removal after sperm washing

BACKGROUND: Human immunodeficiency virus (HIV)/hepatitis C virus (HCV)-seropositive males can now father children safely, avoiding transmission risks to the mother and the children using sperm washing and nested PCR (nPCR) techniques. Nevertheless, we still lack enough data to determine the reasons why approximately 10% of the performed sperm washes remain positive, thus forcing the repetition of the treatment. Semen quality in infected males is also essential to these procedures. We aimed to determine the predictive value of the semen parameters, sperm washing procedure and the infection status for the post-wash viral positivity, as well as the correlation between the semen and the disease features. METHODS: Semen characteristics were evaluated in 136 samples provided from 125 males. We also included a control group of 125 males matched by age and length of sexual abstinence. At the time of semen retrieval, 70 of them were infected with HIV (45 also with HCV, 64.3%), and 55 of them with HCV alone. nPCR for viral detection was performed in each sample. RESULTS: Thirteen out of 136 (9.5%) of the samples were positive for one or more viral detections (HIV RNA, HIV DNA and HCV RNA, when needed). From a total of 240 nPCR viral analyses, 16 were positive (6.6%). None of the seminal parameters were adequate to predict post-wash results, nor was a positive result dependent on the volumes used in the semen wash. A positive correlation was found between post-wash progressive motility and CD4 blood levels, as well as a negative correlation between progressive motility and time of evolution of the disease in HIV-infected males. CONCLUSIONS: Semen analysis, according to the World Health Organization criteria, of HIV- and HCV-affected patients showed no differences from that of non-infected males. Moreover, low CD4 blood levels, and a long evolution of the disease do not negatively affect sperm motility.     

Frequent structural chromosome aberrations in immotile human sperm exposed to culture media

BACKGROUND: The influence of culture media or centrifugation on chromosomes of immotile human sperm was examined using ICSI into mouse oocytes. METHODS: In experiment 1, immotile and motile human sperm retrieved directly from ejaculates were injected into mouse oocytes. In experiment 2, immotile human sperm were exposed to seminal plasma or one of four kinds of culture media (HEPES-BWW, modified-BWW, modified-human tubal fluid (HTF) and Dulbecco's phosphate-buffered saline) for 1.5-2.5 h at 18 degrees C in air before microinjection. In experiment 3, immotile human sperm were centrifuged along with HEPES-BWW before microinjection. In experiment 4, frozen-thawed immotile human sperm washed with seminal plasma or HEPES-BWW were injected into mouse oocytes. The hybrid oocytes were prepared for chromosome slides at first cleavage metaphase and were then examined cytogenetically. RESULTS: In experiment 1, there was no significant difference in the incidences of structural chromosome aberrations between motile and immotile sperm (4.3% versus 5.8%). In experiment 2, culture media caused more frequent structural chromosome aberrations (14.3-32.6%) in immotile sperm than did seminal plasma (5.4%). In experiment 3, structural chromosome aberrations were found in 48.1% of the centrifuged immotile sperm, and a live/dead sperm viability test intimated that the aberrant sperm were probably dead. In experiment 4, the incidence of structural chromosome aberrations in frozen-thawed immotile sperm was significantly higher in HEPES-BWW (62.2%) than in seminal plasma (17.2%). CONCLUSIONS: The results indicate that immotile sperm do not have significantly more DNA lesions than motile sperm, although DNA of immotile sperm appears to be vulnerable to damage caused by different culture media.     

HIV particles detected in spermatozoa of patients with AIDS

In this paper the Authors describe the presence of HIV particles on and in mature spermatozoa either ejaculated by AIDS patients or incubated in vitro with HIV. Both kinds of spermatozoa have particles localized around the sperm organelles. In the first case, the nucleoid of the virus can be enveloped by a membrane-like coat or be devoid of it and form buddings in the plasma membrane. In the in vitro infected spermatozoa, only membrane enveloped nucleoids are present, and no process of budding can be found. The Authors conclude considering that the spermatozoa of the AIDS patients can be penetrated by the virus particles in different moments of their life, and show the HIV particles in different stages of their cycle: some of them have freshly penetrated the sperm, and are still contained in a membrane-like coat, others are replicated and are budding through the sperm plasma membrane. On the contrary, in vitro infected spermatozoa have only freshly penetrated virus particles, and lack buddings and membrane-free nucleoids. The presence of the HIV virus in spermatozoa is substantiated by labelling with monoclonal or polyclonal anti-HIV antibodies.     

Lead, magnesium, selenium and zinc in human seminal fluid: comparison with semen parameters and fertility

The concentrations of lead, magnesium, selenium and zinc inseminal fluid from men with variable semen quality (sperm morphology,density and motility) and fertility were determined by atomicabsorption spectrometer without or with Zeeman background correction.The mean (?SD) concentration of selenium in the samples (n =142) was 28.8 ? 9.5 µg/l, which was about a third of thecorresponding serum value (77.8 ? 13.3 µg/l, n = 140).The serum selenium level was significantly (P < 0.001) higherin infertile than in fertile men, but the seminal fluid didnot show such a difference. No correlation was obtained betweenselenium values in seminal plasma and sperm density or motility.The levels of lead in seminal fluid were very low with no correlationto the levels of magnesium, selenium and zinc or the semen qualities.The seminal fluid lead concentration was significantly (P <0.001) higher in infertile (3.6 ? 3.2 µg/l, n = 79) thanin fertile men (1.7 ? 1.0 µg/l, n = 39). Magnesium (103.5? 49.2 mg/l, n = 90) and zinc (141.1 + 71.7 mg/l, n = 157) concentrationsin seminal fluid were comparable with previous reports. Bothminerals showed a positive correlation to the seminal fluidselenium, while only zinc displayed a borderline correlationwith sperm density. The present findings indicate that the determinationof seminal fluid selenium may not offer any advantages overzinc and magnesium measurement in the fertility assessment andits role in human semen remains obscure. The low lead concentrationsin the present material is a clear indication of low industrialexposure.     

Enhanced survival of ultraviolet-irradiated herpes simplex virus in human cytomegalovirus-infected cells

Infection of primary rabbit kidney cells and Vero cells with human cytomegalovirus (HCMV) enhanced survival of uv-irradiated herpes simplex virus. In PRK cells, maximal enhancement was observed when cells were preinfected with HCMV at multiplicities of 1 to 10 PFU/cell and when HCMV-infected cells were used to assay for uv-irradiated HSV within 2 days postinfection. HCMV-Enhanced reactivation was sensitive to uv-irradiation, although virus reactivation was much more resistant to irradiation than infectivity. Furthermore, the enhancement process was sensitive to caffeine and pretreatment of cells with cycloheximide augmented the enhancement induced by HCMV infection.