Exposure to trichloroethylene and its metabolites causes impairment of sperm fertilizing ability in mice.

Trichloroethylene (TCE) is a prevalent occupational and environmental contaminant that has been reported to cause a variety of toxic effects. Here, we have undertaken studies to test the hypothesis that TCE exposure adversely affects sperm function and fertilization. Sperm retrieved from mice exposed to TCE (1000 ppm) by inhalation for 1 to 6 weeks were incubated in vitro with eggs isolated from superovulated female mice. The number of sperm bound per egg was significantly decreased when mice were exposed to TCE for 2 and 6 weeks but not at exposures of 1 and 4 weeks. In vivo fertilization was also determined in superovulated female mice mated with males exposed to TCE for 2 to 6 weeks. The percentages of eggs fertilized, as assessed by the presence of two pronuclei, were significantly decreased after 2 and 6 weeks of TCE exposure. A slight but insignificant decrease was observed after 4 weeks of TCE exposure. The direct effects of TCE and its metabolites, chloral hydrate (CH) and trichloroethanol (TCOH), on in vitro sperm-egg binding were also investigated. Sperm-egg binding was significantly decreased when sperm were pretreated with CH (0.1-10 microg/mL). Significantly lower levels of sperm-egg binding were also detected with TCOH (0.1-10 microg/mL), although the decreases were not as pronounced as those for CH. These results showed that TCE exposure leads to impairment of sperm fertilizing ability, which may be attributed to TCE metabolites, CH, and TCOH.     

Field evaluation of the ICT Malaria Pf/Pv immunochromatographic test for the detection of asymptomatic malaria in a Plasmodium falciparum/vivax endemic area in Thailand

Rapid antigen assays provide an effective tool for the detection of malaria in symptomatic patients. However, the efficacy of these devices for detecting asymptomatic malaria, where parasite levels are normally significantly lower than in symptomatic patients, is less well established. We evaluated the efficacy of a new combined Plasmodium falciparum-Plasmodim vivax immunochromatographic test (ICT Malaria Pf/Pv) in a cross-sectional malaria survey of the village of Ban Kong Mong Tha, Kanchanaburi Provice, Thailand, from August to December 2000. A total of 1,976 bleeds were made from 559 individuals over the course of the study. Blinded microscopy of thick and thin blood films was used as the gold standard; all discordant and 10% of concordant results were cross-checked. Of 1,976 ICT Malaria Pf/Pv dipsticks tested, 98.3% (n = 1,943) performed as expected, as evidenced by the appearance of the control line. The ICT Malaria Pf/Pv test was both sensitive (100.0%) and specific (99.7 %) for the diagnosis of falciparum malaria with parasitemias of > or = 500 trophozoites/microL; however, only 15.9% (13/82) of infected individuals had parasitemia rates this high. When P. falciparum parasitemia rates were < 500/microL, the sensitivity of the diagnosis was only 23.3%, with a positive predictive value (PPV) and a negative predictive value (NPV) of 76.2 and 97.2%, respectively. The ICT Malaria Pf/Pv test was specific, but not sensitive, for the diagnosis of vivax malaria with parasite rates of > or = 500 trophozoites/microl, with sensitivity, specificity, PPV, and NPV of 66.7%, 99.9%, 66.7%, and 99.9%, respectively. At parasite rates of < 500/microL, corresponding values were 0.0%, 99.9%, 0%, and 95.1%. Because of the relatively high cost of these assays, low parasite rates found in the majority of asymptomatic individuals, and low sensitivity of this assay with rates of < 500/microl, use of this assay as a tool for active case detection is of limited value in western Thailand.     

Remodeling of the plasma membrane in preparation for sperm–egg recognition: roles of acrosomal proteins

The interaction of sperm with the egg''s extracellular matrix, the zona pellucida (ZP) is the first step of the union between male and female gametes. The molecular mechanisms of this process have been studied for the past six decades with the results obtained being both interesting and confusing. In this article, we describe our recent work, which attempts to address two lines of questions from previous studies. First, because there are numerous ZP binding proteins reported by various researchers, how do these proteins act together in sperm–ZP interaction? Second, why do a number of acrosomal proteins have ZP affinity? Are they involved mainly in the initial sperm–ZP binding or rather in anchoring acrosome reacting/reacted spermatozoa to the ZP? Our studies reveal that a number of ZP binding proteins and chaperones, extracted from the anterior sperm head plasma membrane, coexist as high molecular weight (HMW) complexes, and that these complexes in capacitated spermatozoa have preferential ability to bind to the ZP. Zonadhesin (ZAN), known as an acrosomal protein with ZP affinity, is one of these proteins in the HMW complexes. Immunoprecipitation indicates that ZAN interacts with other acrosomal proteins, proacrosin/acrosin and sp32 (ACRBP), also present in the HMW complexes. Immunodetection of ZAN and proacrosin/acrosin on spermatozoa further indicates that both proteins traffic to the sperm head surface during capacitation where the sperm acrosomal matrix is still intact, and therefore they are likely involved in the initial sperm–ZP binding step.     

Molecular basis of hereditary methaemoglobinaemia, types I and II: two novel mutations in the NADH-cytochrome b5 reductase gene

Hereditary methaemoglobinaemia, caused by deficiency of NADH-cytochrome b₅ reductase (b5R), has been classified into two types, an erythrocyte (type I) and a generalized (type II). We analysed the b5R gene of two Thai patients and found two novel mutations. The patient with type II was homozygous for a C-to-T substitution in codon 83 that changes Arg (CGA) to a stop codon (TGA), resulting in a truncated b5R without the catalytic portion. The patient with type I was homozygous for a C-to-T substitution in codon 178 causing replacement of Ala (GCG) with Val (GTG). To characterize effects of this missense mutation, we investigated enzymatic properties of mutant b5R (Ala 178 Val). Although the mutant enzyme showed normal catalytic activity, less stability and different spectra were observed. These results suggest that this substitution influenced enzyme stability due to the slight change of structure. In conclusion, the nonsense mutation led to type II because of malfunction of the truncated protein. On the other hand, the missense mutation caused type I, due to degradation of the unstable mutant enzyme with normal activities in patient's erythrocytes, because of the lack of compensation by new protein synthesis during the long life-span of erythrocytes.     

Identification of Hb Q-India (64 Asp→His) in Thailand

More than 30 different hemoglobin variants either affecting  or β globin chains have been identified in Thailand. The large variety in the different forms of hemoglobinopathy contributes to several complicated interactions, since different types of defective globin alleles are prevalent in Thailand and nearly 30-40% of the population are carriers of either  or β thalassemia (thal). Many rare and novel abnormal globin variants in Thai subjects have been identified in our laboratory within the past few years; including Hb Lepore-Hollandia, homozygous Hb Tak, Hb Dhonburi, Hb G-Makassar, Hb G-Coushatta, Hb New York, Hb Paksè and Hb Pak Num Po. In addition to these, here we report, for the first time, the identification of Hb Q-India, an innocuous  globin variant, in a Thai family with Indian ancestry. This report highlights the complexity associated with identifying unknown globin variants within a population that has a heterogeneous repertoire of globin chain disorders.     

Levels of cholesterol and phospholipids in freshly ejaculated sperm and Percoll-gradient-pelletted sperm from fertile and unexplained infertile men

Cholesterol and phospholipid levels were determined in individual sperm samples obtained from 20 fertile and 20 unexplained infertile men. The determination was performed on both washed freshly ejaculated sperm and Percoll-gradient-pelletted sperm. Although sperm cholesterol levels in unexplained infertile patients were significantly lower, i.e., 10.6 +/- 1.3 (mean +/- SD) nmol/10(7) freshly ejaculated sperm and 5.4 +/- 0.7 nmol/10(7) Percoll-gradient-pelletted sperm as compared with 19.9 +/- 1.9 nmol/10(7) and 12.6 +/- 1.5 nmol/10(7) for corresponding sperm populations in fertile donors. Motility parameters measured in 10 sperm samples of the two groups of fertile and unexplained infertile men revealed increases in the amplitude of lateral head displacement and decreases in percent of straightness in sperm tracks from unexplained infertile men.     

Effects of human sera and human serum albumin on mouse embryo culture

Human proteins normally used to supplement human in vitro fertilization—embryo transfer (IVF-ET) culture media were tested for their effects on mouse embryo development from the zygote stage. These proteins included follicular and luteal-phase maternal sera, fetal cord sera, and both human and bovine serum albumin. Our results revealed that both maternal and fetal cord sera did not permit mouse blastocyst formation. Furthermore, predialysis of the human maternal sera and removal of IgG by protein A column chromatography did not improve their support of mouse embryonic development to the blastocyst stage. Similar detrimental effects were observed with maternal sera from term-pregnant IVF-ET patients. Interestingly, these serum samples had supported the in vitro growth of the human zygotes which resulted in these patients' pregnancies. Only some batches of human serum albumin supported mouse blastocyst formation, whereas all sources of bovine serum albumin were effective in this regard. These results raise the question of the suitability of the mouse embryo culture system as a quality control for the testing of protein supplements for human IVF-ET.     

Comparison of the in vitro fertilization rate by human sperm capacitated by multiple-tube swim-up and Percoll gradient centrifugation

Two sperm preparation methods, a multiple-tube swim-up and Percoll-gradient centrifugation, were employed in our human in vitro fertilization program. The fertilization rate of these two sperm preparation methods was compared when they were employed in semen samples of <60 million motile sperm/ml. The results described here suggest that both of these methods gave a similar fertilization rate in these semen samples, i.e., 72±8% for the Percoll-gradient centrifugation method and 66±8% for the multiple-tube swim-up method.     

Compound heterozygosity for alpha0-thalassemia (- -THAI) and Hb constant spring causes severe Hb H disease

We report two unrelated cases of Hb H-Constant Spring (Hb H-CS) disease caused by a compound heterozygosity for alpha0-thalassemia (--THAI deletion) and Hb CS (alpha142, TAA-->CAA) in Thai patients. Hematological and clinical observations in the probands are more severe than those of deletional type of Hb H disease (- -/-alpha), owing to an early onset of anemia and recurrent episode of anemic crises. These cases also address the importance of the - - THAI deletion which causes a severe clinical phenotype, and that could be missed by routine screening. We suggest that testing for this mutation should be included in the screening program for the prevention and control of thalassemia in Thailand, and possibly in other countries in Southeast Asia, where alpha0-thalassemias are highly prevalent.     

Croblongifolin, a new anticancer clerodane from Croton oblongifolius

A new furoclerodane, croblongifolin, together with one known clerodane, crovatin and one known labdane, nidorellol, were isolated from the stem bark of Croton oblongifolius. Structures were established based on spectroscopic and X-ray analysis. Croblongifolin showed a significant cytotoxicity against various human tumor cell lines including HEP-G2, SW620, CHAGO, KATO3 and BT474.